ABSTRACTS – NUMERICAL LIST Abstract #1 Presenting Author: Yifei Miao, Postdoctoral Fellow, City of Hope Medical Center, Duarte, California, USA Title: Mapping Long Non-coding RNA in Chromatin Remodeling: Implications in Endothelial Gene Expression and Function Abstract #2 Presenting Author: Sangeeta Dhawan, PhD, City of Hope, Duarte, California, USA Title: Epigenetic Dysregulation of β-cell Identity and Function in Diabetes Abstract #3 Presenting Author: Giulio Pasinetti, MD, PhD, Icahn School of Medicine at Mount Sinai, New York, New York, USA Title: Childhood and Adolescent Obesity and the Long-term Cognitive Consequences during Aging Abstract #4 Presenting Author: Giulio Pasinetti, MD, PhD, Icahn School of Medicine at Mount Sinai, New York, New York, USA Title: Epigenetic Mechanisms Linking Diabetes and Cognitive Dysfunction Abstract #5 Presenting Author: Joanna DiSpirito, PhD, Harvard Medical School, Boston, Massachusetts, USA Title: Molecular Diversification of Regulatory Tregs in Parenchymal Tissues Abstract #6 Presenting Author: Assam (Sam) El-Osta, Professor, Monash University, Melbourne, Victoria, Australia Title: Epigenetic Regulatory Network Identifies Pathways Implicated in Diabetic Nephropathy Abstract #7 Presenting Author: Hyun Cheol Roh, PhD, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA Title: Temperature-Dependent Reprogramming of Beige Adipocyte Identity Abstract #8 Presenting Author: Mahua Choudhury, PhD, Texas A&M University, College Station, Texas, USA Title: Dysregulation of Epigenetic Regulators in Diet-induced Diabesity Abstract #9 Presenting Author: Rowan Beck, BS, University of North Carolina Chapel Hill, Raleigh, North Carolina, USA Title: MiRNAs as Potential Markers and Drivers of Arsenic-associated Diabetes Abstract #10

Presenting Author: Munmun Chattopadhyay, PhD, Texas Tech University Health Sciences Center, El Paso, Texas, USA Title: Alterations in Inflammatory Mediators and Histone Acetylation in Diabetic Painful Neuropathy Abstract #11 Presenting Author: Taiyi (Diana) Kuo, PhD, Columbia University, New York, New York, USA Title: An Integrated Genetic Map of Dedifferentiating β cells Identifies Human Diabetes Susceptibility Gene C2cd4a as an Effector of β cell Failure Abstract #12 Presenting Author: Arturo Mensoza, PhD, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA Title: Both TRB1 and NCOR1 Repress a Network of Transcription Factors to Control Lipid Synthesis and Sequestration Abstract #13 Presenting Author: Kristina J. Holme, Graduate Student, The University of Michigan, Ann Arbor, Michigan, USA Title: Reprograming of Feeding Behavior by Diet Abstract #14 Presenting Author: Farnaz Shamsi, PhD, Joslin Diabetes Center, Boston, Massachusetts, USA Title: Transcriptional and Epigenetic Regulation of UCP1 Gene Expression Abstract #15 Presenting Author: Matthew Wortham, Postdoctoral Scholar, University of California, San Diego, California, USA Title: Adaptation of the Insulin Secretory Response to Fasting Requires the Histone Demethylase LSD1 Abstract #16 Presenting Author: Kevin Costello, BS, City of Hope, Duarte, California, USA Title: Transposable Element Contribution to Gene Regulation in Response to Metabolic Pressures Abstract #17 Presenting Author: Ajeet Pratap Singh, Assistant Professor, Cornell University, Ithaca, New York, USA Title: MIR-375 and MIR-7 Control Intestinal Stem Cell Proliferation Abstract #18 Presenting Author: Yu-Han Hung, , Cornell University, Ithaca, New York, USA Title: Discovery of the Molecular Postdoctoral Fellow, Mechanisms that Underlie MicroRNA-29 Mediated Control of Lipogenesis in the Liver Abstract #19 Presenting Author: Dana Avrahami Tzfati, Hebrew University, Jerusalem, Israel Title: Deciphering the Epigenomic Basis of β Cell Dedifferentiation during Diabetes Abstract #20

Presenting Author: Dawn K. Coletta, PhD, University of Arizona, Tuscon, Arizona, USA Title: Epigenetic Modifications of Skeletal Muscle Contribute to the Weight Loss Induced by Roux-en-Y Gastric Bypass Surgery Abstract #21 Presenting Author: Marie-France Hivert, MD, MMSc, Harvad Medical School, Harvard Pilgrim Health Care Institute, Boston, Massachusetts, USA Title: Maternal 2-hour Glucose Levels Are Associated with Placental DNA Methylation at PDE4B, LDLR, and TNFRSF1B Loci with Functional Expression Adaptations Abstract #22 Presenting Author: Rachel Stegemann, PhD Candidate, Case Western Reserve University, Cleveland, Ohio, USA Title: Transgenerational Inheritance of Diet-Induced Obesity Resistance in Mice Abstract #23 Presenting Author: Dennis Pollow Jr., PhD, University of California, San Diego, La Jolla, California, USA Title: Systemic Inhibition of the Histone Demethylase Lsd1 Prevents High Diet-induced Glucose Intolerance and Dysregulated Hepatic Gene Expression Abstract #24 Presenting Author: Yuta Hiraike, MD, PhD, University of Tokyo, Bunkyo-ku, Tokyo, Japan Title: NFIA Co-localizes with PPARgamma and Transcriptionally Controls the Brown Fat Gene Program Abstract #25 Presenting Author: Parijat Senapati, PhD, Beckman Research Institute of City of Hope, Duarte, California, USA Title: Hyperinsulinemia Induced Changes in Chromatin Acetylation and Gene Expression in Triple Negative Breast Cancer Abstract #26 Presenting Author: Yan Chun Li, PhD, University of Chicago, Chicago, Illinois, USA Title: ATP-citrate Lyase Is an Epgenetic Regulator to Promote Nephropathy in Obesity and Type 2 Diabetes Abstract #27 Presenting Author: Amy L. Stockert, PhD, Ohio Northern University, Ada, Ohio, USA Title: Expression Changes of Metabolic Regulator FoxO1 between Early and Mid-stage Differentiation in Cinnamon Treated 3T3-L1 Abstract #28 Presenting Author: Sadhan Das, PhD, Beckman Research Institute of City of Hope, Duarte, California, USA Title: Functional Characterization of Diabetes-induced Long Non-coding RNA Dnm3oS in Macrophages Abstract #29 Presenting Author: Sadhan Das, PhD, Beckman Research Institute of City of Hope, Duarte, California, USA

Title: Regulation of Angiotensin II Actions by Enhancers and Super-enhancers in Vascular Smooth Muscle Cells Abstract #30 Presenting Author: Valerie Gagné-Ouellet, PhD Student, University of Sherbrooke, Quebec, Canada Title: Ppargc1a DNA Methylation Mediates Mitochondrial Biogenesis through Lineage Differentiation Programs in Brown Adipocytes Abstract #31 Presenting Author: Alexander Murashov, MD, PhD, East Carolina University, Greenville, North Carolina, USA Title: Long-term Exercise Induces MICRORNA and MRNA Transcriptome Changes in Murine Epididymal Sperm Abstract #32 Presenting Author: Lauren Sparks, PhD, Translational Research Institute for Metabolism and Diabetes, Orlando, Florida, USA Title: Exercise Response Heterogeneity of In Vivo Muscle Mitochondrial Functcion Is Related to Distinct Myocellular Epigenetic Profiles in Individuals with Type 2 Diabetes Abstract #33 Presenting Author: Weiming Zhang, PhD, University of Colorado Denver, Aurora, Colorado, USA Title: Epigenetic Mediation of In Utero Exposure to Gestational Diabetes (GDM) on Chidhood Adiposity Outcomes Using Bayesian Network Analysis Abstract #34 Presenting Author: Jonathan Haldeman, Graduate Student, Duke University, Durham, North Carolina, USA Title: Creation of a Versatile Adenovirus-based Epigenetic Engineering System for Diabetes Research Abstract #35 Presenting Author: Dario F. De Jesus, MSc, Joslin Diabetes Center, Boston, Massachusetts, USA Title: Epigenetic Reprogramming of Hepatic Steatosis in the Offspring of a Non-dietary Model of Insulin Resistance Abstract #36 Presenting Author: Samantha Day, PhD, The National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, Arizona, USA Title: Whole Genome Sequencing Identifies CpG-SNPs that Associate with Type 2 Diabetes in American Indians Abstract #37 Presenting Author: Bongsoo Park, Research Associate, Johns Hopkins University, Baltimore, Maryland, USA Title: Epigenomic Marks of Air Pollution and Insulin Resistance Abstract #38

Presenting Author: Frankie D. Heyward, PhD, Beth Israel Deaconess Medical Center and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA Title: Cell Type-specific Transcriptional and Epigenetic Profiles from a Rare Population of Neurons within the Arcuate Nucleus Abstract #39 Presenting Author: Chisayo Kozuka, PhD, Joslin Diabetes Center, Boston, Massachusetts, USA Title: Bromodomain-containing Proteins Play a Pivotal Role in Glucose Metabolism in Mice

PRESENTING AUTHOR DISCLOSURES It is the Association’s policy to ensure balance, independence, objectivity, and scientific rigor in

all of its educational activities. All participating planning committee members and faculty are

required to disclose to the program audience any financial relationships related to the subject

matter of this program. Disclosure information is reviewed in advance in order to manage and

resolve any possible conflicts of interest. The intent of this disclosure is to provide participants

with information on which they can make their own judgments.

Rowan Beck, BS Disclosed no conflict of interest. Munmun Chattopadhyay, PhD Disclosed no conflict of interest. Mahua Choudhury, PhD Disclosed no conflict of interest. Dawn K. Coletta, PhD Disclosed no conflict of interest. Kevin Costello, BS Disclosed no conflict of interest. Sadhan Das, PhD Disclosed no conflict of interest. Samantha Day, PhD Disclosed no conflict of interest. Joanna DiSpirito, PhD Disclosed no conflict of interest. Sangeeta Dhawan, PhD Disclosed no conflict of interest. Assam (Sam) El-Osta Disclosed no conflict of interest. Dario F. De Jesus, MSc Disclosed no conflict of interest. Valerie Gagné-Ouellet Disclosed no conflict of interest. Jonathan Haldeman Disclosed no conflict of interest.

Frankie D. Heyward, PhD Disclosed no conflict of interest. Marie-France Hivert, MD, MMSc Disclosed no conflict of interest. Yuta Hiraike, MD, PhD Disclosed no conflict of interest. Kristina J. Holme Disclosed no conflict of interest. Yu-Han Hung Disclosed no conflict of interest. Chisayo Kozuka, PhD Disclosed no conflict of interest. Taiyi (Diana) Kuo, PhD Disclosed no conflict of interest. Yan Chun Li, PhD Disclosed no conflict of interest. Arturo Mensoza, PhD Disclosed no conflict of interest. Yifei Miao Disclosed no conflict of interest. Alexander Murashov, MD, PhD Disclosed no conflict of interest. Bongsoo Park Disclosed no conflict of interest. Giulio Pasinetti, MD, PhD Disclosed no conflict of interest. Dennis Pallow, Jr. PhD Disclosed no conflict of interest. Hyun Cheol Roh, PhD Disclosed no conflict of interest. Parijat Senapati, PhD Disclosed no conflict of interest. Farnaz Shamsi, PhD Disclosed no conflict of interest.

Ajeet Pratap Singh Disclosed no conflict of interest. Lauren Sparks, PhD Disclosed no conflict of interest. Rachel Stegemann, PhD Disclosed no conflict of interest. Amy L. Stockert, PhD Disclosed no conflict of interest. Dana Avrahami Tzfati Disclosed no conflict of interest. Matthew Wortham Disclosed no conflict of interest. Weiming Zhang, PhD Disclosed no conflict of interest.

ABSTRACT #1 MAPPING LONG NON-CODING RNA IN CHROMATIN REMODELING: IMPLICATIONS IN ENDOTHELIAL GENE EXPRESSION AND FUNCTION Y Miao, F-M Lin, C-H Lou, Y-T Wang, Z Chen, City of Hope Medical Center, Duarte, CA Serving as a bioactive interface between the blood flow and the vessel wall, the vascular

endothelium senses mechanical and chemical signals under various physiological and

pathophysiological conditions, such as atherosclerosis and diabetes. Long non-coding RNAs

(lncRNAs) have emerged as novel epigenetic regulators in cardiovascular systems through

mediating diverse molecular mechanisms. In this study, we aim to identify lncRNAs that regulate

endothelial gene expression and function, specifically through chromatin remodeling. We applied

protective (e.g. statins, athero-protective flow) vs. detrimental stimuli (e.g. TNF, high glucose,

athero-prone flow) to endothelial cells and identified differentially regulated lncRNAs through

RNA-sequencing. In combination with histone chromatin immunoprecipitation-sequencing, one

group of these lncRNAs are transcribed from genomic enhancer loci and likely to act in trans.

Mapping chromatin interactome of these lncRNAs using chromatin capture technologies and DNA

FISH suggests that these lncRNAs may regulate key endothelial genes involved in NO

bioavailability, vessel development, and angiogenesis. Inhibition of these lncRNAs using locked

nucleic acid-GapmeRs or CRISPR-Cas9-mediated genomic editing decreases the expression of

several key endothelial markers; complementarily, overexpression of these lncRNAs increases

these gene expressions and improves endothelial function. Mechanistically, these lncRNAs

promote and/or enhance the recruitment of transcription factors, mediators, and RNA polymerase

to form activation centers for transcriptional induction. Additionally, these lncRNAs can be

detected in the circulation, suggesting their potential use as diagnostic markers. We anticipate

that the identification of these lncRNAs can have significant impacts on the understanding of

diseases involving endothelial dysfunction, such as diabetes complications.

ABSTRACT #2 EPIGENETIC DYSREGULATION OF BETA-CELL IDENTITY AND FUNCTION IN DIABETES A Butler, S Dhawan, City of Hope, Duarte, CA. Both type 1 and type 2 diabetes (T1D and T2D) are caused by loss of functional beta-cell mass.

T1D and T2D have in common cellular stress that induces a progressive loss of function,

impaired identity and eventual beta-cell loss. Functional defects in evolving T1D and T2D

recapitulate neonatal beta-cells, with uncoupling of ambient glucose and insulin secretion.

Diabetes has a strong hereditary component, and yet <10% of hereditary T2D can be

accounted for by genetic linkage, implying the involvement of epigenetic inheritance. We have

previously shown that epigenetic mechanisms such as DNA methylation regulate beta-cell

identity and functional maturation during development. We now show that impaired beta-cell

identity and function in diabetic mice and humans is characterized by loss of epigenetic

regulation at specific loci expressing metabolic enzymes and hormones, such as the low Km

hexokinases 1 and 2 (Hk1 and Hk2), Lactate dehydrogenase (Ldha), and Neuropeptide Y (Npy).

Further, we show that diabetic beta-cells are marked by a global shift in epigenetic signatures

such as the loss of DNA 5-hydroxymethylation (5hmC), along with altered expression of the

enzyme Tet2, which regulates this epigenetic mark. Finally, we present data showing a direct

role of Tet2 in regulation of beta-cell function. Together, these data imply that epigenetic

dysregulation is an important mechanism underpinning beta-cell failure in T2D and early T1D.

ABSTRACT #3 CHILDHOOD AND ADOLESCENT OBESITY AND THE LONG-TERM COGNITIVE CONSEQUENCES DURING AGING G Pasinetti, J Wang, R Singh, Icahn School of Medicine at Mount Sinai, New York, NY The prevalence of childhood/adolescent obesity and insulin resistance has reached an epidemic

level. Obesity’s immediate clinical impacts have been extensively studied; however, current

clinical evidence underscores the long-term implications. Our current studies explore the

impacts of brief childhood/adolescent obesity and insulin resistance on cognitive function in later

life. To mimic childhood/adolescent obesity and insulin resistance, we exposed 9-week-old

C57BL/6J mice to a high-fat diet for 15 weeks, after which the mice exhibited diet-induced

obesity and insulin resistance. We then put these mice back on a normal low-fat diet, after

which the mice exhibited normal body weight and glucose tolerance. However, a spatial

memory test in the forms of the Morris water maze (MWM) and contextual fear conditioning at

85 weeks of age showed that these mice had severe deficits in learning and long-term memory

consolidation. Mechanistic investigations identified increased expression of histone

deacetylases (HDAC)5, accompanied by reduced expression of brain-derived neurotrophic

factor, in the brains 61 weeks after the mice had been off the high-fat diet. Electrophysiology

studies showed that hippocampal slices isolated from these mice are more susceptible to

synaptic impairments compared with slices isolated from the control mice. We demonstrated

that a 15-week occurrence of obesity and insulin resistance during childhood/adolescence

induces irreversible epigenetic modifications in the brain that persist following restoration of

normal metabolic homeostasis, leading to brain synaptic dysfunction during aging. Our study

provides experimental evidence that limited early-life exposure to obesity and insulin resistance

may have long-term deleterious consequences in the brain, contributing to the

onset/progression of cognitive dysfunction during aging.

ABSTRACT #4 EPIGENETIC MECHANISMS LINKING DIABETES AND COGNITIVE DYSFUNCTION G Pasinetti, J Wang, R Singh, Icahn School of Medicine at Mount Sinai, New York, NY Emerging studies have demonstrated that diabetes-associated chromatin modifications

pertinent to epigenetic mechanisms play an important role in brain pathophysiology, cognitive

deterioration, and possibly in dementia. In mechanistic studies, using a mouse model of diet-

induced type 2 diabetes we observed changes similar to what was observed in postmortem

brains of diabetic subjects, such as altered expression of proteins involved in synaptic plasticity.

Most importantly, the elevation of epigenetic modifiers by diet induced diabetes was associated

with mitochondrial respiratory dysregulation and synaptic plasticity impairments. Inhibition of

HDAC IIa was able to restore synaptic plasticity and improve mitochondrial function in vitro

neuronal cells. Based on this evidence we continued to examine the changes in chromatin

structure and gene expression of mitochondrial and synaptic proteins in the brains of diet-

induced diabetic mice. Preliminary evidence suggested that diet-induced type 2 diabetic

conditions can causally promote expression of HDAC5 and HDAC9 in the brain of mice

coincidental with mitochondrial respiration compared to mice fed controlled diet. We are

currently exploring whether pharmacologic inhibition of HDAC class IIa with MC1568 can

prevent or treat pathologies of diet-induced type 2 diabetes, such as synaptic impairment and

energy metabolism in vivo. Ultimately, our studies will clarify whether inhibition of HDAC IIa

members can prevent diabetes-induced chromatin structure alterations of targeted genes in the

brains of diet-induced type 2 diabetic mice and lead to improved cognitive function and brain

connectivity assessed by micro MRI.

ABSTRACT #5 MOLECULAR DIVERSIFICATION OF REGULATORY T CELLS IN PARENCHYMAL TISSUES J DiSpirito, D Zemmour, R Zilionis, A Klein, C Benoist, D Mathis, Harvard Medical School, Boston, MA A resident population of regulatory T cells (Tregs) in visceral-adipose tissue (VAT) plays an anti-

inflammatory role in the context of metabolic stress from a high fat diet. In general, VAT and

other parenchymal-tissue-resident Tregs have transcriptomes distinct from those of their

counterparts in lymphoid organs. However, the molecular mechanisms, including epigenomic

modifiers, that regulate the generation, maintenance and specialized functions of tissue-Tregs

are largely unknown. We exploited advances in analyzing the chromatin-accessibility and gene-

expression profiles of small, ex vivo cell populations to explore the diversification of Treg cells in

VAT, skeletal muscle, and the colon. Our major findings were that: the unique phenotypes of

parenchymal-tissue Treg cells reflected layering of regulatory elements, encompassing pan-

tissue, tissue-restricted, and tissue-specific inputs; the chromatin landscape surrounding most

Treg genes induced in parenchymal tissues was already accessible in spleen; rather few,

shared, transcription-factor families appeared to be important drivers of tissue-Treg-restricted

gene expression, but different family members predominated in different tissues; and tissue-

specific transcription factors often collaborated with ubiquitous factors to achieve tissue-

restricted expression via feed-forward loops (FFL). In VAT Tregs, we identified a novel putative

FFL containing AP-1 and ETS family members and connected to adipose-tissue-specific genes,

as well as a peripheral node of PPAR target genes – both of which could be potential targets

for amplifying the metabolically beneficial functions of adipose-tissue Tregs.

ABSTRACT #6 EPIGENETIC REGULATORY NETWORK IDENTIFIES PATHWAYS IMPLICATED IN DIABETIC NEPHROPATHY S El-Osta, Central Clinical School, Monash University, Melbourne, Victoria, Australia To apply systems level understanding of the role of DNA methylation, it is important to

distinguish the essential sequence elements involved in regulating gene expression. This

becomes a particularly challenging task for diabetic kidney disease (DKD) when reliable

epigenetic markers such as DNA methylation are limited. While genome-wide methylation

studies are typically performed using BeadChip array technology, this does not provide

sufficient coverage to construct an epigenetic regulatory network (ERN) important because

diabetic nephropathy is considered a complex polygenic and multifactorial disorder.

Furthermore, much of the heritability of DKD remains unexplained with very few risk variants

showing robust replication in large-scale genome wide association studies. To address this

knowledge gap, we examined DNA methylation using massive parallel sequencing to describe

an ERN in the Finnish Diabetic Nephropathy (FinnDiane) cohort. The case group included 25

FinnDiane participants, and the healthy group consisted of 14 nondiabetic participants.

Methylation sequencing in leukocytes derived from the 39 individuals show differentially

methylated regions (DMRs) are associated with DKD progression. Methylation distribution at

CpG islands, shores and shelves accounted for less than 5% of the total differential count. Gene

body-related regions made up >60% of the methylation differences, with <10% localized to

exons. Integrative methylation analyses reveal 494 DMGs (differentially methylated genes) that

intersect with CTCF binding sites (181 genes with increased- and 313 genes with reduced-

methylation). Furthermore, CTCF binding sites are sensitive to the loss-of-methylation with

MTOR gain-of-function in diabetes. We provide evidence of a direct mechanism by DNA

methylation interfering with gene activity and influencing many genes within a network. We

explore why DNA methylation is important and discuss how the molecular mechanisms that

accomplish this covalent modification involve the assembly of specialized chromatin structures

on methylated DNA and are recognized by transcription factors such as CTCF. Not only does

the ERN presented here strengthen the evidence base against methylation changes merely

being an epiphenomenon but the identification of core pathways and gene targets derived from

the FinnDiane study will be a useful resource and better understanding of regulatory

mechanisms that contribute to the pathogenesis of DKD.

ABSTRACT #7 TEMPERATURE-DEPENDENT REPROGRAMMING OF BEIGE ADIPOCYTE IDENTITY HC Roh, LT Tsai and ED Rosen, Beth Israel Deaconess Medical Center, Boston, MA Beige and brown adipocytes generate heat in response to reductions in ambient temperature.

When warmed, both beige and brown adipocytes exhibit morphological ‘whitening’, but it is

unknown whether or to what extent this represents a true shift in cellular identity. Using cell

type-specific profiling in vivo, we uncover a unique paradigm of temperature-dependent

epigenomic plasticity of beige, but not brown, adipocytes, with conversion from a brown to a

white chromatin state. Despite this profound shift in cellular identity, warm whitened beige

adipocytes retain an epigenomic memory of prior cold exposure defined by an array of poised

enhancers that prime thermogenic genes for rapid response during a second bout of cold

exposure. Warm-induced whitening of beige adipocytes is driven by a transcriptional cascade

involving the glucocorticoid receptor and Zfp423. These studies identify the molecular basis of

an extraordinary example of cellular plasticity in response to environmental signals.

ABSTRACT #8 DYSREGULATION OF EPIGENETIC REGULATORS IN DIET-INDUCED DIABESITY M Choudhury1, CA Powell1*, S Meruvu1*, J Zhang1*, MH Park1, R Sonkar2, K Bhakta3, K Gutiérrez4 1Texas A&M University, College Station, TX 2University of Alabama, Birmingham, AL 3MCPHS/Umass Memorial Hospital, Corpus Christi, TX 4Instituto Potosino de Invertigacion Cientifica y Technologica, San Luis Potosi, S.L.P., Mexico *Equal contribution Modern diets rich in fat and high fructose are contributing to the epidemic of diabesity and

related complications. In this study, we investigated the sex-based temporal effects of low fat

(LFD), high fat (HFD), and high fat-high fructose (HFD-HF) diets on epigenetic regulation of

metabolically active tissues.

Both male and female mice gained significant weight when fed HFD and HFD-HF, but male

mice gained more weight overall. Male mice on HFD and HFD-HF had a worse outcome in

GTTs at 12 and 20 weeks (wks) compared to LFD. At 12 wks, female mice on HFD and HFD-

HF performed worse on GTT compared to LFD, but at 20 wks, there was no difference between

any groups. Only male mice on HFD had impaired ITT performance compared to LFD at 19 wks

while female mice showed no difference in ITT at 10 and 19 wks. HFD-HF male mice showed

decreased liver weight at 4 wks, however, at 20 wks, liver weights were significantly increased

over LFD. In contrast, female mice did not show any changes in liver weights.

Sirt3 gene expression was regulated temporally and in a sex-based manner in metabolically

active tissues. In liver, Sirt3 expression was significantly decreased in HFD-HF male mice at 12

and 20 wks, and in HFD-HF female mice at 20 wks compared to LFD. In brown adipose tissue

(BAT), at 20 wks, Sirt3 expression was significantly decreased in male and females on HFD-HF

compared to LFD and HFD. In liver, HFD-HF significantly increased miR-103/107 expression in

both male and female mice compared to LFD; however, only HFD significantly increased miR-

103/107 expression in female mice. While miR-103/107 expression levels increased significantly

in BAT in males on HFD and HFD-HF, female mice showed no difference compared to LFD. In

the kidney, global DNA methylation was downregulated in HFD-HF male mice. Consistent with

these results, MAT2a gene expression and DNMT activity were also attenuated in HFD-HF

group. Interestingly, a panel of pro-inflammatory genes was significantly increased by HFD-HF

diet in males.

In conclusion, we observed a sex-based and temporal response to HFD and HFD-HF in both

metabolic parameters and epigenetic regulation. While mice exposed to HFD and HFD-HF

exhibited diabesity parameters, there was distinct differential epigenetic regulation occurring

with the presence of fructose in the diet.

ABSTRACT #9 MIRNAS AS POTENTIAL MARKERS AND DRIVERS OF ARSENIC-ASSOCIATED DIABETES R Beck1, M Styblo1, P Sethupathy2 1University of North Carolina, Chapel Hill, NC 2Cornell University, Ithaca, NY Type 2 diabetes (T2D) is a metabolic disorder affecting 9% of the global population. While diet

and exercise can influence the risk for T2D, growing evidence indicates that the rise in T2D

cannot be attributed solely to the increasing rate of obesity. One potential factor, which is much

less studied in the context of T2D, is exposure to environmental obesogens and diabetogens.

Several population studies have found an association between exposure to inorganic arsenic

(iAs) in drinking water and an increased incidence or prevalence of diabetes. Recent mouse

studies demonstrated that iAs exposure leads to impaired fasting glucose and/or glucose

intolerance. The mechanism(s) underlying iAs associated metabolic dysfunction are poorly

understood, but microRNAs (miRNAs) have emerged as attractive candidates. MiRNAs are

environmentally-responsive, short non-coding RNAs that regulate gene expression and are

stably detected in circulation. They have been reported in independent studies as regulators of

insulin secretion and as responders to arsenic exposure in cells and in vivo, but these findings

have not been directly linked. We hypothesize that dysregulation of miRNAs may contribute to,

and/or serve as biomarkers for, defects in insulin secretion upon exposure to iAs. To test this

hypothesis, rat insulinoma cells (INS-1 832/13) were treated with 0, 0.5, of 1.0 µM iAs for 24

hours. We found that iAs significantly inhibited glucose-stimulated insulin secretion (GSIS) in a

dose-dependent manner. We observed significant (P < 0.01) changes in the expression of three

miRNAs: miR-29b (+1.7-fold), miR-146a (+1.7-fold), and miR-217 (-2.1-fold), in INS-1 cells upon

exposure to iAs. We propose that dysregulation of these three miRNAs may be at least partially

responsible for the inhibition of GSIS after iAs exposure. Furthermore, we are performing gain-

and loss-of-function studies in INS-1 cells to determine whether miR-217 over-expression and

miR-29b/miR-146a suppression mitigate the negative effect of iAs on GSIS. In addition, to test

whether miRNAs are candidate markers of iAs-associated diabetes, we are examining miRNA

levels in fasting plasma collected from a cohort of individuals exposed to iAs in drinking water.

The goal is to correlate these miRNAs with already-measured metabolic parameters such as

insulin and glucose, as well as with levels of iAs and its metabolites in plasma and urine.

Results of this study will help characterize the roles of miRNAs in iAs-induced islet dysfunction

and differentiate these from the mechanisms involved in traditional obesity-induced T2D.

ABSTRACT #10 ALTERATIONS IN INFLAMMATORY MEDIATORS AND HISTONE ACETYLATION IN DIABETIC PAINFUL NEUROPATHY V Thakur, J Sadanandan, M Gonzalez, R Solis, P Quesada and M Chattopadhyay, Texas Tech University Health Sciences Center, El Paso, TX, USA Peripheral sensory neuropathy is one of the most common complications of diabetes.

Accumulating evidence suggests that chronic low-grade inflammation is involved in the

pathogenesis of the disease. We hypothesize that hyperglycemia causes changes in histone

acetylation and release of inflammatory mediators in the peripheral nervous system of diabetic

animals with painful neuropathy; therefore, blocking this increase will prevent or delay the

development of neuropathy. High mobility group box 1 (HMGB1), a nuclear protein released by

injured and severely stressed cells, promotes cytokine release via histone acetylation and its

interaction with the Toll-like receptor (TLR). In this study we investigated the changes in

inflammatory mediators and histone modifications in the dorsal root ganglia (DRG) and spinal

cord dorsal horn neurons as well as compared the changes in behavior with treatment. Type 2

diabetic (T2D) animals with pain were treated with HMGB1 inhibitor Glycyrrhizin (GLC) for 2 days

a week for 3 weeks at 50 mg/kg I.P. injection 6 weeks after diabetes. T2D animals demonstrated

significant changes in thermal hyperalgesia manifested by a decrease in withdrawal latency to

heat, mechanical hyperalgesia as measured by the Randall Sellito method of paw pressure at 6

weeks after diabetes and also exhibited marked increases in HMGB1, IL1β, TLR4 and H3K9

acetylation as determined by the Western blot analysis and immunohistochemistry. To determine

whether increased acetylation of H3K9 is responsible for changes in TLR4 level in the

pathogenesis of the painful neuropathy in diabetic animals, we analyzed the T2D animals with

treatment or without treatment at 3 weeks after treatment. Our results show that animals treated

with GLC had significant decrease in thermal hyperalgesia along with changes in histone

acetylation and expression of inflammatory mediators. This preliminary study suggests that

HMGB1 and histone acetylation play an important role in the inflammatory aspect of the painful

neuropathy in T1D animals and may provide a novel treatment approach for this difficult-to-treat

complication of diabetes.

ABSTRACT #11 AN INTEGRATED GENETIC MAP OF DEDIFFERENTIATING ΒETA CELLS IDENTIFIES HUMAN DIABETES SUSCEPTIBILITY GENE C2CD4A AS AN EFFECTOR OF ΒETA CELL FAILURE Taiyi Kuo1, Manashree Damle2, Mitchell A. Lazar2, Domenico Accili1

1Department of Medicine and Berrie Diabetes Center, Columbia University College of Physicians and Surgeons, New York, NY 2The Institute for Diabetes, Obesity, and Metabolism, and Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

Diabetic β cell failure is associated with β cell dedifferentiation. Here, we integrated genome-

wide histone modification and transcriptome data from chemically sorted β cells of multiparity-

induced diabetic mice to identify effectors of dedifferentiation, and their relationship with the

genetic susceptibility to β cell dysfunction in humans. Diabetic β cells demonstrate skewed

Hnf4α and Pax6 enhancer selection, conserved bivalent histone characteristic of human

endocrine progenitors, activation of α cell signature genes, and silencing of the insulin genes.

We detected combined alterations of histone marks and transcript levels encoding C2cd4a, a

human type 2 diabetes susceptibility gene across multiple ethnic groups. We discovered a

FoxO1-associated super-enhancer, as well as a heretofore unknown long-noncoding RNA, that

potentially governs C2cd4a expression. Thus, multiple lines of evidence point to C2cd4a as a

key effector of β cell failure.

Graphical Abstract

Dedifferentiating cell

FoxO1

enhancer

Hnf4

enhancer

Pax6

enhancer

H3K27ac

H3K4me3

Ins1, Ins2 H3K4me3

T2D GWAS gene

C2cd4a

H3K4me3

cell signature gene

Gc

H3K27me3

H3K4me3

bivalent Wnt signaling genes

ABSTRACT #12 BOTH TRB1 AND NCOR1 REPRESS A NETWORK OF TRANSCRIPTION FACTORS TO CONTROL LIPID SYNTHESIS AND SEQUESTRATION A Mendoza, L Al-Sowaimel, A Hollenberg, Beth Israel Deaconess Medical Center, Boston, MA Thyroid hormones (TH) are major modulators of lipid synthesis and oxidation in mammals. T3

actions are mediated by the thyroid hormone receptors (TR) that control the expression of target

genes by modulating their rate of histone acetylation. In the absence of T3, the TR recruits the

nuclear receptor corepressor (NCoR1), which is the scaffold of a multiprotein complex that

includes histone deacetylase 3 (HDAC3). Binding of T3 to the TR, induces the dismissal of the

corepressor and facilitates the interaction with coactivators that increase histone acetylation.

Recently, in our laboratory we have developed a series of animal models that allowed us to test

the actions of T3 in vivo. Thus, we have observed that the liver-specific ablation of NCoR1

caused hepatic steatosis, which was consistent with upregulation of lipogenic T3-targets.

Therefore, we hypothesized that the deletion of NCoR1 increases TR’s ligand-sensitivity. Here,

we tested this by deleting TRB1 and NCoR1 (NTKO) together in liver of mice. Our results

showed that NTKO mice developed hepatic steatosis that could not be ameliorated by T3

treatment. This was consistent with higher activation of lipogenic genes such as Acaca, Scd1,

and Fasn in NTKO in contrast to Wt and NCoR1KO mice. In this context, ChERBP beta was

dramatically induced in NTKO mice and could mediate the hepatic steatosis seen. Moreover,

perilipin2 (PLIN2), which coats lipid droplets, was activated in NTKO. Interestingly, this effect

was secondary to TRB1 deletion, given the lack of response to thyroid status of PLIN2. The

higher gene activation in NTKO in contrast to NCoR1KO suggests that additional nuclear

receptors could have enhanced activity in NTKO. Indeed, several receptors were upregulated in

NTKO, including Rev-erbα and β, NR0b2 and NR1i3, which in turn could mediate the enhanced

gene expression caused by deletion of NCoR1 and TRB1. Also, the deletion of TRB1 may relive

a repression mechanism that was not overturned by deletion of NCoR1 alone. The increased

lipogenesis and lipid sequestration in TNKO mice would be beneficial for glucose metabolism.

Indeed, Glut4 was activated in NTKO mice, suggesting increased insulin sensitivity and glucose

tolerance.

Supported by American Diabetes Association 1-17-PMF-007 (A.M.), and NIH grants DK056123

and DK098525 (A.N.H.).

ABSTRACT #13 REPROGRAMMING OF FEEDING BEHAVIOR BY DIET Kristina J Holme, Christina May, Katty Wu, Daniel Wilinski, Monica Dus, The University of Michigan, Ann Arbor, MI While we understand how changes in the environment such as temperature and light direct

animal behavior by acting acutely on neural circuits, we know less about the how the

environment can lead to persistent changes in brain and behavior. Tackling this question has

been challenging because it requires having a circuit-based understanding of the behavior and

a mechanistic way to study how neural connections are changed by the environment. The

reshaping of circuits that regulate food intake by a hyper-caloric diet in Drosophila melanogaster

provides an attractive model for studying this question because the circuits are mapped, the

behavior is easily quantifiable, and the environmental variables are simple to measure. We

found that animals fed a Western style high-calorie diet show profound deregulation of feeding

states: they incorrectly process the nutritional value of food, eat more, and become obese. We

will present data showing how these behaviors are mediated by the metabolic-transcriptional

reprogramming of distinct feeding circuits by diet and how their effect is persistent even after

animals are returned to the control diet.

ABSTRACT #14 TRANSCRIPTIONAL AND EPIGENETIC REGULATION OF UCP1 GENE EXPRESSION Farnaz Shamsi1, Morten Lundh2, Tian Lian Huang1, Matthew D. Lynes1, Yu-Hua Tseng, 1Joslin Diabetes Center, Boston, MA 2University of Copenhagen, Blegdansvej, Copenhagen, Denmark Brown and beige adipose tissue (BAT) play a central role in the regulation of energy

expenditure in response to environmental changes, such as cold and diet. Therapeutic

approaches targeting brown and white adipose tissue (WAT), and modulating the energy-

dissipating capacity of these tissues, hold great promise for the treatment of obesity and its

comorbidities. Uncoupling protein 1 (UCP1) is the defining marker for brown/beige adipocytes

and plays a key role for BAT-mediated thermogenesis.

To identify novel transcriptional and epigenetic regulators involved in UCP1 expression, we

applied the CRISPR engineered DNA-binding molecule-mediated chromatin

immunoprecipitation (enChIP) in murine brown preadipocytes and adipocytes. Specific genomic

regions of UCP1 were targeted using a flag-tagged, catalytically inactive form of Cas9 (dCas9)

and guide RNAs that recognize the 3kb upstream regulatory region of UCP1. Using this

strategy, we specifically isolated the UCP1 promoter and, subsequently, purified the interacting

proteins. The characterization of the proteins associated with the UCP1 regulatory region using

mass spectrometry allowed us to identify the nuclear receptor co-activator Flightless-1 (FLII)

and its binding partner, Leucine Rich Repeat (in FLII) Interacting Protein 1 (LRRFIP1). In

addition, results showed that histone arginine methyltransferase PRMT1 was enriched on the

UCP1 promoter. Chromatin Immunoprecipitation of histone modifications revealed dynamic

regulation of the histone arginine methylations that modulate UCP1 expression under different

conditions. These novel factors are involved in the local remodeling of the chromatin structure,

independent of the canonical transcription factors previously implicated in UCP1 expression.

Given the unbiased nature of our approach, we uncovered the novel transcriptional and

epigenetic factors that regulate UCP1 gene expression. Considering the key role of BAT in the

regulation of energy expenditure, these findings may offer new therapeutic targets to combat

obesity and insulin resistance through increasing UCP1 expression and, subsequently,

enhancing BAT activity and whole body energy metabolism.

ABSTRACT #15 ADAPTATION OF THE INSULIN SECRETORY RESPONSE TO FASTING REQUIRES THE HISTONE DEMETHYLASE LSD1 M Wortham, M Sander, University of California San Diego, La Jolla, CA The ability of pancreatic β-cells to adapt the insulin secretory response to fluctuating insulin

demand is critical for the maintenance of blood glucose homeostasis. One potential link

between nutrient state and adaptive insulin secretion is the epigenome. As several chromatin-

modifying enzymes utilize metabolic intermediates as substrates, these enzymes could sense

metabolic signals and enact changes to β-cell function. To test this model, we have investigated

the effects of feeding and fasting on chromatin state and β-cell function. Islets isolated from

fasted mice exhibited reduced glucose-stimulated insulin secretion that coincided with extensive

deacetylation of histone H3K27. Genes linked to fasting-regulated H3K27ac sites belonged to

functional categories associated with metabolism and nutrient sensing. Importantly, we found

that fasting-responsive sites were enriched for binding by the histone demethylase Lsd1, which

has been shown to regulate chromatin in response to metabolic stimuli. To assess the role of

Lsd1 in β-cell adaptation, we conditionally deleted the Lsd1 gene in β-cells of adult mice

(hereafter Lsd1Δβ mice). Inactivation of Lsd1 resulted in hyperinsulinemia and hypoglycemia,

which was exacerbated by fasting. Accordingly, islets from Lsd1Δβ mice exhibited basal insulin

hypersecretion in vitro. ChIP-seq analysis revealed increased histone methylation at fasting-

regulated sites in Lsd1Δβ islets. This dysregulation at the level of chromatin was accompanied by

aberrant expression of associated target genes. Particularly striking was the abnormal

expression of glycolytic enzyme genes, resulting in accelerated glucose metabolism. Analysis in

humans further suggested that the role of LSD1 in the regulation of insulin secretion is

conserved. We found that LSD1 genomic binding sites in human islets are enriched for genetic

variants associated with fasting glucose levels in genome-wide association studies. Combined,

our data suggest that Lsd1 functions as a nutrient sensor in the β-cell that adjusts glucose

metabolism and insulin secretion in response to changing insulin demand.

ABSTRACT #16 TRANSPOSABLE ELEMENT CONTRIBUTION TO GENE REGULATION IN RESPONSE TO METABOLIC PRESSURES Kevin Costello1, 2, Juan Du1, Candi Trac1, Dustin E Schones1, 2 1City of Hope, Beckman Research Institute, Duarte, CA 2City of Hope, Irell & Manella Graduate School, Duarte, CA Transposable elements (TEs) are parasitic DNA sequences with the ability to propagate

throughout genomes. These elements have been adapted to be key regulators of many genes

and are involved in the establishment of gene regulatory networks. Gene regulatory networks

are important to activate many genes at once that perform similar functions (i.e. inflammatory

pathways). Many TE families, which are defined by having homologous sequences, are

enriched for transcription factor binding motifs, such as STAT3 binding sites, or insulator

sequences, such as CTCF binding sites. When a TE containing a functional motif inserts near a

gene, the regulation of that gene can be altered. This process can place many genes under the

control of the same transcription factors, leading to the creation of a gene regulatory network.

Additionally, given the deleterious nature of transposition, TEs are regularly targeted by

heterochromatin marks, further impacting gene regulation. Metabolic activity can have a

profound effect on the regulation and expression of many genes. High fat diet in mice can lead

to the altered availability of numerous TEs, as well as alter the availability of the motifs found on

these TEs. We find that numerous LINEs have enriched availability of many inflammatory motifs

when treated with high fat diet compared to a control diet. We have additionally observed

variation in TE accessibility across recombinant inbred mouse strains with unique metabolic

phenotypes. We observed a possible correlation between the motif availability for a number of

TEs and the triglyceride content of the mice. This could indicate that TE variation across mouse

strains could cause a change in lipid metabolism of the mice and be responsible for variation of

weight observed for each strain. The altered patterns of TE availability in response to metabolic

stresses could have a profound effect on genomic expression and be responsible for altered

phenotypic effects across strains of recombinant inbred mice.

ABSTRACT #17

MIR-375 AND MIR-7 CONTROL INTESTINAL STEM CELL PROLIFERATION A Singh1, M Shanahan1, M Czerwinski2, M Kanke1, A Bonfini1, V Rinaldi1, Y-H Hung1, B Peck2, E Curry3, J Schimenti1, N Buchon1, J Spence, and P Sethupathy 1Cornell University, Ithaca, NY 2University of Michigan, Ann Arbor, MI 3University of North Carolina, Chapel Hill, NC The intestinal epithelium (IE) is a rapidly renewing tissue in the body, with a turnover rate of ~4-

5 days. This rapid renewal, and therefore IE homeostasis, depends upon a small population of

actively cycling intestinal stem cells (ISCs) located in the IE crypt. Several factors can influence

the behavior of ISCs and thereby regulate IE function. However, the underlying molecular

mechanisms are poorly understood. Recently we demonstrated that miRNAs in ISCs are highly

sensitive to the presence of commensal microbes, leading to the hypothesis that they are

important players in the regulation of IE homeostasis. In the present study, we focus on two

specific miRNAs, miR-375 and miR-7. We first demonstrated that these miRNAs are robustly

expressed in ISCs according to four independent methods of ISC isolation, including FACS-

based sorting of Lgr5+ cells. We found that miR-375 in particular is highly enriched in ISCs

relative to the other cell types of the IE, except for enteroendocrine cells where its expression is

the highest. We then showed that a 5-month obesogenic high-fat diet (HFD), which boosts ISC

proliferation, leads to a significant reduction in the levels of miR-375 and miR-7 in ISCs.

Moreover, integrative transcriptomic analysis revealed that miR-375 is a candidate master

regulator of genes that are significantly up-regulated by HFD. Robust suppression of miR-375 or

miR-7 in 3-D mouse enteroids established from the jejunum of wild-type, Sox9-EGFP, or Lgr5-

EGFP reporter mice, led to a hyper-proliferative phenotype. Notably, similar loss-of-function

studies are ongoing in 3-D human enteroids in order to validate these findings. Data from

CRISPR/Cas9 genetic deletion studies showed that miR-375-/- mice exhibit significant increases

in jejunal IE crypt proliferation. Currently, functional studies are being performed in Drosophila

melanogaster (Dme), a species that is evolutionarily distant from mouse and human, but in

which miR-7 and miR-375 are conserved. Early findings suggest that miR-7 may regulate ISC

proliferation also in the Dme midgut. The next steps are to define the key targets of miR-375

and miR-7 and also to determine whether these too are as evolutionarily conserved as the

miRNAs themselves. Our study has identified critical miRNAs in the response of the intestinal

crypt to environmental stimuli.

ABSTRACT #18 DISCOVERY OF THE MOLECULAR MECHANISMS THAT UNDERLIE MICRORNA-29 MEDIATED CONTROL OF LIPOGENESIS IN THE LIVER Y-H Hung1, C Kurtz2, M Kanke2, M Hussain3, P White4, X Li5, P Sethupathy1

1Cornell University, Ithaca, NY 2University of North Carolina, Chapel Hill, NC 3NYU Winthrop, Mineola, NY 4Duke Molecular Physiology Institute, Durham, NC 5NIH/National Institute of Environmental Health Sciences, Research Triangle Park, NC

MicroRNAs (miRNAs) are important regulators of diverse metabolic pathways. Emerging

evidence suggests their roles as etiological factors and/or potential therapeutic targets for a

wide spectrum of diseases including type 2 diabetes (T2D) and hyperlipidemia. Previously, we

reported that miR-29 is aberrantly elevated in the liver of rodent models of obesity or overt T2D.

Furthermore, we demonstrated that inhibition of miR-29 leads to a significant reduction in

circulating lipids (~40% for cholesterol and ~15% for triglycerides) in chow-diet fed wide type

(WT) C57BL/6J mice. However, the molecular mechanisms underlying this dramatic lipid-

lowering effect remains unclear. We hypothesized that miR-29 controls lipid synthesis in the

liver in part through regulation of Sirtuin-1 (Sirt1), which we have previously reported as a

putative target of miR-29, and which others have shown is involved in the suppression of

hepatic lipid synthesis. In the present study, we first show that miR-29 is aberrantly elevated in

the liver of Zucker Fatty (ZF) obese rats compared to lean littermates. Furthermore, treatment of

ZF obese rats with 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2), which improves

insulin sensitivity and reduces hepatic lipid content, restores the levels of hepatic miR-29. Next,

we demonstrate that suppression of miR-29 by systemic administration of locked nucleic acid

(LNA) anti-sense inhibitors (20 mg/kg) in chow-fed WT C57BL6/J mice leads to a robust

reduction in de novo hepatic lipid synthesis. To determine whether this result is dependent on

Sirt1, we have repeated the experiment in mice with Sirt1 liver-specific knock-out (SLKO). The

findings from this study will help elucidate the centrality of Sirt1 in the molecular network through

which miR-29 regulates lipid synthesis and potentially other metabolic processes in the liver.

ABSTRACT #19 DECIPHERING THE EPIGENOMIC BASIS OF BETA CELL DEDIFFERENTIATION DURING DIABETES D. Avrahami1, D. Bernstein, M. Golson, A Ackerman J.Schug, L. Hee-Woong KJ Won, A. Naji, C Liu, B Glaser and KH Kaestner2 1Hebrew University, Jerusalem, Israel 2University of Pennsylvania Perelman School of Medicine, Philadelphia, PA Beta-cell dysfunction is a hallmark of diabetes. Recent single cell RNA-Sequencing studies of

human islet cells from diabetic and non-diabetic donors revealed partial de-differentiation of beta–

cell towards a premature state and a shift in the expression profile towards other islet cell types

such as alpha cells. This apparent islet cell plasticity in response to a chronic metabolic stress

might enable a new therapeutic approach if we can develop methods for its reversal. However, it

is still unclear to what extent de-differentiation of beta cells can be reversed, and whether it

involves semi-stable epigenomic changes such as DNA methylation. To investigate this issue, we

profiled the methylome and transcriptome of human alpha and beta-cells across various ages and

in diabetics using RNA-Seq and whole-genome bisulfite sequencing (WGBS). We discovered

remarkable differences between the methylome of alpha and beta cells as early as three months

of age. Strikingly, while distal regulatory elements of beta-cell function genes become

demethylated with maturity, alpha cells show the opposite trend of de novo methylation of distal

elements. Next, we employed our map of differential methylated regions during the human alpha

and beta-cell maturation process to analyze the methylation changes that occur in alpha and beta

cells of type 2 diabetics. These novel epigenomic datasets allow us to identify specific loci for

targeted epigenetic modifications that might prove useful for the reversal of de-differentiation in

β-cells from T2D patients, thus restoring functionality.

ABSTRACT #20 EPIGENETIC MODIFICATIONS OF SKELETAL MUSCLE CONTRIBUTE TO THE WEIGHT LOSS INDUCED BY ROUX-EN-Y GASTRIC BYPASS SURGERY LR Roust, SE Day, LA Garcia, RL Coletta, LE Campbell, TR Benjamin, EA De Filippis, LJ Mandarino, JA Madura II, DK Coletta, University of Arizona, Tucson, AZ The mechanisms of weight loss and metabolic improvements following Roux-en-Y gastric bypass

(RYGB) surgery are incompletely understood but epigenetic modifications are likely to contribute.

The aim of our study was to investigate skeletal muscle DNA methylation after weight loss induced

by bariatric surgery. Muscle biopsies were obtained basally from seven insulin-resistant morbidly

obese (BMI >40 kg/m2) female subjects (45.1±3.6 years) pre- and 3 months post-RYGB with

euglycemic hyperinsulinemic clamps to assess insulin sensitivity. Four lean (BMI <25 kg/m2)

females (38.5±5.8 years) served as controls. We performed the next generation methylation

reduced representation bisulfite sequencing on DNA isolated from the vastus lateralis muscle

biopsies. We focused our methylation analysis on sites that fell within the promoters and

untranslated regions (5’ and 3’UTR), since these regions are typically involved in the initiation of

gene transcription. MethylSig analysis identified 20 differentially methylated cytosines (DMCs)

that were significantly altered in methylation post-RYGB versus pre-RYGB (Benjamini Hochberg

q<0.05). Of these, 7 DMCs (chr1:91184127 [BARHL2]; chr3:113160431 [WDR52]; chr11:628172

[CDHR5/SCT]; chr14:24666733 [TM9SF1]; chr15:62457572 [C2CD4B]; chr17:45330989 [ITGB3]

and chrX:41782532 [CASK]) post-RYGB surgery were normalized to levels similar to those

observed in lean controls. Transcription factor binding analysis of the DMCs identified various

transcriptional regulators (AhR:Arnt, AP-2alphaA, Pax-5, p53). Our results demonstrate that

weight loss after RYGB alters the epigenome through DNA methylation, and highlights novel

genes that may contribute to the metabolic improvements observed post-RYGB.

ABSTRACT #21 MATERNAL 2-HOUR GLUCOSE LEVELS ARE ASSOCIATED WITH PLACENTAL DNA METHYLATION AT PDE4B, LDLR, AND TNFRSF1B LOCI WITH FUNCTIONAL EXPRESSION ADAPTATIONS MF Hivert1, A Cardenas1, V Gagné-Ouellet2, P. Perron3, L. Bouchard2

1Harvard Medical School, Harvard Pilgrim Health Care Institute, Boston, MA 2Université de Sherbrooke, Quebec, Canada 3CIUSSS de l’Estrie – CHUS, Sherbrooke, Quebec, Canada Background: Maternal hyperglycemia during pregnancy is associated with fetal growth and adverse perinatal and developmental outcomes. Placental adaptations to maternal hyperglycemia have been hypothesized to be part of the pathophysiology explaining these associations, yet exact mechanisms remain unknown. Methods: We conducted an epigenome-wide association study of prenatal maternal glucose and placental DNA methylation among 448 mother-infant pairs in Gen3G, a prospective birth-cohort. Women without pre-existing diabetes were recruited during 1st trimester of pregnancy and followed until delivery. All women performed a standard 2h 75-gram oral glucose tolerance test at 24-28 weeks. At delivery, we collected placenta samples and measured DNA methylation using Illumina MethylationEPIC BeadChip. We analyzed 791,131 autosomal CpGs using robust

linear regression models (controlling for FDR q<0.05). We quantified gene expression in 104

randomly selected placenta samples from the same cohort using real-time quantitative PCR. Results: Maternal 2h-glucose was strongly associated with lower DNA methylation levels of 4

CpGs within a regulatory region of PDE4B (top signal at cg07734160: β= -1.16, CI: [-1.5, -0.8];

P=1.2x10-9). We also observed that DNA methylation at all four CpG sites was significantly

correlated with PDE4B mRNA levels in placenta (r= -0.35 to -0.26, P<0.01). Furthermore, CpG

sites located within LDLR, TNFRSF1B and BLM were associated with maternal 2-hour glucose. Methylation at LDLR and TNFRSF1B loci were correlated with mRNA levels of their respective

gene (LDLR r: -0.33, P<0.001; TNFRSF1B r: 0.21, P=0.003). Conclusions: Maternal glucose response is associated with placental DNA methylation at several genomic loci, particularly within the PDE4B gene. PDE4B encodes a cAMP-specific phosphodiesterase that is a key regulator of many physiological processes and has been postulated to have a role in placental inflammatory pathways and prematurity.

ABSTRACT #22 TRANSGENERATIONAL INHERITANCE OF DIET-INDUCED OBESITY RESISTANCE IN MICE R Stegemann, D Buchner, Case Western Reserve University, Cleveland, OH Obesity affects over one billion people worldwide. Individuals that are overweight are at greater

risk for other disorders, such as type 2 diabetes and cardiovascular disease; making obesity the

sixth leading risk factor for loss of health and life worldwide. Obesity is a complex disease with

heritable factors accounting for 45-75% of body weight differences among individuals. Although

risk variants have been identified, they account for a small portion of the heritability. We

hypothesize that transgenerational epigenetic effects account for some of the “missing

heritability” associated with obesity.

Transgenerational epigenetic inheritance refers to heritable phenotypes, not caused by DNA

sequence changes, being transmitted across multiple generations without the transmission of

the original triggering event. Our lab used a congenic mapping approach to identify quantitative

trait loci (QTL) that confer resistance to diet-induced obesity between the obesity susceptible

mouse strain C57BL/6J and the obesity-resistant mouse strain A/J. A critical benefit of congenic

strains is the ability to generate genetically identical offspring from genetically different parents.

This provides a robust mechanism such that epigenetic inheritance can be studied in the

complete absence of any confounding genetic variation. This enabled us, in contrast to many

epigenetic studies, to focus on transgenerational inheritance that is triggered by non-transmitted

genetic variants rather than environmental exposures. Using this approach, we previously

identified the QTL Obrq2a, and demonstrated that it was inherited in a transgenerational

manner via the paternal germline. Eight new sub-congenic strains spanning Obrq2a were

generated and are being used for higher resolution QTL mapping. These strains will provide the

resources to identify epigenomic and transcriptomic modifications associated with the

transgenerational inheritance of diet-induced obesity resistance and will also identify a new

gene(s) that control this atypical inheritance. Collectively these studies will provide new

mechanistic insight into the genetics of epigenetics, which is a poorly understood and

underappreciated process.

ABSTRACT #23 SYSTEMIC INHIBITION OF THE HISTONE DEMETHYLASE LSD1 PREVENTS HIGH FAT DIET-INDUCED GLUCOSE INTOLERANCE AND DYSREGULATED HEPATIC GENE EXPRESSION Dennis Pollow, Matthew Wortham, Maike Sander, University of California, San Diego, CA Emerging evidence suggests that epigenetic mechanisms contribute to dysregulation of

gluconeogenesis in the liver and development of fatty liver disease as a result of chronic nutrient

excess. Therefore, the identification of epigenetic regulators that link nutrient cues to gene

regulation in the liver could further our understanding of disease pathogenesis and provide

novel therapeutic targets for the treatment of metabolic disease. We sought to determine the

contribution of the nutrient-responsive histone demethylase Lsd1 to high fat diet (HFD)-induced

glucose intolerance. To this end, male C57BL/6J mice were fed a chow diet or HFD (60% fat)

for 8 weeks with or without daily administration of an irreversible and selective Lsd1 inhibitor.

Systemic inhibition of Lsd1 prevented HFD-induced weight gain and attenuated glucose

intolerance and hyperinsulinemia observed in response to HFD feeding. HFD-induced hepatic

steatosis was also prevented by Lsd1 inhibition. Comparison of liver RNA-seq profiles from

HFD- and chow-fed mice revealed 2,116 differentially expressed genes. Systemic Lsd1

inhibition normalized expression levels of 958 genes altered by HFD feeding, including critical

gluconeogenic and lipogenic genes such as G6pc, Pck1, Pklr, Foxo1, Lpin1, Plin2 and Gpat4.

These results indicate a critical role for Lsd1 in mediating gene expression changes in the liver

that are associated with HFD-induced metabolic dysfunction. Thus, targeting Lsd1 could be a

promising approach to prevent liver disease in response to chronic overnutrition.

ABSTRACT #24 NFIA CO-LOCALIZES WITH PPARgamma AND TRANSCRIPTIONALLY CONTROLS THE BROWN FAT GENE PROGRAM Y Hiraike, H Waki, T Yamauchi, T Kadowaki, University of Tokyo, Tokyo, Japan Brown fat dissipates energy in the form of heat by means of the uncoupling protein-1 (UCP1) on

the mitochondrial inner membrane. In humans, brown fat activity is inversely correlated with

body mass index. And several pilot studies have shown that therapeutic interventions such as

chronic cold exposure successfully recruit human brown fat and increase systemic energy

expenditure. Therefore, stimulating development and/or function of brown fat would be a novel

strategy for the treatment of obesity and its complications. However, global landscape of brown

fat development is not entirely understood. Here, we identified nuclear factor I-A (NFIA) as a

novel transcriptional regulator of brown fat. The binding motif for Nuclear factor I (NFI)

transcription factor is enriched within brown-fat-specific open chromatin regions. Of the four

isoforms of NFI family, NFIA is highly expressed in brown fat compared to white fat or muscle.

Introduction of NFIA into myoblasts results in lipid accumulation, activation of the brown fat gene

program and suppression of muscle gene program. Conversely, the brown fat of NFIA knockout

mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of

muscle genes. Moreover, human perirenal brown fat of patients with pheochromocytoma show

concurrent increase in NFIA and UCP1 expression. Mechanistically, NFIA selectively co-localize

with PPARγ at the brown-fat-specific enhancers, and co-localization of NFIA facilitates the

binding of PPARγ, leading to increased chromatin accessibility and active transcription.

Collectively, these results indicate that NFIA is a novel key transcription factor that co-localizes

with PPARγ and activates the brown fat gene program.

ABSTRACT #25 HYPERINSULINEMIA INDUCED CHANGES IN CHROMATIN ACETYLATION AND GENE EXPRESSION IN TRIPLE NEGATIVE BREAST CANCER P Senapati1, J Cordova1, DK Ann2, V Seewaldt1, DE Schones1

1Beckman Research Institute of City of Hope, Duarte, CA 2CoH National Comprehensive Cancer Center, Duarte, CA Obesity associated hyperinsulinemia or insulin-resistance is considered as a poor prognostic

factor for Triple negative breast cancer (TNBC), a particularly aggressive breast cancer subtype,

constituting about 15-20% of all breast cancers. Insulin-resistance occurs when cells stop

responding to insulin and there is an abnormally high level of insulin in the blood. Insulin signaling

can activate the PI3K/Akt/mTOR pathway that is associated with increased proliferation rate of

TNBC cells. However, the mechanistic role of insulin resistance in promoting TNBC is unclear.

We are particularly interested in studying how insulin signaling impinges on mitochondrial

dysfunction and nuclear processes through the mTOR pathway. mTOR signaling enhances

mitochondrial biogenesis and activity thereby potentially increasing acetyl-CoA precursors such

as pyruvate and citrate. Nuclear histone acetyltransferases utilize acetyl-CoA as a substrate to

acetylate histones, leading to increased chromatin accessibility and gene expression. We show

here that insulin treatment to the MDA-MB-231 TNBC cell line leads to increases in global histone

acetylation levels, in particular H3K9ac, through PI3K/AKT/mTOR signaling. Quantitative ChIP-

seq analyses confirmed the global increase in histone acetylation at gene promoter regions that

could potentially alter chromatin accessibility and/or gene expression. Future studies will be

focused on studying the detailed molecular mechanism and effects of insulin induced chromatin

hyperacetylation and possible reversal by metformin.

ABSTRACT #26 ATP-CITRATE LYASE IS AN EPIGENETIC REGULATOR TO PROMOTE NEPHROPATHY IN OBESITY AND TYPE 2 DIABETES Y Chen, DK Deb, YC Li, University of Chicago, Chicago, IL Obesity and type 2 diabetes are leading causes of chronic kidney disease, but the mechanism

whereby obesity and diabetes promote renal injury remains poorly understood. Hyperglycemia,

hyperlipidemia and chronic inflammation are key features of obesity and type 2 diabetes. We

hypothesize that hyperglycemia and hyperlipidemia drive excess nutrient flows into kidney cells

and alter cellular metabolism. As a result, increased intracellular acetyl-CoA provides the

substrate for lipid biosynthesis as well as for histone acetylation in the genome. We used ob/ob

BTBR mice, an obese model of susceptible diabetic nephropathy, to test this hypothesis. The

ob/ob mice developed age-dependent hyperglycemia and albuminuria. Histology revealed

marked glomerular expansion, glomerulosclerosis and ectopic lipid accumulation in the kidney

compared with ob/+ mice. Total lipids, triglyceride and cholesterol contents in the renal cortex

were markedly elevated. The expression of key enzymes involved in fatty acid and cholesterol

biosynthesis (ACC, FAS, HMGCR) and pro-fibrotic factors (TGF-1, fibronectin (FN)) was also

markedly increased in the cortex, accompanied by a dramatic up-regulation of ATP-citrate lyase

(Acly), an enzyme that converts citrate to acetyl-CoA. Histone acetylation at H3K9/14 and

H3K27, but not H3K18, was also dramatically elevated in the cortex. In ex vivo kidney tissue

cultures, inhibition of Acly activity by SB-204990 not only blocked histone acetylation but also

suppressed the expression of TGF-1, FN, ACC, FAS and HMGCR in ob/ob kidneys. ChIP

assays confirmed that the promoters of all these genes were hyperacetylated at the H3K9/14

and H3K27 sites. We further used mesangial cell (MC) cultures to study the roles of Acly in

renal fibrogenesis and ectopic lipid biosynthesis. To mimic the condition of hyperglycemia,

hyperlipidemia and chronic inflammation, we exposed MCs to high glucose (HG), sodium

palmitate (SP) and TNF-. A combination of HG, SP and TNF- dramatically stimulated Acly

expression and enzymatic activity, increased H3K4/19 and H3K27 acetylation, up-regulated

TGF-1, FN, ACC, FAS and HMGCR expression and promoted histone acetylation in these

gene promoters. Blockage of Acly by SB-204990 or siRNA attenuated these regulatory

processes, whereas overexpression of Acly enhanced these regulations. The stimulation of Acly

expression by HG, SP and TNF- was mediated by CREB and NF-B at Acly gene promoter.

These data strongly suggest that Acly is a critical epigenetic regulator to promote renal injury in

obesity and type 2 diabetes.

ABSTRACT #27 EXPRESSION CHANGES OF METABOLIC REGULATOR FOXO1 BETWEEN EARLY AND MID-STAGE DIFFERENTIATION IN CINNAMON TREATED 3T3-L1 A Stockert, Ohio Northern University, Ada, OH The ramification of obesity and type 2 diabetes is broadening within the United States to include

detrimental effects to health of patients and the fiscal stability of healthcare. Over 9% of the US

population has some form of diabetes, costing approximately $254 billion in the year 2012.

Development of new first line treatment is expensive; discovering effective and safe alternatives

will result in significant cost savings globally as well as improving quality life for the potentially 1

in 3 individuals expected to develop type 2 diabetes by the year 2050. Key mechanistic links

between type 2 diabetes and obesity in multiple cell types will translate into streamlined

treatment efficiency and establishment of new preventative therapies.

Clinically we have demonstrated significant reduction in blood glucose values with

supplementation of cinnamon, while other researchers have reported reduction in lipid values.

Our lab has examined differentiation of pre-adipocytes treated with cinnamon and have

identified significant effects on cell size and lipolysis.

Among other cinnamon effects observed, research suggests cinnamon alters expression of

glucose transporter 4 (GLUT4) in adipose cells. Likewise, peroxisome proliferating-activated

receptor gamma (PPAR) is involved in adipose cell differentiation. Results in our lab indicate

an influence of cinnamon on adipose differentiation, likely via modulation of PPAR. Expression

of PPAR and GLUT4 are both mediated by metabolism modulator, forkhead box 1 (FoxO1), a

transcription factor highly sensitive to epigenetic modifications. Here we identify differences in

FoxO1 expression of cinnamon treated 3T3-L1 pre-adipocytes during early and mid-stage

chemical differentiation induction. Our data clearly show a significant increase in FoxO1

expression during mid stage differentiation when treated with cinnamon.

ABSTRACT #28 FUNCTIONAL CHARACTERIZATION OF DIABETES-INDUCED LONG NON-CODING RNA Dnm3oS IN MACROPHAGES S Das1*, MA Reddy1*, P Senapati1, K Stapleton1, L Lanting1, M Wang1, R Ganguly1, V Amaram1, S Devaraj2, R Natarajan1 1Beckmaan Research Institute of City of Hope, Duarte, CA 2Texas Children’s Hospital & Health Center, Houston, TX Diabetic complications are associated with enhanced macrophage activation and inflammation.

Long noncoding RNAs (lncRNAs) regulate gene expression in multiple ways ranging from

modulation of chromatin structure to post-transcriptional mechanisms. Dysregulation of lncRNA

function is implicated in several diseases, but their role in diabetes complications is not very clear.

Using RNA-sequencing, we profiled the transcriptome including lncRNAs in bone marrow derived

macrophages from type 2 diabetic (T2D) db/db mice versus control db/+ mice. We further

characterized the function of lncRNA Dynamin3 opposite strand (Dnm3os), which was

upregulated in macrophages from db/db mice. Interestingly, Dnm3os was also increased in

macrophages from type 1 diabetic C57BL/6 and ApoE-/- mice, high fat diet-induced insulin-

resistant mice, and in monocytes from human T2D patients. Dnm3os was also induced in mouse

macrophages treated with diabetogenic agents palmitic acid (PA) and High Glucose. Promoter

reporter assays showed PA regulates Dnm3os via NF-kB. RNA-seq analysis in mouse

macrophages stably overexpressing Dnm3os followed by in silico DAVID analysis showed that

genes differentially regulated by Dnm3os are associated with phagocytosis, inflammation and

immune response. Conversely siRNAs targeting Dnm3os attenuated inflammatory and immune

response genes. RNA-FISH showed that Dnm3os is predominantly localized in the nucleus and

its overexpression altered global histone modification marks in macrophages. In vitro RNA pull

down assays using biotinylated Dnm3os combined with mass spectrometry in macrophages

showed that Dnm3os interacts with nucleolin, ILF2, ARP3 and other RNA binding proteins. RNA-

FISH coupled with Immunofluorescence further confirmed the nuclear co-localization of Dnm3os

with nucleolin and ILF2. Furthermore, nucleolin knockdown in macrophages stably expressing

Dnm3os enhanced inflammatory gene expression and H3K9ac at their promoters. These results

reveal that Dnm3os promotes macrophage inflammation/activation in diabetes via novel nuclear

mechanisms involving interactions with nucleolin and ILF2. These findings could lead to lncRNA-

based therapies for inflammatory diabetes complications.

ABSTRACT #29 REGULATION OF ANGIOTENSIN II ACTIONS BY ENHANCERS AND SUPER-ENHANCERS IN VASCULAR SMOOTH MUSCLE CELLS S Das, P Senapati, Z Chen, MA. Reddy, R Ganguly, L Lanting, V Mandi, A Bansal, A Leung, S Zhang, X Wu, DE. Schones, R Natarajan, Beckman Research Institute of City of Hope, Duarte, CA Increased Angiotensin II (AngII) actions are associated with vascular and renal complications of

diabetes. AngII promotes cardiovascular diseases (CVDs) like hypertension and atherosclerosis

by activating growth-promoting and pro-inflammatory gene expression in vascular smooth

muscle cells (VSMCs). Enhancers and Super-enhancers (SEs), key genomic regulatory regions,

play critical roles in driving disease-associated gene expression. However, enhancers/SEs

mediating VSMC dysfunction in CVDs remain uncharacterized. Enhancer and SE repertoires

involved in AngII-induced gene expression were profiled by ChIP-seq analysis using antibodies

against enhancer marks histone H3-lysine4-monomethylation (H3K4me1) and H3-lysine-27-

acetylation (H3K27ac), and SE mark BRD4 in rat VSMCs with and without AngII treatment.

Results show that AngII treatment increases enrichment of H3K27ac in cultured VSMCs, in ex

vivo treated rat aortas, and aortas from AngII-infused mice. AngII-induced enhancers and SEs

are enriched in binding sites for signal-dependent transcription factors like AP-1, and are

dependent on AngII receptor (AT1R) activation and key downstream signaling kinases.

Moreover, CRISPR-Cas9 mediated deletion of candidate enhancers/SEs, pharmacological

inhibition of SE binding protein BRD4, or knockdown of overlapping long noncoding RNAs

(lncRNAs) can block AngII-induced genes associated with growth-factor signaling and

atherosclerosis. Furthermore, the BRD4 inhibitor JQ1 attenuates AngII-induced enhancers and

inflammatory genes in vitro in VSMC, and ameliorates AngII-induced hypertension, medial

hypertrophy and inflammation in vivo in mice. Interestingly, human genomic regions orthologous

to AngII-regulated H3K27ac rat enhancers harbor known GWAS/SNPs for AngII-related CVD

diseases/traits. These results demonstrate that AngII-induced signals integrate epigenetic

enhancer/SEs and lncRNAs to promote the expression of genes involved in VSMC dysfunction,

and could uncover novel therapeutic targets for CVDs in diabetes.

ABSTRACT #30 PPARGC1A DNA METHYLATION MEDIATES MITOCHONDRIAL BIOGENESIS THROUGH LINEAGE DIFFERENTIATION PROGRAMS IN BROWN ADIPOCYTES V Gagné-Ouellet1, K Thibeault1, M Mayhue1, M-F Hivert2, S Ramanathan1, S M Labbé3, R Guérin4, L Bouchard1

1University of Sherbrooke, Quebec, Canada 2Harvard Medical School, Boston, MA 3IDS Therapeutique Inc., Sherbrooke, Quebec, Canada 4CIUSSS Saguenay Lac St-Jean, Hôpital Chicoutimi, Quebec, Canada Background: Obesity is an overwhelming problem as its worldwide prevalence is constantly

increasing. Improving understanding of fat biogenesis and maintenance is now critical as existing

treatments have only been associated to short-term beneficial effects and more than often have

safety concerns. Brown adipose tissue (BAT) is well known for its involvement in energy

expenditure through thermogenesis. Diet and cold exposure are among the BAT-activation

inducing factors, along with some inflammatory factors such as IL15, but exact mechanisms

remain poorly understood. Indeed, we and others reported that modulation of DNA methylation

(DNAm) at specific loci is likely to be involved in BAT genesis. This study thus aimed at

investigating the cell-specific DNAm profile in different adipose tissue depots in a mouse model

and to determine whether DNAm profile is altered in IL15 KO mice and impacted by diet and

chronic cold exposure.

Methodology: We measured DNAm of adipogenesis genes (i.e. Ppargc1a, Prdm16, Bmp7,

Ctbp2 and Ucp1) in BAT, white subcutaneous (SAT) and visceral (VAT) adipose tissues and in

muscle from male C57BL/6 and IL15-/-mice, submitted or not to a high fat diet (60 kcal % fat)

(n=8 by group). C57BL/6 mice were also exposed to cold (10°C, 14 day; n=8). The mitochondrial

ND1/B2M copy number ratio was calculated as a mitochondrial DNA content proxy.

Results: Proximal promoter of Ppargc1a showed lower DNAm in BAT (Δ14%; p<0.001) and in

muscle (Δ30%; p<0.001) as compared to SAT and VAT. Ppargc1a DNAm and mRNA levels were

inversely correlated with each other (r=-0.5; p=0.001). Interestingly, Ppargc1a DNAm levels were

then showed to mediate (p<0.0001) the strong correlation between mRNA levels and

mitochondrial biogenesis (r=0.641; p<0.001). Neither IL-15 deficiency, high-fat diet or chronic cold

exposure were associated with DNAm variations within the epigenotyped tissues.

Conclusion: This study provides novel evidence that Ppargc1a DNAm is involved in BAT genesis

and muscle cells differentiation and activation.

ABSTRACT #31 LONG-TERM EXERCISE INDUCES MICRORNA AND MRNA TRANSCRIPTOME CHANGES IN MURINE EPIDIDYMAL SPERM A Murashov and E Pak, East Carolina University, Greenville, NC Thrifty phenotype hypothesis explains current obesity epidemic as an epigenetic changes in

metabolic phenotype due to low physical activity and a high caloric diet. Several lines of

evidence suggest that changes in paternal nutrition affect offspring predisposition to obesity. We

were the first to show that paternal long-term exercise programs offspring for lower energy

expenditure and increased risk for obesity. The transmission of epigenetic information to the

next generation could occur via changes in histone modifications, DNA methylation and RNA

levels. In the current study we asked question if paternal long-term exercise may affect sperm

miRNA and messenger RNA transcriptome.

All experiments were approved by IACUC at East Carolina University. CD-1 four-week-old male

mice were randomly assigned to sedentary and exercise groups. Animals were individually

housed with unlimited access to food and water. Animals in exercise group had unlimited

excess to the free running wheels during a 12-week period. At the end of the 12 weeks, male

mice were mated with control age-matched females to obtain F1 generation. F0 and F1 male

mice were euthanized at 16 weeks of age and epididymal sperm was collected using swim-up

procedure. Total RNA was isolated using Ambion RNAqueous-Micro Kit. RNA-seq

methodologies and initial analyses were conducted by LC Sciences using Illumina 2000 and 2 ×

100 bp paired-end sequencing. Paired-end reads were mapped to the mouse genome using

Bowtie2 v2.2.9. The abundance was estimated using RSEM v1.3.0 and the differential

expression analysis was done using EdgeR v3.12.1 in the Bioconductor package. Genes

showing significant differences (p-value < 0.05) were selected for enrichment analysis using

GAGE v2.20.1. miRNA targets were predicted with microRNA.org, DIANA Tools and miRWalk.

miRNA-gene relationships with negative correlation were further subjected to GO enrichment

and KEGG analyses.

Integrated analysis of miRNA and mRNA expression profiles identified significant changes in

miRNA-mRNA regulatory network centered around miR-10/100 an evolutionarily ancient and

conserved miRNA family located in the Hox gene clusters.

ABSTRACT #32 EXERCISE RESPONSE HETEROGENEITY OF IN VIVO MUSCLE MITOCHONDRIAL FUNCTION IS RELATED TO DISTINCT MYOCELLULAR EPIGENETIC PROFILES IN INDIVIDUALS WITH TYPE 2 DIABETES L Sparks, Translational Research Institute for Metabolism and Diabetes, Orlando, FL Exercise training does not benefit all individuals with type 2 diabetes (T2DM). We classified

individuals with T2DM in Non-Responders (NR) or Responders (R) based on whether in vivo

mitochondrial function (PCr recovery) decreased or increased with exercise training. Individuals

with T2DM discontinued glucose lowering medication during 10 weeks of exercise training. PCr

recovery, insulin sensitivity (M-value) and HbA1c were determined before and post-intervention,

and before intervention in healthy individuals (non-T2DM). Global DNA methylation and mRNA

expression profiles were obtained from NR and R primary human skeletal muscle cells (HSkMC)

before intervention. Before intervention, NR had better PCr recovery than R. R, but not NR, had

lower M-value than non-T2DM. NR and R had elevated HbA1c compared to non-T2DM. By

design, NR decreased, while R increased, PCr recovery with exercise training. R improved M-

value, while NR increased HbA1c. Changes in M-value and HbA1c correlated with changes in

PCr recovery. HSkMC established from NR versus R displayed significantly distinct DNA

methylation and gene expression patterns. Differentially methylated regions of DNA in the HSkMC

significantly correlated with in vivo changes in PCr recovery, M-value, and HbA1c. Exercise

response heterogeneity of in vivo muscle mitochondrial function in individuals with T2DM related

to blunted effects on insulin sensitivity and glycemic control, and distinct myocellular epigenetic

profiles. Gaining a deeper understanding of these cell autonomous molecular profiles that

underpin exercise response heterogeneity, particularly in individuals with T2DM, will shift the

paradigm by allowing exercise prescriptions to be targeted to those most likely to benefit and

identifying novel approaches to treat those who do not.

ABSTRCT #33 EPIGENETIC MEDIATION OF IN UTERO EXPOSURE TO GESTATIONAL DIABETES (GDM) ON CHILDHOOD ADIPOSITY OUTCOMES USING BAYESIAN NETWORK ANALYSIS Weiming Zhang1*, Katerina Kechris1,2*, Elizabeth J Davidson2, Tasha E Fingerlin1,2,4, Ivana Yang2,3,4, Dana Dabelea3 1Department of Biostatistics and Bioinformatics, Colorado School of Public Health, University of Colorado Denver, Aurora, CO 2Division of Biomedical Informatics and Personalized Medicine, Department of Medicine, University of Colorado School of Medicine, Aurora, CO 3Department of Epidemiology, Colorado School of Public Health, Aurora, CO 4Center for Genes, Environment and Health, National Jewish Health, Denver, CO *These authors contributed equally We have previously shown that exposure to maternal gestational diabetes mellitus (GDM) is

associated with adiposity-related outcomes in pre-pubertal youth in the Exploring Perinatal

Outcomes in CHildren (EPOCH) Study. Using genome-wide methylation analysis we identified

13 candidate loci from 9 genes (PTPRN2, DAPL1, RNF39, SH3PXD2A, ST5, NPHP4, E2F6,

SHMT1, POU5F1) that were differentially methylated in GDM- exposed vs non-exposed

offspring, while some were also associated with adiposity-related outcomes (body mass index,

waist circumference, visceral and subcutaneous adipose tissue by MRI, skinfolds) at an average

age of 10 years. For these loci, we validated methylation changes using pyrosequencing at this

time point (average 10 years) and later in adolescence (average 15 years), in addition to

performing gene expression profiling for the corresponding genes. Using Bayesian network

analysis to assess probabilistic relationships, we found a suggestive causal relationship

between GDM exposure, methylation, gene expression and a variety of adiposity-related

outcomes for two of these genes: POU5F1 and SHMT1. POU5F1 encodes a transcription factor

and has been previously identified as a type 2 diabetes (T2D) genetic susceptibility locus by a

trans-ethnic genome-wide association meta-analysis. SHMT1 encodes the cytosolic form of

serine hydroxymethyltransferase and has been implicated in neural development and increased

cardiovascular risk. In summary, our findings suggest that epigenetic marks and corresponding

gene expression changes may provide a critical link between in utero exposure to GDM and

obesity in childhood.

ABSTRACT #34 CREATION OF A VERSATILE ADENOVIRUS-BASED EPIGENETIC ENGINEERING SYSTEM FOR DIABETES RESEARCH J Haldeman1, M Arlotto1, T Becker2, C Newgard2 1Duke University, Durham, NC 2Duke University Medical Center, Durham, NC For over 20 years our lab has pioneered the use of recombinant adenoviruses (Ad) to

manipulate gene expression in primary pancreatic islets via transgene overexpression and

RNAi-mediated gene repression. During this time, Ad vectors have proven to be an invaluable

tool to gain insights into an otherwise difficult model system. However, major shortcomings

which hamper the utility of Ad are the laborious and time-consuming methods required to

generate and customize these vectors. To overcome this, we created a plasmid-based modular

vector platform (pMVP) utilizing Gateway Multisite Pro cloning to allow for the rapid generation

of unique vectors containing a gene of interest along with an array of promoters, epitope tags,

inducible systems, and/or fluorescent reporters. Using pMVP, an Ad can be constructed in 3

days with <2 hours of benchwork.

Studies have found that T2D-associated SNPs are enriched in putative pancreatic islet

regulatory elements; however, characterizing the function of these elements within their native

chromatin context is difficult. Several groups have shown that dCas9 fusions with epigenetic

modifiers can be utilized to interrogate regulatory elements, but these tools are spread across

various platforms and some are too large for efficient delivery to living cells. The large

packaging limit and broad tropism of Ad make them uniquely suited for this application.

Therefore, using pMVP as a foundation, we assembled a modular Ad for genomic interrogation

with Cas9 (pMAGIC). We generated an Ad backbone with an 8.3kb insert capacity to permit the

packaging of 3 gRNAs alongside a nuclease-dead Staphylococcus aureus Cas9 fused to one of

the following: VPR, a transcriptional activator; KRAB, a repressor domain which recruits H3K9

methyltransferases; p300core, the H3K27 acetyltransferase domain of p300; LSD1, a H3K4me1/2

demethylase; or Dnmt3a-3L, a DNA methyltransferase. To validate pMAGIC, we generated Ad

vectors to target enhancer IV of the homeobox transcription factor Pdx1 (MODY4). Targeting of

either dSaCas9/KRAB or LSD1 to this element in the INS1 832/13 beta-cell line significantly

decreased Pdx1 gene expression (37% and 55% respectively, p<0.05), and depleted H3K27ac

enrichment. In conclusion, we have created and validated unique Ad platforms, pMVP and

pMAGIC, to rapidly generate vectors with diverse promoters, epitope tags, and fluorescent

markers (pMVP), and for manipulating the epigenetic status of regulatory elements to unravel

their roles in the etiology of T2D (pMAGIC). (Supported by NIH grant DK46492)

ABSTRACT #35 EPIGENETIC REPROGRAMMING OF HEPATIC STEATOSIS IN THE OFFSPRING OF A NON-DIETARY MODEL OF INSULIN RESISTANCE DF De Jesus and RN Kulkarni, Joslin Diabetes Center, Harvard Medical School, Boston, MA NAFLD prevalence is increasing worldwide despite the deceleration of the obesity epidemic.

Few studies have focused on investigating the effects of early nutritional insults that increase

the likehood of developing NAFLD in the offspring. Virtually none include the use of non-dietary

models manifesting hyperglycemia and hyperinsulinemia – two hallmarks of gestational and

T2D. We aimed to determine the genetic and epigenetic effects of paternal versus maternal

genetic insulin resistance on the developmental programming in the offspring of the liver-

specific insulin receptor knockout (LIRKO) mice. Male control F1 offspring from father LIRKO

(FL), mother LIRKO (ML) or control mothers and fathers (C) were weaned on a chow or high-

fat-diet (HFD) and followed for 3 months. FL and ML showed impaired growth until 2 months of

age on chow. After 3 weeks on HFD, ML but not FL exhibit increased body weights compared to

C. FL and ML exhibited insulin resistance on chow and HFD. FL and ML developed prominent

hepatic steatosis compared to C when challenged with HFD. These mice exhibited increased

hepatic gene expression of fatty acid transporters and lipogenesis-associated genes. Hepatic

transcriptomic and genome-wide DNA methylation analysis of FL and ML on chow presented

enriched-pathways associated with TGF-β signaling and lipid synthesis. Six of the most

significant altered genes belonged to the TGF-β superfamily. Among them, gene “A” is currently

being investigated as a biomarker of diabetes and metformin usage. Gene “B” on the other hand

is involved in the development of kidney fibrosis. FL and ML on chow presented decreased

hepatic mRNA levels of A and increased levels of B. Interestingly, on HFD FL and ML presented

increased hepatic gene expression of A while a 50% reduction in B mRNA levels compared to

C. In-vivo and In-vitro modeling of hepatic steatosis increased A and decreased B mRNA and

protein levels. Gene A promoter methylation was increased in FL and ML on chow and B

presented several altered individual CpGs spanning its promoter region compared to C. Knock-

down experiments performed in HepG2 cells revealed that A and B act by modulating PPARγ,

SREBP1c and CD36 gene expression in an AKT>mTOR dependent manner. Finally, patients

with clinical defined hepatic steatosis revealed a 45% reduction in B mRNA levels. Our data

suggests that prenatal insulin resistance epigenetically regulates 2 novel genes that have

detrimental effects on the metabolic adaptation and transcriptional regulation of hepatic

metabolism that may contribute to the development of NAFLD.

ABSTRACT #36 Whole Genome Sequencing Identifies CpG-SNPs that Associate with Type 2 Diabetes In American Indians SE Day, YL Muller, P Chen, S Kobes, WC Knowler, RL Hanson, C Bogardus, LJ Baier The National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, AZ Type 2 diabetes (T2D) is a heritable disease. Yet, the established single nucleotide

polymorphisms (SNPs) associated with T2D from prior genome-wide association studies

(GWASs) do not account for a large portion of the liability for this disease. Therefore, an

additional layer contributing to the risk for T2D may be epigenetic factors. DNA methylation, for

example, can regulate gene expression through the addition of a methyl group to a cytosine

preceding a guanine, termed CpG dinucleotides. Interplay between epigenetic regulation and

genetic nucleotide variation can occur through methylation site availability, whereby a CpG site

can either be created or abolished by the presence of a SNP (termed CpG-SNP). The goal of

the current study is to identify CpG-SNPs in the genomes of American Indians and to assess

association with T2D in this high prevalence population. Whole genome sequence data from

335 American Indians identified over 2 million CpG-SNPs. Genotypic data for these SNPs were

available from a GWAS of 7,701 American Indians, and were either genotyped using a custom

Affymetrix Axiom array or imputed. Among these CpG-SNPs, 30 achieved genome-wide

significance for their associations with T2D after adjusting for age, sex, birth year and the first 5

genetic principle components (significance in this American Indian population requires P≤5×10-

7). These 30 CpG-SNPs mapped to a region on chromosome 11 spanning approximately 700

kilobases. In our and other GWASs, this region containing the genes INS-IGF2-TH and KCNQ1,

harbors several distinct signals for T2D. To prioritize CpG-SNPs in this region for further

functional analysis, databases were used to identify those with regulatory potential. PROMO

version 3.0.2 predicted 11 sites that overlap potential transcription factor binding motifs.

Furthermore, Ensemble Variant Effect Predictor identified 6 CpG-SNP sites in regulatory

regions, such as CTCF binding (rs2000993 in INS-IGF2; rs10770142 in TH) and enhancer

regions (rs2237896 and rs2237897 in KCNQ1). CTCF can regulate the interaction between

promoter and nearby enhancer regions, and is sensitive to DNA methylation. In the future,

methylation will be measured at these sites in peripheral blood leukocytes. The sites

demonstrating differential methylation will be functionally characterized for their potential role in

T2D. In summary, our preliminary data identified CpG-SNPs associated with T2D in or near

established T2D loci, and future functional studies may determine whether methylation is a

contributing mechanism.

ABSTRACT #37 EPIGENOMIC MARKS OF AIR POLLUTION AND INSULIN RESISTANCE B Park1, P Rengasamy2, J Yin1, X Rao2, H Reyes-Caballero1, S Rajagopalan2, S Biswal1 1Department of Environmental Health and Engineering, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 2Cardiovascular Research Institute, Case Western Reserve University, Cleveland, OH Air pollution, particularly exposure to particulate matter that is < 2.5 micrometers (PM2.5) is a

leading global risk factor for cardiovascular morbidity and mortality. Air pollution aggravates the

development of metabolic syndrome which increases the risk of diabetes and cardiovascular

disease. Prevalence of diabetes has increased at an alarming rate in India and China where air

pollution is high. Understanding the environmental underpinnings of this multifactorial disease is

critical for developing population level preventive approaches and novel biomarkers. We have

previously demonstrated that chronic exposure to PM2.5 causes insulin resistance in the mouse

model. As a part of the NIEHS supported TaRGET (Toxicant Exposures and Responses by

Genomic and Epigenomic Regulators of Transcription) Program, we are investigating the

alteration of epigenomic marks associated with this phenotype. Adolescent male and female

C57BL/6 mice (3 weeks) were exposed to 2 and 14 weeks of PM2.5 or filtered air (FA) for

perturbation using normal chow diet or high-fat diet (60% kcal fat). Fourteen weeks of PM2.5

exposure (~10x ambient level/~ 70-100ug/m3, 6 hours/day, 5 days/week) caused a significant

increase in insulin resistance in male mice but not females as measured by GTT (Glucose

Tolerance Test) and ITT (Insulin Tolerance Test). While the female mice on high-fat diets alone

experienced less diabetes risk, the mixture of perturbations from high-fat diets and PM2.5 gave

rise to a significant risk to the female mice. We identified important differentially expressed

genes that are linked to phenotypic changes. By incorporating ATAC-seq and WGBS, we are

identifying potential risk epigenome changes such as nucleosome repositioning and DNA

methylation profile modifications. This integrated analysis using an environmental exposure

model can support the further discovery of the molecular mechanisms underlying progression of

type 2 diabetes due to exposure to air pollution and a high-fat diet. This work was supported by

NIEHS U01ES026721.

ABSTRACT #38 CELL TYPE-SPECIFIC TRANSCRIPTIONAL AND EPIGENETIC PROFILES FROM A RARE POPULATION OF NEURONS WITHIN THE ARCUATE NUCLEUS Frankie D Heyward, Hyun Roh, Danielle Tenen, Christopher Jacobs, John N Campbell, Bradford B Lowell, Linus T Tsai, Evan D Rosen Division of Endocrinology, Beth Israel Deaconess Medical Center and Department of Genetics, Harvard Medical School, Boston, MA The arcuate nucleus (ARC) of the hypothalamus is home to a wide assortment of cell-types,

many of which uniquely contribute to the regulation of energy homeostasis. Given the high

degree of cellular heterogeneity present within the ARC, solutions are needed to achieve a

better appreciation for the distinct molecular characteristics of relevant cell types. Past

approaches enabling cell-type specificity are beset with limitations involving time- and labor-

intensiveness, low cellular throughput, and processing-induced changes in cellular state.

Moreover, to-date, there exist a paucity of techniques that allow for epigenetic profiling of rare

cell-types within the brain. The recently developed Nuclear tagging and Translating Ribosome

Affinity Purification (NuTRAP) provides an unprecedented ability to obtain either cell type-

specific transcriptional or epigenetic profiles. Here we examine the capacity of NuTRAP to

obtain cell-type specific transcriptional and epigenetic profiles in a relatively small population of

energy state-sensitive neurons within the ARC.

ABSTRACT #39 BROMODOMAIN-CONTAINING PROTEINS PLAY A PIVOTAL ROLE IN GLUCOSE METABOLISM IN MICE C. Kozuka1, V. Sales1, Y. Yuchi1, S. Osataphan1, J. Desmond, C. Mulla2, C. Smith, J. Qi3, J. Dreyfuss1, H. Pan, M.E. Patti1

1Joslin Diabetes Center, Boston, MA 2Joslin Diabetes Center, Harvard Medical Center, Boston, MA 3Dana-Farber Cancer Institute, Boston, MA

Epigenetic regulators can be modified by environmental factors and may contribute to risk for

obesity and diabetes. However, the impact of interventions targeting epigenetic modifications on

diabetes-related systemic metabolism remain poorly understood. Bromodomain-containing

proteins (BRDs) recognize acetylated lysine residues; to determine the impact of this protein

family on metabolism, we treated male ICR mice (age 31 weeks) with the BRD inhibitor, JQ-1

(50 mg/kg/day, ip, n = 6/group) for 16 days, and assessed glucose and lipid metabolism.

Body weight tended to be reduced in JQ-1-treated mice (12% vs. 9% for JQ-1 vs. vehicle).

Fasting blood glucose levels tended to be increased in JQ-1 treated mice (160 vs. 139 mg/dl,

p=0.1), but JQ-1 induced severe glucose intolerance (2 g/kg intraperitoneal GTT, AUC: 74,544

vs. 46,184 mg·dl-1·min, p < 0.001). Plasma insulin levels did not differ in the fasting state but

were numerically lower at 15 minutes after glucose injection in JQ-1-treated mice (28%, p=0.2);

islet number and size did not differ. Insulin sensitivity, as assessed by insulin tolerance testing

(0.5 U/kg), did not differ. JQ-1 treatment significantly decreased plasma cholesterol levels (79

vs. 165 mg/dl for JQ-1 vs. vehicle, p = 0.01), but differences in triglyceride levels were not

significant (60 vs. 45 mg/dl). We next determined the effect of JQ-1 on hepatic glucose and lipid

metabolism. Expression of the gluconeogenic gene Pepck was increased by 1.6-fold with JQ-1

(p < 0.01), with similar pattern for G6Pase. Although hepatic cholesterol content was decreased

(27 %), expression of genes regulating cholesterol metabolism was increased by JQ-1 (Ldlr,

Srebp2, Hmgcs and Hmgcr, 1.2-2.0-fold, p < 0.05). Expression of genes related to bile acid

synthesis and metabolism (CYP7A, FXR, and SHP) were also increased by 1.4 to 2-fold.

Collectively, these results indicate that BRDs can regulate systemic glucose metabolism,

potentially via effects on hepatic glucose and lipid metabolism.