Epigenomics and transcriptomics regulation of human ESC and iPSC : A correlation Epigenomics and transcriptomics regulation of human ESC and iPSC : A correlation study of study of pluripotencypluripotency markers, DNMT and KDM familymarkers, DNMT and KDM family

Tharvesh M. Liyakat Ali, Ashish S. Tomar, Dr. Subhasini Srinivasan* and Dr. Vibha Chaudhury*

Institute of Bioinformatics and Applied Biotechnology, E-City Phase I, Bangalore-560100

TARGET GENESTARGET GENES

MATERIALSMATERIALS

METHODOLOGYMETHODOLOGY

Chromatin Remodeling

ES Vs iPS

Mapping Chip-seq(ES and iPS) on

Hg19 using Bowtie

Correlation

Mapping BS-seq (ES and iPS) on Hg19

using BSMap

C-methylation Analysis CpG, CHH,CHG

ES Vs iPS

Chromatin accessible

regions

Differential Expression

ES Vs iPS

Mapping mRNA(ES and iPS) on Hg19 using Bowtie

Expression of transcripts involved in

reprogramming

MACS Protocol

DEseq2 Package

Annotation and Peak analysis

GBSA

GENES FUNCTION

OCT4 Self renewal of stem cells by forming heterodimer with SOX2

NANOG Maintain plueripotency - works with OCT4 and SOX2 to establish ES identity

KLF4 Key transcription factor in ESC development & prevent differentiation

SOX2 Maintain plueripotency with OCT4

DNMT3A Tranfer methyl group to specific CpG in DNA, reduce expression

DNMT3B Denovo methylation

DNMT3L Required for differentiation of embryonic stem cells

PHF19 Bind to H3K36Me3 & recruits PRC2 which demethylates leads to repression

DNMT1 Maintanance methyl transfer enzyme

KDM2A Histone H3 Lysine 36 demethylase

KDM2B Promotes iPSC generation

KDM4A Upregulated during muscle differentiation, Demethylates H3K9 and H3K36

KDM6A H3K27 demethylase & endodermal differentiation

KDM1B H3K4me2 demethylase & role in modelling iPSC as ESC

KDM3A H3K9Me2 demethylase & Essential in progression with endodermaldifferentiation

ABSTRACTABSTRACT

Generation of iPSC has provided immense potentialin regenerative medicine and study of diseasemodels. iPSC have been shown to be highly similar toESC at gene expression, chromatin modification andc-methylation level, the latter two being involved inepigenetic regulatory mechanisms. Thesemechanisms involve epigenetic barriers that mightrestrict the transition from somatic to inducedpluripotent state and play a critical role in decidingcell’s fate. Hence it is important to understand thefactors involved in this transition to gain betterinsights in to the reprogramming mechanism of cells.Here, we report analyses of hESC and iPSC cell linesusing an integrated approach comprising ChIP-seq,RNA-seq and BS-seq data. Expression correlationwith chromatin remodelling and c-methylation forcertain epigenetic and reprogramming factors wasalso performed with special emphasis on KDM2B, asit is an important factor in iPS generation.

RESULTSRESULTS• Analysis of RNA-Seq, BS-Seq and ChIP-Seq shows correlation in gene expression, methylation and histone modification of hESC and iPSC.

• One of the genes KDM2B which is essential for promoting iPSC generation was not expressed and also extensive methylation(Fig. 9) could be found all over the gene.

• Similar level of expression is observed between iPSC and fibroblast, except Oct4, Nanog, Sox2, Myc which are only expressed in iPSC because of reprogramming.

• KDM1A gene which is necessary for cell differentiation is expressed more in iPSC than in hESC (Table.3).

• Although pluripotent factor is expressed in iPSC, several other epigenetic factor like DNMT3b, KDM2b etc. exhibits different chromatin position and methylation than hESC.

• This shows that iPSC was not efficiently reprogrammed or dedifferentiaed to ES like state, but it is seemingly in transient state.

Table 1: List of targeted reprogramming and epigenetic regulators and its function.

Fig 1. Histogram representing expression level from RNA-seq data.Comparison of epigenetic regulator between fibroblast cell(Control), hESC and iPSC . KDM2B promoting pluripotent stem cell was not expressed, other KDM’s also expressed in iPSC as similar to the parental fibroblast cell i.e. not fully transformed

REFERENCESREFERENCES

Fig 3. Whole chromosome wide localization of H3K36Me3 in hESC and iPSC, in ESC it is spanned across the genes, while iPSC has sharp peak at TSS and less at TES

Fig 4. Whole chromosome wide localization of H3K27Me3 in hESC and iPSC, in ESC it is localized at TSS shows less expression while iPSC shows inverse of it and localization is maintained over the genebody.

Fig 2. Heatmap: Expression of epigenetic and reprogramming factor in hESC and iPSC

Fig 8. Localization of H3K27Me3 at KDM2B(a and b), in (b) might be responsible

for repression of KDM2B in iPSC where as it is present in hESC

RNA-SeqRNA-Seq

ChIP-SeqChIP-Seq

BS-SeqBS-Seq

Fig 9. Methylation at CpG sites of KDM2B of iPSC(a) and hESC(b). In KDM2B(a) methylation over genebody is seen but not at promoter gene, although we could find no expression of KDM2B in iPSC and no methylation found in hESC clearly we could find expression.

Fig 10. Methylation at CpG sites of DNMT3A of iPSC(a) and hESC(b). In DNMT3A of iPSC(a) promoter region is methylated , therefore it shows no expression, whereas no methylation is found in hESC(b) results in expression of DNMT3A, that helps in maintaining pluripotency

Fig 5. Localization of H3K36Me3 at OCT4(a and b) , SOX2(c and d), NANOG(e and f) and MYC(g and h) in hESC and iPSC respectively shows slight difference in chromatin position compared to other factors and fold enrichment

Fig 6. Localization of H3K36Me3 at KDM3A(a and b), KDM1A(c and), KDM2B(e and f), KDM4A(g and h), KDM3B(i and j), KDM4B(k and l) in hESC and iPSC respectively shows difference in chromatin position at genebody and fold enrichment. Although in (f) KDM2B H3K36Me3 is spread it shows less expression in iPSC might be repression because of H3K27me3 shown.

Fig 7. Localization of H3k36Me3 at DNMT1(a and b) and DNMT3B(c and d) in hESC and iPSC respectively shows high expression of DNMT3B in both but no expression of DNMT1 in iPSC because of less localization.

CONCLUSIONCONCLUSION• iPSC was not fully transformed to embryonic stem celllike state.

• Along with reprogramming factors like OCT4,KLF4,SOX2 and NANOG it is essential to induce/repressother epigenetic factors like KDM2b, DNMT3b etc., forbetter transformation.

•More biological replicates are required to obtainstatistical significant inference.

•Accessibility to more stem-cell NGS data will aidresearch in regenerative medicine.

FUTURE WORKFUTURE WORK1) Explore ncRNAs like microRNA and lncRNA and its

differential expression between hESC and iPSC and validating

2) Build transcription factor and epigenetic network that regulates pluripotency.

Cell Lines RNA-seq Chip-seq Bs-seq

hESC GSM438361 GSM667641 GSM675542

iPSC GSM706050/51/52 GSM752993, GSM752971/75

GSM706057/58

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSWe would like to acknowledge IBAB, Prof. N.Yathindra for providing an eminent work environment and a best place for nurturing brain. We would like to thanks Dr. Srivatsan for giving us the project idea, Abdullah khan for his support in tackling computer related issues.

•Lister R, Pelizzola M, Dowen RH, Hawkins RD et al. Human DNA methylomes at base resolution show widespread epigenomicdifferences. Nature 2009•Hawkins RD, Hon GC, Lee LK, Ngo Q et al. Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 2010•Lister R, Pelizzola M, Kida YS, Hawkins RD et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 2011

Table 2: GEO accession id for downloaded datasets.

(a) (b)

(c)

(e) (f)

(d)

(g) (h)

(a)

(f)(e)

(d)(c)(b)

(j)(i)

(h)(g)

(k) (l)

(d)(c)

(b)(a)

(a) (b)

(a) (b) (a) (b)

Chromatin

Modification

Factor iPS Generation ES Self

renewal

ES Differen-

taition

Gain Of

Function

Loss Of

Function

Gain Of

Function

Loss Of

Function

H3K27me KDM6b/ Utx

No effect Reduced efficiency

No effect No effect

H3K36me KDM2a/Fbx110

Enhanced Efficiency

Reduced Efficiency

Self renewal ,differentiation

Lineage bias

H3K36me KDM2b Promotes IPS generation

Impaired ability

Is highly expressed

Differentiation

DNAmethylation

Dnmt3a/b NA No effect No effect Differentiation defect

DNAmethylation

Dnmt1 Required for pluripotency

Reduced efficiency

De novo methylation, imprinting

Leads to impaireddevelopment

Ref. zhanglab Harvard univ.

Table 3. Epigenetic Modulators. List of major chromatin regulators which are necessary in reprogramming and stem cell like state maintenance.

ABBREVIATIONSABBREVIATIONShESC – human Embryonic Stem Cells

iPSC – induced Pluripotent Stem Cells