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

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.

Steve Munger

May 14, 2018
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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

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  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

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  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.

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  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

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  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.

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  6. Evidence of genetic variability in
    maintenance of the pluripotent ground state
    in ES cells.
    Anne Czechanski
    Permissive
    Recalcitrant

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  7. N = 185
    N = 192
    Predictive
    Biology, Inc.
    Experimental Design: Profiling chromatin
    accessibility and gene expression in DO mESCs.

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  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

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  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

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  10. QTL location
    Shared trans-bands

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  11. QTL location
    eQTL or caQTL
    specific trans-bands

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  12. QTL location

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  13. Genetic dissection of an eQTL trans-band
    Chr 15: 7-8.5Mb
    202 Target genes
    Many target genes
    associated with
    pluripotency
    pathways.

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  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 _

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  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

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  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

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  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

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  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

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  19. PWK
    CAST
    NOD
    WSB
    B6
    A/J
    NZO
    129S1
    Founder Allele
    Target Gene
    Wild-derived Founders + NOD = WD-like

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  20. Lifr genotype is associated with self renewal
    capacity in CC-RIX mESC lines.
    Lifr Genotype
    Percent self renewal
    Anne Czechanski

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  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

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  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

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  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 _

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  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 _

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  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

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  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.

    View full-size slide

  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.

    View full-size slide

  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.

    View full-size slide

  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.

    View full-size slide

  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

    View full-size slide