1. Epigenomics and transcriptomics regulation of human ESC and iPSC : A correlationEpigenomics and transcriptomics regulation of human ESC and iPSC : A correlation
study ofstudy 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 endodermal
differentiation
ABSTRACTABSTRACT
Generation of iPSC has provided immense potential
in regenerative medicine and study of disease
models. iPSC have been shown to be highly similar to
ESC at gene expression, chromatin modification and
c-methylation level, the latter two being involved in
epigenetic regulatory mechanisms. These
mechanisms involve epigenetic barriers that might
restrict the transition from somatic to induced
pluripotent state and play a critical role in deciding
cell’s fate. Hence it is important to understand the
factors involved in this transition to gain better
insights in to the reprogramming mechanism of cells.
Here, we report analyses of hESC and iPSC cell lines
using an integrated approach comprising ChIP-seq,
RNA-seq and BS-seq data. Expression correlation
with chromatin remodelling and c-methylation for
certain epigenetic and reprogramming factors was
also performed with special emphasis on KDM2B, as
it 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 cell
like state.
• Along with reprogramming factors like OCT4,
KLF4,SOX2 and NANOG it is essential to induce/repress
other epigenetic factors like KDM2b, DNMT3b etc., for
better transformation.
•More biological replicates are required to obtain
statistical significant inference.
•Accessibility to more stem-cell NGS data will aid
research in regenerative medicine.
FUTURE WORKFUTURE WORK
1) 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
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
We 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 epigenomic
differences. 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/F
bx110
Enhanced
Efficiency
Reduced
Efficiency
Self renewal
,
differentiatio
n
Lineage bias
H3K36me KDM2b Promotes IPS
generation
Impaired
ability
Is highly
expressed
Differentiation
DNA
methylation
Dnmt3a/b NA No effect No effect Differentiation
defect
DNA
methylation
Dnmt1 Required for
pluripotency
Reduced
efficiency
De novo
methylation,
imprinting
Leads to
impaired
development
Ref. zhanglab Harvard univ.
Table 3. Epigenetic Modulators. List of major
chromatin regulators which are necessary in
reprogramming and stem cell like state maintenance.
ABBREVIATIONSABBREVIATIONS
hESC – human Embryonic Stem Cells
iPSC – induced Pluripotent Stem Cells