deciphering hoxa9 function in multipotential progenitor biology and b cell development
DESCRIPTION
Deciphering HoxA9 function in multipotential progenitor biology and B cell development Kay L. Medina, PhD Associate Professor Dept. of Immunology Mayo Clinic College of Medicine. Overview of adult hematopoiesis in the mouse. PMN. Mac. GMP. Meg. Ery. MEP. CMP. cDC. pDC. CDP. HSC. - PowerPoint PPT PresentationTRANSCRIPT
Deciphering HoxA9 function in multipotential progenitor
biologyand B cell development
Kay L. Medina, PhDAssociate Professor
Dept. of ImmunologyMayo Clinic College of Medicine
HSC STRC
CMP
ILC2 NKP
GMP Mac PMN
MEP Ery Meg
Thymus
ETP
ELP
GMLP
ALP BLPPre-
Pro-BPro-B
Overview of adult hematopoiesis in the mouse
CDP pDC cDC
Sequentially-acting regulatory circuits orchestrate B cell fatespecification and commitment
HSC STRC
ELP
GMLP
ALP BLPPre-
Pro-BPro-B
HoxA9IkarosPU.1
Flt3E2A
E2A, Flt3
IL-7R
c-Myb
EBF1
FOXO1
IL-7R
Pax5
IL-7R
Pax5
E2A B220+CD19-
B220+CD19+
DH-JH
VH-DJH
Hoxa9 regulates Flt3 in lymphohematopoietic progenitors
Gwin and Medina, JI, 2010
hoxa9
ebf1
Inverse expression of HoxA9 and EBF1 in lymphoid/B cell precursors
CLP/BCP
Myeloid-Lymphoidprogenitor
All-Lymphoidprogenitor
B-Lymphoidprogenitor
Pro-B cell
HSC/MPP program Alternate lineage programs
B cell programTranscription factors, signaling molecules, chemokines, microRNAs
Hoxa9Flt3
Hoxa9Flt3IL7R
Hoxa9Flt3IL7
EBF1
IL7REBF1
hoxa9-/- flt3l-/- mice exhibit a severe lympho-hematopoietic block
Gwin and Medina, JI, 2013
HSC STRC GMLP
hoxa9-/- flt3l-/-
WT-RAG1-GFP+ Hoxa9-/- RAG1-GFP+ Flt3L-/- RAG1-GFP+ Hoxa9-/-Flt3L-/- RAG1-GFP+F
lt3
GFP GFP GFP GFP
ELP ELP ELP ELP
HoxA9 and Flt3 signaling function together to establish themultipotential progenitor pool competent to undergo lymphoid priming
The role of HoxA9 in B cell fate specification.
ebf1 foxo1 gfi1b
EBF1FOXO1
GFI1b
HoxA9-/- hematopoietic progenitors have impaired IL-7 signaling#
cells
x 1
05
B6 HoxA9-/- IL-7R
MF
I IL-
7R
Lin- ckit+ Flt3+
B6HoxA9
Culture model to identify HoxA9 target genes critical for B cell development
WT
HoxA9-/-
c-kit Sca-1 CD34 CD150 Flt3 IL-7R
Lin- c-kit cells cultured for 17 days in SCF+FL+Tpo+IL-6
B
# ce
lls x
106
B22
0
CD19 CD19
WT MPPs HoxA9-/- MPPs
Day 17 MPP
24 hr CLP
IL7,SCF,FL
Harvest
Switch
RNA-Seq Platform
Sequencing
WT MPP-1WT MPP-2HoxA9-/- MPP-1HoxA9-/- MPP-2
Sequencing
WT CLP-1WT CLP-2HoxA9-/- CLP-1HoxA9-/- CLP-2
2 independentexperiments
Summary of RNA-Seq Platform
Hoxa9-/- MPP
42
Hoxa9-/- MPP
73
Hoxa9-/- CLP
16
Hoxa9-/- CLP
102
13
MPP CLP
29 3 17
MPP CLP
56 86
Sample Total Reads
Hoxa9ko-CLP-1 89,662,470
Hoxa9ko-CLP-2 86,812,194
Hoxa9ko-MPP-1 79,030,174
Hoxa9ko-MPP-2 102,001,936
WT-CLP-1 108,111,316
WT-CLP-2 118,748,182
WT-MPP-1 98,958,636
WT-MPP-2 99,110,526
Read length:50bp
40-50X depth of coverage/gene
Differential expression cutoff: absolute fold change 1.4
False discovery rate of ≥0.1; p>0.05
Hoxa9-/- MPPs
64%HSC/MPP
myeloid/DC
Hoxa9-/- MPPs
54%Lymphoid
Hoxa9-/- CLPsHoxa9-/- CLPs
93%HSC/MPP
myeloid/DC
63%Lymphoid
Hoxa9-deficiency impairs lymphoid priming and downregulation of HSC/MPP and/or alternative lineage gene programs
Shared upregulated Lcn2 – lipocalin 2 – neutrophilMgam – maltase-glucoamylase - neutrophilClec4b – ATP binding cassette - DCAdam23 – disintegrin and metallopeptidase - DCPglyrp1 – peptidoglycan recognition protein - neutrophilElane – elastase – macrophage/HSC/MPPAbca13 – ATP binding cassette - neutrophilEhd3 – EH domain containing 3 – T cellsDio2 – deiodinase – HSC/MPPMpo – myeloperoxidase - monocytesVcam1 – vascular cell adhesion molecule 1 – HSC/MPPDach1 – dachshund - neutrophilPpic – peptidylprolyl isomerase C – HSC/MPPRab38 – member of RAS family - macrophageMgl2 – macrophage galactose N-acetyl-galactosamine specific Ikzf2 – ikaros zinc finger 2; Helios – HSC/T cells/T regsGata2 – HSC/MPP
Shared downregulatedRag1 – recombinase activating gene – lymphocytesGfra1 – sperm stem cell self renewal - BCPsRag2 - recombinase activating gene - lymphocytesPpfia4 – protein tyrosine phosphatase – spl DC / MacsSlc15a2 – H+/peptide transporter - BCPsP2rx3 – purinergic receptor P2X - BCPsCdh17 – cadherin 17 - BCPsCecr2 – cat eye syndrome - BCPsGimap4 – GTPase - BCPsLin28b - regulator adult to fetal HSC; let7 biogenesis - BCPsCd27 – member of TNF receptor family – MPPs/BCPsIgfbp3 – insulin growth factor 2 binding protein - BCPs
B22
0
CD19 CD19
WT MPPs HoxA9-/- MPPs
17 day MPP switched to SCF+FL+IL7 and cultured for an additional 6-8 days
Mac
-1
CD19 CD19
Gated onGFP+ cells Hoxa9-/- d17 MPP in SCF+FL+IL7
6-8 days after retroviral transduction
MigR1-GFP Mig-EBF-GFP
Ectopic expression of EBF1 is sufficient to restore the generation of BCPs from HoxA9-/- MPPs
Linking RNA-Seq and Microarray data to the IL-7R signaling pathway
Conclusions:
We developed a short-term in vitro culture model to expand MPPs from WT and HoxA9-/- mice that will be informative in the identification, characterization, and validation of HoxA9 regulated genetic circuits in early hematopoiesis.
RNA-Seq confirmed decreased lymphoid priming in MPPs and under B cell differentiation conditions due to HoxA9 deficiency independent of Flt3 signaling.
HoxA9 deficiency impairs IL-7R expression and signaling – enforced expressionof EBF1 can bypass the IL-7 signaling defect.
Gfi1b
Runx1 IL7R
E2A* → FOXO1→ EBF1→Pax5
Flt3 → PI3KMeis1Hoxa9
MAPK
STAT5
HSC program
c-Myb Gfra1Ctr9Ccr9Scdn4
Underway:
1. Identify novel HoxA9 target genes : compare results to microarray platform of genes upregulated in EBF1-/- multipotent cells that express endogenous Hoxa9 - underexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional activation) - overexpressed in EBF1-/- MPPs in Affymetrix Array Chd17, CD27, CCR9, Ppfia4, Sorcs2, RAG2, GPR25, Jup, Igf2bp3
- overexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional repressor) - underexpressed in EBF1-/- MPPs in Affymetrix Array Mgst1, Tgm2, Enpp4, Plekha8, Muc13, Cpne2, Tmem176a, Mreg, Adcy6, STAT4, Sulf2, SpiB
2. Identify novel EBF1 target genes : compare results to microarray platform of genes underexpressed in EBF1-/- multipotent cells to genes underexpressed in HoxA9-deficient MPPs or CLPs P2rx3, RAG1, Rgs8, Cecr2, Egfl7, Uaca, Lax1, Dntt, Slc15a2, Gimap4
3. Perform gain-of-function and loss-of-function experiments in WT or HoxA9-/- MPPs to evaluate gene function in B cell development followed by molecular validation.
4. Determine if HoxA9 target genes are relevant to leukemogenesis.
Acknowledgements
Medina LabKim GwinZhihui (Cherry) Xu, MDZhixin (Jason) Hua, PhDJoe Dolence, PhDMike BellElena FrankMariya Shapiro
Flow Cytometry CoreGene Expression Core
Funding by NHLBI R01096108Eagles FoundationConcern FoundationDept of Immunology