Spatial partitioning of the regulatory
landscape of the
X-inactivation centre
Elphège P. Nora, Bryan R. Lajoie, Edda G. Schulz, Luca
Giorgetti, Ikuhiro Okamoto, Nicolas Servant, Tristan
Piolot, Nynke L. van Berkum, Johannes Meisig, John
Sedat, Joost Gribnau, Emmanuel Barillot, Nils Blüthgen,
Job Dekker & Edith Heard
Zhouyan Lu, MengMeng Wu, Peng Zeng
2013/4/26
X-chromosome inactivation (XCI)
• X-inactivation is a process by which one of the
two copies of the X chromosome present in
female mammals is inactivated.
• The X-inactive specific transcript (Xist) gene
encodes a large non-coding RNA that is
responsible for mediating the specific
silencing of the X chromosome from which it
is transcribed.
X-inactivation centre (Xic)
• Contains Xist ,its repressive antisense
transcript Tsix and the regulator of Tsix, Xite,
and many other elements.
• Xic is thought to affect choice, counting and
cis-inactivation during the initiation of X
inactivation
Spatial partitioning
• Analysed the spatial organization of a 4.5megabases (Mb) region including Xist.
• Discovered a series of discrete 200-kilobase to
1 Mb topologically associating domains (TADs)
on this region.
Methods
• chromosome conformation capture carboncopy (5C)
• fluorescent in situ hybridization (FISH)
Topologically associating domains
• 5C2 analysis in
undifferentiated
mouse embryonic
stem cells (ESCs)
• Long-range ( >50 kb)
contacts preferentially
occur within a series of
discrete genomic
blocks.
3D DNA FISH
Whether the size and location of these
TADs is identical…
…in male and female mouse ESCs?
…during differentiation?
…during XCI?
What might drive chromatin folding in
TADs?
• H3K27me3 and H3K9me2
Lack H3K27me3
Lack H3K9me2
Whether folding in TADs is driven by boundary
elements at their borders?
Summary
• The partitioning is stable throughout
differentiation, X inactivation and in cell lines
with impaired histone-modifying machineries.
• TAD boundaries can have a critical role in highorder chromatin folding but still remain
further investigating.
What's the relationship between
TADs and gene expression?
• The gene expression within the same domain
have higher correlation.
• downregulated : TAD D,F
• upregulated : TAD E
• Jpx,Ftx,Xpr,Rnf12 from TAD E are positive
regulators to Xist, which can be intergrated
into a similar cis-regulatory network.
• Predict that TAD E(~550k) represents
minimum 5' regulatory region for Xist
expression, which maybe the reason why
transgenes tested so far(covering 150kb 5' to
Xist) can't recapitulate Xist expression.
• Xist promoter and positive regulators are
located in TAD E.
• Tsix promoter located in TAD D, in addition to
Xite enhancer, more distant elements within
TAD D may regulate Tsix.
• Tg53: covering TAD D
• Tg80:just covering Xite
• some elements within TAD D regulate Tsix
Summary
• TAD have significant correlation with gene
expression.
• TAD can help us to discover gene expression
pattern.
• TAD can help us to discover gene regulation
elements.
Significant looping events involving the Tsix promoter or its enhancer Xite
Peak-calling of chromosomal looping events at Xist, Tsix and the Xite enhancer
Chromatin signatures of enhancers
Enhancer signatures in the Xist/Tsix region
Linx identification
a transcript initiating approximately 50 kb upstream of the Ppnx promoter
Linx identification
a transcript initiating approximately 50 kb upstream of the Ppnx promoter
Linx RNA shares several features with non-coding RNAs, such as accumulation around
its transcription site, nuclear enrichment and abundance of the unspliced form.
Linx and Tsix are co-expressed in the inner cell mass of blastocysts from
E3.5–4.0 onwards, as well as in male and female mouse ESCs.
Linx RNA is not detected earlier in embryogenesis, nor in extra-embryonic
lineages, implying an epiblast-specific function.
Linx expression is frequently monoallelic, even before Xistupregulation, revealing a
transcriptional asymmetry of the two Xic alleles before XCI
Triple RNA FISH for Linx, Tsix and Xist in differentiating female mouse ESCs revealed that
before Xist upregulation, the probability of Tsixexpression from alleles coexpressing Linx is significantly higher than from alleles that do not express Linx.
Triple RNA FISH for Linx, Tsix and Xist in differentiating female mouse ESCs revealed that
before Xist upregulation, the probability of Tsixexpression from alleles coexpressing Linx is significantly higher than from alleles that do not express Linx.
Discussion