The document discusses X chromosome inactivation (XCI) in mammals, detailing both random and imprinted mechanisms, and the role of regulatory factors such as Xist and Tsix. It explains the processes involved in XCI including counting, choice, spreading, and maintenance across different developmental stages in both mice and humans. Additionally, it highlights findings from studies on chromatin modifications during XCI and potential implications for future research.

1. X Inactivation in Mammals

2.  Definitions.
 Imprinted X Chromosome Inactivation.
 XIST Regulator.
 Why only the Xp is inactivated?
 Reactivation X Chromosome.
 Random X Chromosome Inactivation.
 Non-random X Chromosome Inactivation.

3. XCI is a dosage-compensation mechanism that evolved to
equalize expression levels of x-linked genes in female (2x) and
male (1x) by transcriptional silencing of one x-chromosome in
female mammalian cells.
It is responsible for initiating X inactivation in cis: an Xchromosome fragment that carries a Xic can become
inactivated, whereas one in which the Xic is missing cannot.
The Xic is also involved in „counting‟, whereby only a single X
is kept active per two sets of autosomes in a cell, and all other
Xic-carrying chromosomes are inactivated.

4. Normal
Reciprocal translocation
Deletion

5. Conserved structure, low sequence conservation (except for
repea).
 17 kb – 19 kb spliced, untranslated, nuclear transcript
Xist is essential for the initiation of inactivation in cis
 RNA expressed from and “coats” the inactive X chromosome in
cis.
Xist can only induce silencing during an early developmental
time.
 Conserved “A” repeats ensure silencing function... How?
Multiple Xist domains required for coating… How?

7. In mouse
 This process occur in the early stage of embryo development
approximately in 2-cell stage until the blastocyst stage and
continues in the trophectoderm and the primitive endoderm..
The cell undergo to inactivate x parental only and the x maternal
stay active..

8. Why only the imprinted X inactivation found in XP not in XM
??
because of Rnf12 that inherited only from mother germ line and
this factor act as XIST activator and work in trans ..

10. 
 In this step of imprinting x- inactivation the non-genic repeat
regions, such as long interspersed elements (LINEs) and
short interspersed repetitive elements (SINEs), is silenced.
 The repeat silencing precedes genic silencing in imprinted Xinactivation.
 The X-linked repeat elements preinactivated and inherited
from the paternal germ line.
 Imprinted X-inactivation originates from meiotic sex
chromosome inactivation (MSCI) in male spermatogenesis
and that the pre-inactivated X-chromosome is inherited from
father to daughter.

11. Meiotic silencing is a general silencing
mechanism, which represses unsynapsed
chromatin in male germ cells and has been
termed meiotic silencing of unsynapsed
chromatin (MSUC).

12. MSCI consists of two
genetically separable steps:
1
recognition of the unsynapsed axis,
in which the ATR–TOPBP1 complex phosphorylates H2AX
2
chromosome-wide spreading of the
ATR–TOPBP1 complex and the cH2AX signal to the entire
chromatin.

14. gH2AX signal occurs at the unsynapsed axes of sex
Chromosomes.
MDC1 (mediator of DNA damage checkpoint protein 1)
mediates the spreading of the gH2AX signal over the
chromosome-wide domain through directly recruits the
ATR–TOPBP1 complex to amplify the gh2ax signals in
meiotic silencing.
XY body formation The chromosome-wide accumulation
of gH2AX on sex chromosomes is concomitant with the
formation of a distinct heterochromatin domain called
the XY body.

15. In mouse
 This step Occurs at the four cell stage.
 The XIST expression lead to chromosome XP-inactivation.
What happens in this step?
XIST expressed from XP .
XIST starts coating the XP .
2
3 The number of XIST recruit the Polycomb repressive
complex 2 (pcr2) to the xi.
1

16. 4
5
PCR2 recruite histon h2 lysine 27 trimethylation
(H3k27 me3).
TSIX RNA expressed from XM .

17. In human
They thought that imprinted x-inactivation doesn‟t occur in
human, human x chromosome was investigated(Ilse M.
van den Berg.,et al 2009) in three levels:
1
.
In
At the morula stage only a fraction of unstable
male XIST signals was expressed and this indicating
absence of XCI male preimplantation.

18. In female
Majority of cells had pinpoint signals for XIST RNA at
the 8-cell stage. The XIST signal gradually accumulated
to a full cloud on one of the X chromosomes at the late
morula and blastocyst stages.

19. Transcriptional activity on the XIST-coated chromosomal
region.
activity was investigated by Cot1 RNA FISH staining,
which highlights areas of ongoing hnRNA transcription;
trancriptionally silent nuclear compartments, such as
an Xi chromosome, are devoid of Cot1 RNA.
In
male
!

20. In female
(A–C) Cells of a female lastocyst
embryo stained for Cot1 RNA (red
in A) and XIST RNA (green in B)
showing depleted regions of Cot-1
RNA around the XIST signals
indicating the position of the Xi
chromosome (merged in C).
(D–F) Representative cell of a
female blastocyst with staining for
the X centromeres and XIST RNA
(D; Xcen in magenta, XIST in
green) together with Cot1 (red in E).
Transcription of Cot1 RNA
was absent in a region that
overlaps with XIST RNA staining
(F), whereas the active X without
XIST staining overlaps with
a Cot1-positive region.
(G–J) Female blastocyst cell with
two X centromeres (cyan in G) has
a single XIST cloud on one X
chromosome (green in H) and
monoallelic expression of CHIC1 on
the other X chromosome (red in I,
merged in J).

21. Chromatin conformational changes on the inactive
X -chromosome to further explore.
1. The chromatin state of X chromosomes was carried out with
antibodies that detect
 Hypoacetylation of lysine 9 on histone H3 (H3K9ac)
 accumulation of trimethylation of lysine 27 on H3 (H3K27Me3).
 The enrichment of the histone variant acroH2A.
2. The gender identification of the embryo used DNA FISH.
4
Embryos did not show
accumulation Of
In
male
 H3K27Me3 or
 macroH2a
And no specific exclusion of
H3K9ac was
observed.

22. In female
double staining showed a single region where accumulated
H3K27Me3 formed an exact overlay with the region of H3K9 hypoacetylation.
(A–C) indicating the presence of an Xi. Furthermore,macroH2A enrichment and
accumulation of H3K27Me3
(D–F) colocalized exactly in blastocyst cells. Up to 30% of the analyzable cells in
blastocysts showed a double immunostaining of chromatin marks that are specific
for XCI (either H3K27Me3 together with macroH2A or H3K27Me3 in a depleted
region of H3K9ac). The other 70% of the cells had no visible accumulation
(or depletion, in the case of H3K9ac) of either antibody, and single accumulations
were rarely found (<5%).

23. In marsupials
Imprinted XCI is found in all
tissues of marsupials
XCI is not random but is
paternally imprinted
.Marsupials exhibit dosage
compensation by wide
silencing of the X.
 No hypermethylation is
found Only hypoacetylation.

24. This reactivation process allows both Xp and Xm get an
equal chance to be subjected to random XCI in the future
embryo-proper.
The over expression of TSIX is sufficient for reactivation
of XP .. The experiment done by transgenic TSIX in extraembryonic tissue placenta (which always have imprinting
inactivation..This cell was reactivated by TSIX.

25.  The first instance of X-reactivation occurs at the blastocyst stage between
embryonic day (E)3.5 and E4.5. This coincides with the time when blastocysts
implant into the uterus and shortly after the first distinct cell lineages become
apparent.
 X-chromosome reactivation doesn‟t occur in the trophectoderm and primitive
endoderm, which will later give rise to extra-embryonic tissues like the placenta.
 The characteristic signs of the X-reactivation process are
 the downregulation of Xist expression.
 disappearance of the accumulation of Polycomb proteins Ezh2 and Eed and their
associated histon H3 lysine 27 di/tri-methylation mark (H3K27me2/3) from the
paternal X-chromosome.
 A new study suggests that reactivation of some X-linked genes and of repeat
sequences might even occur before the chromosome-wide removal of Xist RNA and
H3K27me3 from the inactive X-chromosome.

26.  Binding of Oct4, Sox2 and Nanog has been demonstrated to
Xist intron 1 in ES cells lead to downregulation of Xist
expression from the inactive X-chromosome.
 Furthermore, Oct4, Klf4, c-Myc and Rex-1 bind DXPas34
and Oct4, Sox2 and Klf4 bind Xite, both of which are
enhancers of Tsix, the non-coding antisense regulator gene
of Xist during X-inactivation.
 Depletion of Oct4 from ES cells by RNAi knockdown or
inducible downregulation results in upregulation of Xist from
both X-chromosomes in female cells and depending on
experimental conditions even from male ES cells.
 Conversely Tsix is downregulated after Oct4, Rex1 or c-Myc
knockdown.

27. Nanog in particular seems to be important for Xreactivation
in blastocysts, as Nanog-mutant female embryos
fail to erase the characteristic H3K27 trimethylation from
the inactive X in the inner cell mass .
Nanog-mutant ES cells show some Xist upregulation
albeit at lower levels than after Oct4 depletion.

28. Xist regulatory factors
Xist activators
JPX. RNF12. H19. RepA (recruits PRC2)..
(RepA-PCR2)..Target the H3k27me3 modification on the Xist
promoter to facilitate Xist transcription.
Xist inhibators
(Plulripotency factors )
Tsix
TSIX expression blocks loading of the
RepA-PRC2 complex onto the 5 end of
Xist.
Implicated in the direct regulation of
DNA methylation.
Recruiting the RNAI machinery.
Tsix activators (Xite, DXPas34).

30. Random X inactivation occurs in the early female embryo, where
both the maternal and the paternal X chromosome have an equal
chance of becoming inactivated.

32. Initiation
 Sensing: Xpr which is a heterochromatic
region brings the XICs together.
 Counting/choice: The number of XICs are
counted, and one X chromosome is
randomly chosen to remain active while
the other is targeted for inactivation. This
was done by XICs pairing.

33. Pairing .. Counting.. Choice
 X-chromosome pairing via the interaction between oct4 and ctcf that lead to
distribution of tanscription and pluripotent factors on the two X–
chromosome this lead to:
 Xist expression from one X-chromosome and Tsix repression from the same
allele.
 In the other chromosome Tsix recruits the DNA methylatransferase dnmt3a
which methylate Xist promoter leading to Xist silencing and the cell now
have X activation . In the active X the Tsix regulate the Xist silencing by
directly suppress Xist or indirectly by suppress Xist activators
 RepA RNA is expressed from the future Xi and recruits the PRC2 complex.
 Jpx and Rnf12 are upregulated from both the Xi and Xa.

34. Spreading
Spreading is the process of compacting and inactivating the X
chromosome.
A gene designated XIST with the X inactivation center encoded
an RNA that coat the X chromosome and promotes compactionbeginning at the XIC and progressing toward both ends until the
entire x chromosome is inactivated, when spreading is finished
the compacted factor could bar body.
factors that found during spreading process
YY1(YingYang1)
 (hnRNPu)RNA –U (heterogeneous nuclear riboncleo protein U )
SATB1 (special A+T rich binding protein 1)
 LINE(long interspersed elements )
PCL2 (Polycomblike 2)

35. Factors
1
YY1
(YingYang1)
Figures
1,5
2
(hnRNPu) RNA –U
(heterogeneous nuclear
riboncleo protein U )
3
SATB1 (special A+T
rich binding protein 1)
4
LINE(long interspersed
elements )
5
PCL2 (Polycomblike 2)
2
3

36. Maintenance
The bar body is replicated just like other chromosome,
but both copies of the replicated bar body remain highly
compacted and essentially inactivated.
Thus, once an embryonic cell undergoes X-inactivation,
all cells that drive from it will have the same chromosome
inactivation.

37. Primary
Non random
Xinactivation
Primary non random x-inactivation cause by
the mutations in Xist or Tsix.
 When the mutation found in Tsix the wild type
become always the activated chromosome.
 When the mutation found in Xist gene the wild
type become always x-inactivated chromosome.
 According to experiment the mice that
inherited mutation in Tsix from their father, born
healthy, but those inherited mutation from their
mother only 18% of them survived.

38. Secondary
Non-random
X-inactivation
Secondary nonrandom inactivation whereby
cells expressing one of the two x-chromosome
are selected against that the selective death of
cells that inactivate the incorrect number of Xchromosome because the fate of X-chromosome
doesn't determined before silencing.

39. References
Panning, B. 2008 X-chromosome inactivation: the molecular basis of
silencing: Journal of Biology.7:30
Ilse, M. den Berg, V. Joop ,S. E, Laven. Stevens, M. Jonkers, I. Jan
Galjaard, R Gribnau, J. and J. Hikke van Doorninck 2009 X Chromosome
Inactivation Is Initiated in Human Preimplantation Embryos:The American
Journal of Human Genetics. 84: 771–779.
Kalantry, S. 2011 Recent Advances in X-Chromosome Inactivation: NIH
Public Access Author Manuscript. 226(7): 1714–1718.
Payer, B. Jeannie T, Lee . Namekawa H, S. 2011 X-inactivation and Xreactivation: epigenetic hallmarks of mammalian reproduction and pluripotent
stem cells: Hum Genet. 130: 265–280.

40. References
Jeon, Y. Sarma, K. and T Lee, J. 2012 New and Xisting regulatory
mechanisms of X chromosome inactivation:
Current Opinion in Genetics & Development. 22:62–71.
Valerie Gendre, A. and Heard, E. 2011Fifty years of X- activation
Research:Development .138: 5049-5055.
Augui, S. Elphège P. Nora. and Heard, E. 2011 Regulation of X-chromosome
inactivation by the X-inactivation centre: Nature Reviews Genetics. 12: 429442 .
E Senner, C.Brockdorff, N. Xist gene regulation at the onset of X inactivation
2009:Current Opinion in Genetics & Development.19(2): 122-126.
T. Lee, J. 2011 Gracefully ageing at 50, X-chromosome inactivation becomes
a paradigm for RNA and chromatin control: Nature Reviews Molecular Cell
Biology. 12: 815-826.