Genetic dissection of chromatin accessibility and transcript abundance underlying ground state pluripotency in mouse embryonic stem cells.

Many adult-onset diseases are caused by genetic variants that have proximal effects at the earliest stage of development – the pluripotent, ground state. In order to better understand the role of genetics in maintenance of pluripotency, we performed genetic and genomic analysis on a panel of undifferentiated embryonic stem cell lines derived from genetically heterogeneous Diversity Outbred mice (DO mESCs). We profiled chromatin accessibility (ATAC-seq) and transcript abundance (RNA-seq) of each DO mESC line maintained in cell culture conditions that promote the pluripotent state. We mapped thousands of loci with genetic variants that alter chromatin accessibility (caQTL) and transcript abundance (eQTL). Many distant QTL co-localize and appear as prominent trans-bands, suggesting that a common regulator may drive them. One locus on chromosome 15 altered the expression of 208 genes including many with known functions in maintenance of pluripotency. We applied mediation analysis and identified Lifr (leukemia inhibitory factor receptor) transcript abundance as the causal intermediate for these eQTL. Interestingly, sex-specific differences in many of these genes suggest a nonlinear response to Lifr dosage. Joint mediation analysis of eQTL by chromatin accessibility revealed a variable region of open chromatin upstream of the Lifr gene containing a single SNP that predicts the allelic effects on Lifr expression and its downstream targets. This suggests a causal chain of molecular events starting from a single SNP that modulates chromatin state in a Lifr enhancer that in turn affects Lifr transcription, which ultimately regulates transcript abundance of 208 target genes, including both known and novel pluripotency-associated genes. To validate these predictions and demonstrate their effects on ground state pluripotency and early lineage commitment, we have developed complimentary genetic resources including F1 hybrid mESC lines from Collaborative Cross strains and CRISPR-modified inbred, founder mESC lines. Future studies will integrate additional cellular assays within this renewable systems genetic resource to expand our understanding of genetic influence on differentiated cell types and ultimately to fetal and adult in vivo phenotypes.

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1. Genetic dissection of chromatin accessibility and transcript abundance underlying ground state pluripotency in mouse embryonic stem cells Steven Munger The Jackson Laboratory Bar Harbor, Maine USA @stevemunger
2. The Cellular Systems Genetics Consortium Dan Skelly Laura Reinholdt Anne Czechanski Chris Baker Catrina Spruce Candice Byers Steve Munger Selcan Aydin Alex Stanton Gary Churchill Partners: Ted Choi – Predictive Biology, Inc. Alison Harrill – NIEHS NTP Laura Reinholdt Chris Baker Steve Munger Gary Churchill Dan Skelly Anne Czechanski Catrina Spruce Candice Byers
3. Outline The mouse is the best model system. Advantages of genetically diverse mice for gene discovery. Extending the power of DO/CC mice to embryonic stem cells (mESCs). Multi-scale genome-wide phenotyping of chromatin accessibility and transcript abundance in DO mESCs. Genetic dissection and experimental validation of an eQTL trans-band underlying variation in mESC self-renewal.
4. Diversity Outbred and Collaborative Cross mice Powerful orthogonal resources for gene discovery and validation • Balanced population structure • 400+ recombinations per animal • High heterozygosity • Each animal is unique 5 common lab + 3 wild-derived strains 45 million+ SNVs, 2 million+ indels • Reproducible genomes • High genetic diversity • Fewer recombinations per line
5. Embryonic stem cells Pluripotent o Can differentiate into many cell types o Can functionalize/validate genetic variation in vivo. Renewable genetic resource o Effectively immortalize unique (normally transient) DO genomes.
6. Evidence of genetic variability in maintenance of the pluripotent ground state in ES cells. Anne Czechanski Permissive Recalcitrant
7. N = 185 N = 192 Predictive Biology, Inc. Experimental Design: Profiling chromatin accessibility and gene expression in DO mESCs.
8. Goal: Identify genetic variants that affect chromatin accessibility (caQTL), transcript abundance (eQTL), and their downstream consequences. RNA Chromatin QTL Ground state maintenance Differentiation potential 1 3 5 7 9 11 13 15 17 19 2 4 6 8 10 12 14 16 18 X 0 5 10 15 Chromosome LOD score
9. Genetic variation underlies much of the variability in transcript abundance and chromatin occupancy. • • • • • • Position of ATAC peak Position of gene 0.25 0.30 0.35 0.40 Correlation Correlation between transcript abundance and chromatin accessibility Position of ATAC peak Position of gene
10. QTL location Shared trans-bands
11. QTL location eQTL or caQTL specific trans-bands
12. QTL location
13. Genetic dissection of an eQTL trans-band Chr 15: 7-8.5Mb 202 Target genes Many target genes associated with pluripotency pathways.
14. The Chr 15 trans-band contains fewer than 10 genes. Scale chr15: Multiz Align 500 kb mm10 7,100,000 7,200,000 7,300,000 7,400,000 7,500,000 7,600,000 7,700,000 7,800,000 7,900,000 8,000,000 8,100,000 8,200,000 8,300,000 8,400,000 Basic Gene Annotation Set from GENCODE Version M14 (Ensembl 89) RefSeq Genes Placental Mammal Basewise Conservation by PhyloP Vertebrate Multiz Alignment & Conservation (60 Species) Gm38282 Lifr Lifr Lifr Egflam Egflam Gm16029 Gm37743 Gdnf Wdr70 Gm27529 Nup155 2410089E03Rik Gm24144 Nipbl Lifr Lifr Egflam Egflam Egflam Gdnf Gdnf Gdnf Gdnf Wdr70 Nup155 2410089E03Rik Nipbl Nipbl Placental Cons 3.296 _ -3.94 _
15. Searching for transcript and chromatin mediators of distant eQTL è Mediation Analysis RNA QTL trans cis RNA Chromatin Target Causal Intermediates RNA trans QTL cis Target Target Gene ~ eQTLdistant Target Gene ~ eQTLdistant + MediatorRNA Target Gene ~ eQTLdistant + MediatorChromatin X
16. Mediation analysis example – Serping1 0 10 20 30 40 50 0 2 4 6 8 10 12 Chr 15 position LOD score Before including Lifr After including Lifr Chick, Munger et al. 2016
17. Mediation identifies Lifr expression as the causal intermediate conferring the eQTL effect Ripk2 Arrdc3 Bhmt Srsf1 Matn1 Tdg Klf4 Ifitm1 Elf4 Mpp6 Dtd1 Slc52a3 Sema5b T rp53i11 Plekha6 Lama1 Zfand5 Rtkn Slc2a8 4930444M15Rik Fam20c Gm16233 Eng Rock1 T raf3ip2 Ccpg1 Rprm Tmem173 Acot8 Agpat2 Cnn2 Kcnj12 Lockd Nid1 T om1l1 AI464131 Hsf2 Atp10a Fam102a Alpk3 Pde7a Duxbl2 B3gnt3 Sigirr Clca4a Timp1 Nt5dc2 Creb1 Fam169a Cnot2 Ttc21b Sesn3 Islr2 9030617O03Rik Ube2k Tpm1 Pgc Susd2 T agln Gramd3 Tfcp2l1 Apom Cyp4f13 6430573F11Rik Emb Nectin1 Uqcrc1 Bcl3 Fetub Skil Htra1 Map4k5 Ncam1 Smchd1 Duxbl3 Hmgn5 Kbtbd2 Rps6kl1 Fgf8 Socs3 Cacna1a Cdk14 Aes Hap1 Cxcl12 Pak1 Slc40a1 Fzd5 Serping1 Fndc3c1 Gtdc1 Tns1 Rbp1 Cmklr1 Laptm5 Tmem54 Wscd2 Nsmaf Slmo1 Pbx1 Slco4a1 Mapt Fcgrt Pyroxd1 Arhgef10l Tmem55a Hmcn2 Mst1 C2cd2l Mtap7d3 Nmt2 Atp2a3 Nrg2 Ypel2 Igsf21 Rragb Zfp946 Tmsb15a Cxxc1 Otud6a T rap1a Xlr3c Armc10 Lin28a Timm8a2 Gm364 Hormad1 Xlr3b T rim46 Xlr3a Swap70 Qser1 Tle1 Kpna4 Npl Eya3 March8 Fermt3 Plekha1 Man2b2 Pros1 Esrp1 Pcbp4 Gpr160 Kras Nr6a1 Cdkn1c Il34 Serpinb1a Tnfaip8 Wnt8a Cdk17 Ppp2cb Eml4 Ina Slc29a3 Hfe Myb Fry Gm38999 Cadm4 Ephx1 2510039O18Rik C77080 Bcorl1 Rab11fip5 Cdk2ap1 Cdyl Slc1a1 Hnrnph1 Crabp2 Kis2 Osbpl10 T rib2 T rim50 Vrtn Xlr5c Fam161a Xlr5b Foxp1 Speg Gpat2 Nxf3 Xlr4c Xlr4a Xlr4b Xlr5a H2−Eb1 Tmem39a Rab22a Foxi3 4930591A17Rik Cyp2s1 Rac3 Tdrd5 2610035F20Rik Cdyl2 Cnn1 Fam133b Lap3 Iqcg Zbtb1 Tmem231 Pla2g16 Efnb2 Mageh1 Ankrd50 Cd84 Hus1b Abhd6 Cox5a 2610307P16Rik Fbxo6 Sulf1 Pten Hps3 Mpc1 T omm70a Timm17b Ube2g1 Gla Bcl10 Nxf2 Lifr Gm27529 Wdr70 2410089E03Rik Nipbl Nup155 mediator target 0 1 2 3 4 5 6 LOD diff Wscd2 Nsmaf Slmo1 Pbx1 Slco4a1 Mapt Fcgrt Pyroxd1 Arhgef10l Tmem55a Hmcn2 Mst1 C2cd2l Mtap7d3 Nmt2 Atp2a3 Nrg2 Ypel2 Igsf21 Rragb Zfp946 Tmsb15a Cxxc1 Otud6a Trap1a Xlr3c Armc10 Lin28a Timm8a2 Gm364 Hormad1 Xlr3b Trim46 Xlr3a Swap70 Qser1 Tle1 Kpna4 Npl Eya3 target 0 1 2 3 4 5 6 LOD d Lifr Gm27529 Wdr70 2410089E03Rik Nipbl Nup155 Target Genes Candidate Mediators LOD Drop
18. The causal variant(s) on Chr15 appears to segregate in a 4:4 Founder strain pattern. PWK CAST NOD WSB B6 A/J NZO 129S1 Founder Allele Target Gene
19. PWK CAST NOD WSB B6 A/J NZO 129S1 Founder Allele Target Gene Wild-derived Founders + NOD = WD-like
20. Lifr genotype is associated with self renewal capacity in CC-RIX mESC lines. Lifr Genotype Percent self renewal Anne Czechanski
21. The Chr 15 genomic locus under this trans-band Scale chr15: Multiz Align 500 kb mm10 7,100,000 7,200,000 7,300,000 7,400,000 7,500,000 7,600,000 7,700,000 7,800,000 7,900,000 8,000,000 8,100,000 8,200,000 8,300,000 8,400,000 Basic Gene Annotation Set from GENCODE Version M14 (Ensembl 89) RefSeq Genes Placental Mammal Basewise Conservation by PhyloP Vertebrate Multiz Alignment & Conservation (60 Species) Gm38282 Lifr Lifr Lifr Egflam Egflam Gm16029 Gm37743 Gdnf Wdr70 Gm27529 Nup155 2410089E03Rik Gm24144 Nipbl Lifr Lifr Egflam Egflam Egflam Gdnf Gdnf Gdnf Gdnf Wdr70 Nup155 2410089E03Rik Nipbl Nipbl Placental Cons 3.296 _ -3.94 _ >20,000 SNPs in this region among Founder strains 185 SNPs with 4:4 pattern
22. 1 SNP in this region with 4:4 pattern Scale chr15: Multiz Align 50 kb mm10 7,110,000 7,120,000 7,130,000 7,140,000 7,150,000 7,160,000 7,170,000 7,180,000 7,190,000 UCSC Genes (RefSeq, GenBank, tRNAs & Comparative Genomics) Basic Gene Annotation Set from GENCODE Version M14 (Ensembl 89) RefSeq Genes Placental Mammal Basewise Conservation by PhyloP Vertebrate Multiz Alignment & Conservation (60 Species) AK037505 Lifr Lifr Lifr Lifr Lifr Lifr Lifr Lifr Lifr Placental Cons 3.296 _ -3.94 _ chr15:7116944 rs50454566
23. Candidate SNP overlaps ESC-specific open chromatin peak. Scale chr15: 10 kb mm9 7,060,000 7,065,000 7,070,000 7,075,000 7,080,000 7,085,000 7,090,000 UCSC Genes (RefSeq, GenBank, tRNAs & Comparative Genomics) 3134 DNaseI HS Signal Rep 1 from ENCODE/UW B-cell (CD19+) DNaseI HS Signal Rep 1 from ENCODE/UW Cerebellum DNaseI HS Signal Rep 1 from ENCODE/UW Cerebrum DNaseI HS Signal Rep 1 from ENCODE/UW ES-CJ7 DNaseI HS Signal Rep 1 from ENCODE/UW MEL DNaseI HS Signal Rep 1 from ENCODE/UW Whole Brain Adult 8 Weeks DNaseI HS Signal Rep 1 from ENCODE/UW Lifr Lifr Lifr Lifr 3134 S 1 200 _ 1 _ Bcell (CD19+) S 1 200 _ 1 _ Cerebellum S 1 200 _ 1 _ Cerebrum S 1 200 _ 1 _ ES-CJ7 S 1 200 _ 1 _ MEL S 1 200 _ 1 _ 200 _
24. Mediation of Lifr expression on ATAC peaks Scale chr15: DO 5 kb mm10 7,115,000 7,120,000 7,125,000 7,130,000 DO PB357.07_chr15 PB357.12_chr15 PB357.19_chr15 PB358.07_chr15 PB358.18_chr15 PB361.76_chr15 PB368.06_chr15 table browser query on snp142Common wgEncodeUwDnaseEscj7S129ME0SigRep1 Basic Gene Annotation Set from GENCODE Version M14 (Ensembl 89) rs50454566 Lifr Lifr PB357.07_chr15 60 _ 0 _ PB357.12_chr15 60 _ 0 _ PB357.19_chr15 60 _ 0 _ PB358.07_chr15 60 _ 0 _ PB358.18_chr15 60 _ 0 _ PB361.76_chr15 60 _ 0 _ PB368.06_chr15 60 _ 0 _ wgEncodeUwDnaseEscj7S129ME0SigRep1 201 _ 1 _
25. 1 2 3 4 5 50 60 70 80 90 Preliminary Validation: SNP swap in Founder mESC lines confirms the causal SNP in the Lifr enhancer. 10x 1x 1/10 1/100 1/1000 LIF Concentration in Culture Media % Pluripotent Cells High Lifr strain (129) Low Lifr strain (WSB) Anne Czechanski
26. 1 2 3 4 5 50 60 70 80 90 10x 1x 1/10 1/100 1/1000 LIF Concentration in Culture Media % Pluripotent Cells High Lifr strain (129) Low Lifr strain (WSB) High strain with Low SNP Preliminary Validation: SNP swap in Founder mESC lines confirms the causal SNP in the Lifr enhancer.
27. 1 2 3 4 5 50 60 70 80 90 10x 1x 1/10 1/100 1/1000 LIF Concentration in Culture Media % Pluripotent Cells High Lifr strain (129) Low Lifr strain (WSB) Low strain with High SNP Preliminary Validation: SNP swap in Founder mESC lines confirms the causal SNP in the Lifr enhancer.
28. 1 2 3 4 5 50 60 70 80 90 10x 1x 1/10 1/100 1/1000 LIF Concentration in Culture Media % Pluripotent Cells High Lifr strain (129) Low Lifr strain (WSB) Low strain with High SNP High strain with Low SNP Preliminary Validation: SNP swap in Founder mESC lines confirms the causal SNP in the Lifr enhancer.
29. Genetic dissection of pluripotency One SNP in NOD & wild-derived strains in ATAC peak, exerts effects through chromatin Peak (partially) mediates Lifr eQTL Lifr expression regulates 200+ other transcripts …and affects pluripotency.
30. Thank you Dan Skelly Laura Reinholdt Anne Czechanski Chris Baker Catrina Spruce Candice Byers Steve Munger Selcan Aydin Alex Stanton Gary Churchill Partners: Ted Choi – Predictive Biology, Inc. Alison Harrill – NIEHS NTP @genomequant @Laura_Reinholdt @stevemunger