x-chromosome inactivation

gene dosage compensation

• dimorphic sex chromosomes lead to dosage imbalances between homogametic and heterogametic sexes

• organisms evolved different strategies to deal with it:

  • insects: upregulated expression of male X chromosome

  • mammals: inactivation of one X chromosome in females

properties of inactive X chromosome (Xi)

• compact structure

• nuclear periphery

• epigenetically inherited

• DNA methylation (promoters)

• H3K27me, H3K9me, hypoacetylation of histones

• Xist RNA

coincidence of X-chromosome inactivation and differentiation

• XCI occurs first specifically in trophectoderm (extra-embryonic tissue) → specifically affects paternal X (imprinted)

• XCI occurs in embryo after implantation, when epiblast cells begin to differentiate (gastrulation)

• choice of which X is activated seems to be random

• reactivation occurs in primordial germ cells

random X-chromosome inactivation

• accounts for mosaic expression of X-linked genes in females

• Mary Lyon proposed random X inactivation (1961)

• mosaicism is epigenetic, not genetic

X-chromosome inactivation counting

only one active X in female embryonic cells for every diploid complement of autosomes

X-chromosome inactivation choice

• one particular X is chosen for inactivation in each female embryonic cell

• once the choice is made, this X is maintained in inactive state through cell division

X inactivation center (Xic)

• defined by study of X chromosome truncations and translocations → X chromosomes missing Xic cannot be inactivated

• key part of Xic is Xist → silencing caused by expression of Xist

Xist

• long (17kb) non-coding RNA

• spliced and polyadenylated but retained in nucleus

• coats Xi and causes silencing in cis

  • silencing occurs on chromosome it’s expressed from

• repeat sequences are binding sites for important regulators

  • silencing (A), PRC1 recruitment (B,C), localization (E)

cultured embryonic stem cells

• good model system for studying XCI

• derived from blastocyst stage embryos before germ layers are formed (pluripotent)

• can be cultured and differentiated in vitro

Xist regulation

• involves interplay between cis and trans factors

  • repressed by pluripotency transcription factors

• regulated by Tsix

Tsix

• regulates expression of Xist

• normally, XCI in differentiating female ES cells occurs randomly on either X

• removal of Tsix promoter from one X causes Xist to be activated only from this chromosome → X lacking Tsix is always inactivated

  • monoallelic expression corresponding to future Xi

  • only Tsix expressed in males

regulation of Xist by interplay between pluripotency transcription factor Rex1 (trans) and Rnf12 (cis)

• before differentiation: lots of Rex1, less Xist, no XCI

• after differentiation: Rex1 degraded by Rnf12 through ubiquitin proteasome system, more Xist, XCI

• differentiation switch does not work in males because there is only one X

  • not enough Rnf12 expressed to get rid of Rex1 and allow Xist expression

• potential mechanism for ensuring proper ratio of autosomes to active X

genetic approach for defining functions of Xist RNA

• delete parts of RNA and ask how Xist function is altered

• delete A repeat → no repression of transcription, but Xist can still localize to X chromosome

• delete C repeat → no PRC association or H3K27me on inactive X, but repression can still happen

• delete E repeat → no Xist localization to inactive X, repression compromised

biochemical approach for defining functions of Xist RNA

• identify proteins that interact with Xist RNA (or parts of Xist RNA)

• put pieces of RNA into cell extracts and see what binds to them

removal of Xist E repeat or proteins that bind to it

• leads to Xist dispersal from Xi and loss of X inactivation

• multiple RNA binding proteins interact with E repeat and with each other to tether Xist to Xi

• E repeat is necessary for inactive state but inactivation also requires A repeat independently

removal of Xist C repeat

• prevents recruitment of PRC1 and H2K27me to inactive X

• inactive X is enriched in H3K27me

• PRC1/2 complexes localize to inactive X when Xist expression is upregulated

• localization is Xist-dependent (B and C repeats)

• PRC1/2 seem to have a role in stabilizing silent state of inactive X but are not absolutely required for silencing

removal of Xist A repeat

• compromises silencing function of Xist and recruits SPEN and other chromatin proteins

• SPEN associates with HDAC3 (→ histone deacetylation)

• prevents X inactivation even though Xist can still localize to Xi

formation of inactive X chromosome

• Xist doesn’t spread from one location to another

• nucleates foci of heterochromatin proteins at specific locations that drag in and turn off neighbouring genes

escape from XCI

• not all genes on X are subject to inactivation

• explains genetic disorders caused by X-chromosome aneuploidy (Turner’s syndrome, Klinefelter’s syndrome)

• escapee genes often have homologs on the Y chromosome

  • makes them critical for balanced gene dosage

maintenance of XCI

• can generally be maintained in absence of Xist expression → once formed in early embryo, stably propagated without Xist

• stable maintenance in embryo requires DNA methylation (by DNMT1 and DNMT3B) and hypoacetylation of histones

• CGI methylation at promoters is common on inactive X (compared to rest of genome)

• occurs at later stages of XCI

inactive X reactivation (XCR)

• occurs in primordial germ cells (parallel with erasure of imprinting)

• also important for reprogramming somatic cells to induced pluripotent stem cells (iPSCs)

• failure of XCR in reprogrammed cells seems to correlate with lower pluripotency

• impetus to develop strategies to reactivate inactive X

• implications for X-linked genetic disorders

types of hiPSCs

• depends on state of XCI

• XaXa (no Xist) → XaXi (class I, mouse-like)

• XaXi (with Xist) → XaXi (class II, Xist on)

• XaXe (no Xist) → XaXe (class III, Xist off)

  • Xe chromosome with aberrant or eroded XCI