PXR in zebrafish: preliminary RNA-Seq analysis

SRP Project 5:Developmental Toxicity of

non-Dioxin-like PCBs and Chemical Mixtures

John Stegeman, PIJared Goldstone, co-Investigator

Rationale

• To identify and understand developmental effects of chemicals abundant at Superfund sites.

• Focus on Ortho-PCBs; implicated in neurological deficits in mammals, but still not clearly understood.

• Little knowledge of which congeners (non-ortho, mono-ortho or poly-ortho) interact with receptors other than AHR, and no data on CYPs induced or mechanisms of toxicity by non-DL congeners in zebrafish.

• Studies will be extended to populations of Fundulus to address the possibility of resistance to ortho-PCBs

2

6

2'

6'

Cl

Cl

Other organic contaminants

‘Personal care products’

Pesticides, herbicides Drugs, hormones

RotenoneCarbarylDieldrenAldicarbDiazenonFenthion

estrogen

triclosan

Synthetic musksMetal chelators (from shampoo)DetergentsPlastic breakdown (bisphenol A)

caffeineAnalgesicsAnticancer drugsAntidepressantsSteroid drugsSedativesAntianxiety…

DDT

maf

AHRArnt

NRF2

Keap

1

AHR

Ch

ap

NRF2

CAR/PXRC

hap

CAR/PXRRXR

Xenobiotic-metabolizing enzymes / transporters (e.g. CYP, GST, SOD, MDR)

ADAPTIVE ROLES

Target genes

Cl

Cl

Cl

Cl

OH O

N

ClCl

ClCl

N

Cl

Me

Cl

Cl

Altered cell growth& differentiation

TOXIC ROLES

Altered cell growth& differentiation

DEVELOPMENTALROLES

M. Hahn

Gene expression and regulation:Ligand-activated transcription

PXR-CAR network

Oxidizing

Receptors, etc.

Transporters

Conjugating

CAR PXR

Bainy et al Aquatic Tox (2013)

Zebrafish PXR

74% 56%

CN DBD hinge LBD

45 128 233

RXR PXR

coactivators

corepressors

ligandligand

PXRRE

??????{N}2-5??????

Gene response to pregnenolone (PN)

Kubota et al. (unpublished)

µM Concentrations

• PN induces expression of PXR, CYP2AA1, 3A65, 3C1• Are these target genes for PXR?

Kubota et al. (unpublished)

PXR is a reliable target gene for PXR: PXR knockdown and pregnenolone (PN)

exposures

Ctl-MODMSO

PXR-MODMSO

PXR-MOPN

Ctl-MOPN

Normal effect of PN

Ctl-MODMSO

PXR-MODMSO

PXR-MOPN

Ctl-MOPN

Effects of PNbackground of reduced PXR

Background :Role of PXR in

normal homeostasis

Effects of PNBackground also changing

Details

• Standard MO injection at 1 cell stage• PN treatment 48 72 hpf• Sampled at 72 hpf

• Standard Illumina Tru-Seq mRNA selection• 50 nt single end (SE), 4 samples per lane

• 2 batches of experiments (2 replicates/batch)

Zv9 Ensembl 74+ annotations

16 samples

* Map to transcriptome only

Tophat -T -- transcriptome-index

Tophat -T -- transcriptome-index

Cuffdiff Danio_rerio.Zv9.74.gtf Ctr1.bam,Ctr2.bam,…Trt1.bam,Trt2.bam,…

Tophat and Cuffdiff settings• Tophat (default settings)

--read-mismatches = 2--read-gap-length=2--splice-mismatches=0--transcriptome-only

• Cuffdiff (default settings)--FDR = 0.05Pooled dispersion (4 replicates per sample, 2 conditions)

per-condition dispersion possibleGeometric library normalization

‘classic-fpkm’ or poisson possible

Sample Total reads Mapped readsPercent mapped

Ctr-MO_D1_transcriptome 27,601,435 18,641,486 67.5%

Ctr-MO_D2_transcriptome 26,271,248 17,823,335 67.8%

Ctr-MO_D3_transcriptome 23,283,511 14,804,298 63.6%

Ctr-MO_D4_transcriptome 31,387,740 21,612,686 68.9%

Ctr-MO_PN1_transcriptome 26,855,354 18,487,708 68.8%

Ctr-MO_PN2_transcriptome 27,393,801 18,553,578 67.7%

Ctr-MO_PN3_transcriptome 25,737,244 16,736,913 65.0%

Ctr-MO_PN4_transcriptome 27,709,251 19,132,564 69.0%

PXR-MO_D1_transcriptome 28,555,850 19,560,222 68.5%

PXR-MO_D2_transcriptome 29,387,173 20,881,062 71.1%

PXR-MO_D3_transcriptome 30,275,205 19,711,304 65.1%

PXR-MO_D4_transcriptome 23,168,627 15,749,058 68.0%

PXR-MO_PN1_transcriptome 26,511,140 18,071,404 68.2%

PXR-MO_PN2_transcriptome 22,620,334 14,313,274 63.3%

PXR-MO_PN3_transcriptome 25,146,055 16,202,337 64.4%

PXR-MO_PN4_transcriptome 39,004,311 27,451,375 70.4%

Average 27,556,767 18,608,288

Ctl-MODMSO

PXR-MODMSO

PXR-MOPN

Ctl-MOPN

6↑18↓

PXR 0.2x control

347↑115↓

4↑18↓

226↑127↓

PXR 2.7x control

PXR 0.16x control

PXR not sig (2.1x control)

RNA-seqfragments

qPCR

RNA-seq and qPCR results match

Kubota et al. (unpublished)

Control-MO; DMSO vs. PN, subset 347↑115↓

gene_id gene CtlMO_D CtlMO_PNfold_

changeENSDARG00000079227 plekhs1 0.19 8.01 213.5ENSDARG00000038371 cyp2k6 0.10 1.41 47.5ENSDARG00000093640 ugt5a2 0.22 1.07 10.0ENSDARG00000051914 slc14a2 0.44 2.11 9.5ENSDARG00000027852 plekhf1 4.99 22.58 8.8ENSDARG00000070420 cyp24a1 2.65 11.70 8.5ENSDARG00000043587 srd5a2a 4.40 18.51 7.9ENSDARG00000092052 GSTK1 (3 of 4) 0.47 1.86 7.3ENSDARG00000027088 ptgdsb 57.17 204.38 6.3ENSDARG00000018621 slc6a19a 1.30 4.36 5.7ENSDARG00000019532 fads2 5.67 17.02 4.9ENSDARG00000028396 fkbp5 72.19 213.07 4.8ENSDARG00000033170 sult2st1 5.94 17.23 4.6ENSDARG00000061274 lss 1.47 3.86 4.0ENSDARG00000027529 hmox1 1.91 4.41 3.3ENSDARG00000021787 abcb5 10.70 23.94 3.2ENSDARG00000045627 cyp3a65 2.07 4.61 3.2ENSDARG00000033160 nr1d1 20.34 43.27 3.0ENSDARG00000038366 cyp2k18 0.67 1.38 2.9ENSDARG00000042641 cyp51 9.47 19.40 2.8ENSDARG00000014916 slc10a2 1.82 3.69 2.8ENSDARG00000057262 CYP27A1 (3 of 6) 1.36 2.73 2.7ENSDARG00000029766 nr1i2 5.15 10.26 2.7ENSDARG00000028367 sult2st3 1.89 3.72 2.7

ENSDARG00000089177 CYP46A1 (4 of 5) 1.06 2.08 2.7ENSDARG00000059035 POR (2 of 2) 10.29 19.07 2.4ENSDARG00000045414 elovl2 2.03 3.33 2.0ENSDARG00000009852 cyp19a1b 1.82 2.94 2.0ENSDARG00000041169 hif1al 107.85 168.54 1.9

gene_id gene CtlMO_D CtlMO_PN fold

changeENSDARG00000062632 duox 1.59 0.14 0.03ENSDARG00000094901 ABCC6 (3 of 3) 0.93 0.31 0.20ENSDARG00000090403 cyp2aa8 0.85 0.29 0.21ENSDARG00000087120 SLC5A8 2.65 1.09 0.28ENSDARG00000063475 abcg1 3.55 1.53 0.30ENSDARG00000002981 cyp2aa4 2.43 1.20 0.36ENSDARG00000074635 abca1a 14.95 7.69 0.38ENSDARG00000068603 slc22a5 2.52 1.32 0.39ENSDARG00000068910 nos1 2.29 1.30 0.44ENSDARG00000023537 ahr1b 11.56 7.12 0.50ENSDARG00000033498 rorb 13.56 8.38 0.50ENSDARG00000042533 gstm 42.10 26.45 0.51

Ctl-MO vs PXR-MO, DMSO

gene_id gene CtlMO_D PXRMO_Dlog2(fold_change)

ENSDARG00000034211 capn2l 3.0 7.9 1.38ENSDARG00000092358 si:ch211-114l13.11 4.1 10.3 1.31ENSDARG00000078342 zgc:194125 9.0 20.6 1.19ENSDARG00000091444 TUBB8P7 (3 of 3) 4.9 10.5 1.10ENSDARG00000009890 CR936442.1 2.3 4.5 0.97ENSDARG00000063330 mgat4a 12.7 24.9 0.97ENSDARG00000082753 AC024175.17 218.4 137.7 -0.66ENSDARG00000086583 apoa1b 64.7 40.2 -0.69ENSDARG00000038153 lgals2b 84.1 50.4 -0.74ENSDARG00000015866 apoa2 110.4 60.0 -0.88ENSDARG00000089679 UIMC1 (1 of 2) 5.8 2.6 -1.17ENSDARG00000058476 stc1l 7.5 3.2 -1.23ENSDARG00000088436 CT956064.3 17.5 6.7 -1.39ENSDARG00000081270 rn7sk 37.0 12.8 -1.54ENSDARG00000029766 nr1i2 5.2 1.7 -1.57ENSDARG00000078069 rrm2 4.7 1.6 -1.59ENSDARG00000096403 si:dkey-153m14.1 2220.5 717.3 -1.63ENSDARG00000091744 BX296557.7 102.2 32.5 -1.65ENSDARG00000088951 AL935186.6 95.9 27.6 -1.79ENSDARG00000087345 CABZ01059415.2 2.1 0.6 -1.84ENSDARG00000070212 zgc:158463 1279.8 325.7 -1.97ENSDARG00000087732 Metazoa_SRP 102.2 24.0 -2.09ENSDARG00000044566 fabp6 18.5 4.0 -2.19ENSDARG00000096145 si:dkey-111b14.2 155.3 33.9 -2.20

gene_id geneCtl-MO_PN

PXR-MO_PN

log2(fold_change)

ENSDARG00000088891 slc23a3 0.5 9.4 4.30

ENSDARG00000092358si:ch211-114l13.11 4.6 13.1 1.50

ENSDARG00000074210 zgc:110286 31.7 70.8 1.16ENSDARG00000059529 BX255936.1 28.3 48.7 0.78ENSDARG00000014745 epd 53.1 31.1 -0.77ENSDARG00000036834 zgc:109868 70.3 39.1 -0.85ENSDARG00000015866 apoa2 131.5 71.4 -0.88ENSDARG00000045180 acta2 22.0 11.0 -1.00ENSDARG00000088951 AL935186.6 164.6 76.7 -1.10ENSDARG00000045592 tnni2a.1 19.3 9.0 -1.11ENSDARG00000021787 abcb5 23.7 11.0 -1.11ENSDARG00000091744 BX296557.7 193.4 88.5 -1.13ENSDARG00000052905 zgc:165423 11.9 5.3 -1.17ENSDARG00000038153 lgals2b 113.4 49.8 -1.19ENSDARG00000096145 si:dkey-111b14.2 221.4 89.1 -1.31ENSDARG00000052815 alad 15.9 6.1 -1.38ENSDARG00000014916 slc10a2 3.7 1.3 -1.44ENSDARG00000029766 nr1i2 10.1 2.9 -1.81ENSDARG00000068181 DPEP1 1.3 0.3 -2.29ENSDARG00000055539 epdl2 13.4 2.5 -2.44ENSDARG00000044566 fabp6 41.7 3.6 -3.54ENSDARG00000067570 ctsbb 1.4 0.1 -3.63

Ctl-MO vs PXR-MO, PN

si:ch211-114l13.11lincRNA

lgals2blectin, galactoside-binding, soluble, 2b(human SNP increases myocardial infarction)

apoa2apolipoprotein A2

(hdl protein, hypercholesterolemia)nr1i2 (pxr)fabp6

fatty acid binding protein 6(also binds bile acids, bile acid pathways)

si:dkey-111b14.2lincRNA

Next steps

• Mapping to genome, not just transcriptome– So far, samples produce 91 % mapping– Alternative mapping software (STAR) that is

possibly 10x faster• cummeRbund plots• Meme analysis of upstream region of 20-30

genes for PXR-RE discovery