X inactivation in mammals


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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 X-chromosome 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.

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X inactivation in mammals

  1. 1. X Inactivation in Mammals
  2. 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. 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. 4. Normal Reciprocal translocation Deletion
  5. 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?
  6. 6. 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..
  7. 7. 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 ..
  8. 8.   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.
  9. 9. 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).
  10. 10. 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.
  11. 11. 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.
  12. 12. 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
  13. 13. 4 5 PCR2 recruite histon h2 lysine 27 trimethylation (H3k27 me3). TSIX RNA expressed from XM .
  14. 14. 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.
  15. 15. 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.
  16. 16. 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 !
  17. 17. 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).
  18. 18. 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.
  19. 19. 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%).
  20. 20. 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.
  21. 21. 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.
  22. 22.  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.
  23. 23.  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.
  24. 24. 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.
  25. 25. 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).
  26. 26. 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.
  27. 27. 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.
  28. 28. 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.
  29. 29. 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)
  30. 30. 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
  31. 31. 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.
  32. 32. 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.
  33. 33. 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.
  34. 34. 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.
  35. 35. 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.