X-chromosome Inactivation

In mammals, the sex determination system confers males with one X and Y chromosome, Y
being from the male parent. In case of females there are two X chromosomes, one X
chromosome from each parent. Hence there is a necessity for dosage compensation for
mostly X linked genes, by silencing any one of the X chromosomes. Inactivation of X
chromosome happens during embryonic development at the blastocyst stage, in XX one of
the X undergoes inactivation, either paternal or maternal X chromosome is randomly
silenced within all the non-germline cell of the embryo proper. Upon achieving this
inactivation, the same inactive X chromosome continually is present in all future daughter
cells

Role of lnRNA and chromatin

While the complete understanding of X Chromosome Inactivation (XCI) is not yet achieved,
it is known that a 500 kb stretch of DNA located at Xq 13 called X-inactivation center (XIC) is
of great importance. Located within the loci there is a 100kb core region which consists of
various long nucleotide RNAs (lnRNAs), one of the more important lnRNAs is the X-inactive
specific transcript , also called Xist. Xist has antisense transcript named Tsix along with X-
inactivation intergenic transcription elements Xite, Xist activator called Jpx all of which play
an important function in X Chromosome Inactivation[2].
X-inactive specific transcript (Xist) is a 19 kb long transcript expressed by the X chromosome
(Xi) undergoing inactivation. Only X chromosome undergoing inactivation produces Xist[3].
Tsix on the other hand is 40 kb long and tasked with negatively regulating Xist. X-inactivation
intergenic transcription elements (Xite) is the transcriptional enhancer of Tsix[2]. while Jpx is
necessary for transcriptional expression of Xist[4].

Fig1: Genes associated with XIC. Green box shows expressed genes while grey bx shows
silenced genes.
In case of mouse Xist expression is visible by the 8-cell stage in mouse embryos, in case of
humans it is during the same stage[7].
Process
Tsix and Xite function in counting mechanism in embryonic pluripotent cells at the onset of
differentiation. During this time the two homologous X chromosomes are pulled together in
close proximity to each other’s X-inactivation center (XIC). This chromosome proximity or
‘chromosome kissing’ as it is called, requires Tsix and Xite and is brought in presence of RNA
polymerase II. This chromatin insulator factor CTCF is quite important for this process as
well. This CTCF plays the role of binding Tsix and Xite genomic loci[5].
1)Active X chromosome
Once this binding has been achieved, the molecular cross-talk across the RNA-protein bridge
occurs, where transcription factors such as OCT4 present on the Tsix promoters of both X
chromosomes are put onto one of the X chromosomes thermodynamically. The X
chromosome onto which this transcription factors are dumped becomes the active X
chromosome (Xa) through continued expression of Tsix. Thereafter DNA methyltransferase
3A (DNMT3A-responsible for DNA methylation) carries out stable silencing of Xist on the
active chromosome (Xa) [6].
2)Inactive X chromosome
On the other X chromosome, that is the chromosome to be silenced (Xi), Tsix is
downregulated. This results in de-repression of Xist in cis. The Xist transcript undergoes
capping, splicing and further is polyadenylated yet escapes export.
The increasing levels of Xist RNAs start getting accumulated and are leashed in cis to the Xi
which is brought upon with the assistance of DNA and RNA associations of the
transcriptional regulator YY1, which further allows Xist RNA to spread and cover the Xi
chromosome with its RNA cloud. Xist recruits a chromatin modifier to the Xi, called the
Polycomb Repressive Complex 2 (PRC2)[4]. This spread of the Xist cloud initiates with
around 150 PRC2 binding sites, that is CpG islands arounf the chromosome , which are then
followed by 3000-4000 binding sites.
Initially the PRC2 is recruited towards the X-inactivation center (XIC) and to the Xist
transcripts. This is due to the help of another long nucleotide RNA called RepA . Along with
YY1 and RepA, the Xist also collaborates with hnRNP U (Heterogeneous Nuclear
Ribonucleoprotein U) which is a nuclear matrix-binding protein. This hnRNP U consists of
bivalent (DNA and RNA) binding domains that are necessary for Xi formation[9]. After it
settles alongside the Xi with spreading Xist transcripts, PRC2 is tasked with marking the
chromatin with H3K27 trimethylation. This trimethylation is associated with downregulation
of genes via formation of hetero-chromatic regions. The trimethylation is brought upon H3
histone proteins.
Besides, DNA methylation occurs within the binding sites (CpG islands) on the silenced
genes on the inactivated X chromosome. This methylation along with the histone
modifications in the daughter cells result in silencing of most of the genes on the Xi.
X Chromosome Inactivation is reversible at the beginning, but turns stable after the
initiation phase, which is when Xi is condensed into a heterochromatic form which is
transcriptionally inactive. Xist RNA is not required necessarily for X Chromosome
Inactivation (XCI), although Xist is extremely necessary for Xi formation. Xist RNA does
however provide stability along with PRC2.
Fig2: Steps in X Chromosome Inactivation
In the above figure
A) Both Tsix and RepA lead to complex formation by recruiting PRC2
B) Xist expression takes place due to loss of Tsix
C) Recruitment of PRC2 by RepA to the Xist promoter takes place and Xist co-
transcriptionally gets the PRC2 to form a complex
D) PRC2 leads to H3K27 methylation by binding to chromatin
E) The complex spreads along the chromosome causing further methylation

Summary
While there are some unknown variables , yet to be established as playing a role in XIC, such
as the role of XAST, which is a lnRNA whose expression is postulated to protect Xa from
inactivation, we do however have little conflict of interest on the X inactivation itself. Long
nucleotide RNA called Xist plays a huge role in the X chromosome inactivation , by directing
methylation of the chromosome. Chromatin insulating factors aid Xist in this inactivation.
The chromatin modifications are due to H3K27 methylation on histones and deactylation
caused by Xist. It is the prevention of acetylation and demethylation that leads to formation
of condensed chromosome and it becoming transcriptionally inactive.

References

1]Long Noncoding RNAs in Imprinting and X Chromosome Inactivation, Stephen Pirnie;Joseph

Autuoro;Gordon Charmichael. 2013

2] Kanduri, C. Long noncoding RNA and epigenomics. Adv. Exp. Med. Biol. 2011

3] Brown, C.J.; Ballabio, A.; Rupert, J.L.; Lafreniere, R.G.; Grompe, M.; Tonlorenzi, R.; Willard, H.F. A gene
from the region of the human X inactivation centre is expressed exclusively from the inactive X
chromosome. Nature 1991

4] Lee, J.T. Epigenetic regulation by long noncoding RNAs. Science 2012

5] Umlauf, D.; Fraser, P.; Nagano, T. The role of long non-coding RNAs in chromatin structure and gene
regulation: Variations on a theme. Biol. Chem. 2008

6] Lee, J.T. Lessons from X-chromosome inactivation: Long ncRNA as guides and tethers to the
epigenome. Genes Dev. 2009

7] Van den Berg, I.M.; Laven, J.S.; Stevens, M.; Jonkers, I.; Galjaard, R.J.; Gribnau, J.; van Doorninck, J.H. X
chromosome inactivation is initiated in human preimplantation embryos. Am. J. Hum. Genet. 2009

8] Perk, J.; Makedonski, K.; Lande, L.; Cedar, H.; Razin, A.; Shemer, R. The imprinting mechanism of the
Prader-Willi/Angelman regional control center. EMBO J. 2002

9] Hasegawa, Y.; Brockdorff, N.; Kawano, S.; Tsutui, K.; Tsutui, K.; Nakagawa, S. The matrix protein hnRNP
U is required for chromosomal localization of Xist RNA. Dev. Cell 2010