Advanced Molecular Genetics-Biology 566 Xist RNA and the Mechanism of X Chromosome Inactivation by K. Plath, S Mlynarczyk-Evans, D.A. Nusinow, and B Panning
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Introduction

X-linked gene dosage between male and female animals proposed over 40 years ago by Mary Lyon.

Silencing of one of the female X-chromosomes (Xi) occurs at random early in development and coincides with differentiation of pluripotent cells to restricted lineages.

Silencing initiated at Xic/XIC and spreads along chromososme.

Once established the Xi is clonally propagated such that females are functionally mosaic for X-linked traits.

Xist/XIST gene is located in the Xic/XIC

Figure 1. Mouse and Human Xic/XIC and Xist/XIST. A. area surrounding the XIST/Xist gene on human and mouse X-chromosomes. Human domain is inverted from mouse relative to telomere. The identification of a human Tsx homolog is unclear. B. Comparison of the mouse Xist and human XIST genes. * = alternative splicing sites. Mouse has a second promoter that has not been found in other Xist/XIST genes analysed to date. Extensive alternative splicing of the human gene has been described yielding isoforms that lack exon 4, half of exon6, exon7 or include the last two introns.

Longest Xist 17.9 kb. Longest XIST 19.3 kb.

Mouse and human Xist/XIST show 49% sequence identity which is lower than 5' & 3' UT regions but slightly higher than introns.

Several short stretches of high homology and six repeated elements A-F.

No open reading frame. Must operate as polyadenylated RNA.

Xist/XIST Localization and function

Retained in nucleus of female somatic cells.

Highly particulate and localize to length of Xi but only pinpoint site on Xa.

Xist/XIST particles released from Xi during mitosis.

Xist levels increase drammatically in female but not male cells during developmental window in which X chromosome silencing occurs.

Xist required in cis for silencing. (deletion studies)

Xist RNA silencing can be blocked by antisense RNA.

Overproduction of Xist RNA can spread to autosomal sequences and silence genes even in males.

Xist transcripts can always coat the chromosome in cis but the RNA can only mediate silencing in a narrow window during differentiation.

There is a 10-20 fold increase in the amount of Xist RNA somatic cells vs embryonic stem (ES)cells in mice. Mature Xist RNA also has a 10-20 fold longer half-life in somatic cells than in ES cells.

Additional developmentally regulated factors, that are as yet unidentified, are necessary for the silencing activity of Xist upon differentiation.

The Xist promoter is methylated on the silent Xist allele on the Xa in somatic cells and less methylated on the Xist-expressing Xi in somatic cells and on both X chromosomes in undifferentiated ES cells.

Choosing the active X Chromosome

Cells must randomly make a differentiating mark on only one X-chromosome, the Xa. This diffusible mark is called the blocking factor.

Blocking factor is autosomally encoded.

The region of the Xic required to mediate blocking Factor activity is referred to as the counting element.

Modification of the counting element by the blocking factor leads to interference with Xist RNA's silencing function on this chromosome.

Additional cis-elements in the Xic region, termed choice elements, can skew the Xa choice such that one chromosome becomes Xa more frequently than the other. Thus, a combination of cis-elements within Xic are empolyed to allow cells to differentiate between Xa from Xi formation.

Figure 2. Xa choice by blocking factor. A. Blocking factor is autosomally encoded. B. Blocking factor can bind either X-chromosome's cis element. C. Binding of the blocking factor prevents the spread of silencing by Xist RNA.

the Counting elements

Figure 3. Xist region and the localization of counting elements. Only multiple tandem copies of the 35 kb transgene carry counting element activity. The 65 kb deletion removed Xa conversion (perhaps related more to Tsix activity). One 80 kb transgene had counting element activity (+) and the other did not (-).

The counting element not likely to be contained in Xist domain since Xist cDNA clones showed no counting element activity.

Xist or Xist RNA acts as a choice element that reduces the affinity of the cis-linked counting element for blocking factor.

Increased transcription of Xist RNA has a negative influence on Xa choice probably by regulating the accessibility of the closely linked counting element for the blocking factor.

Tsix and its function

Two promoters and two polyadenylation sites.

Tsix transcripts of up to 4 kb can be produced by splicing.

Tsix has no significant open reading frames.

Tsix RNA found at Xist pinpoint expression and is extinguished upon differentiation but only several days after Xist is extinguished.

Tsix expression extinguised and then Xi established by Xist spreading.

Tsix is not the counting element.

Normal role of Tsix RNA is to be antisense to Xist and reduce its steady-state level while subsequently promoting Xa choice by increasing the affinity of the cis-linked counting element for blocking factor.

Higher than normal Tsix can not further promote Xa choice.

The spliced form of Tsix RNA contains only 2 kb of overlap with the mature Xist transcript. This overlap occurs within a domain of Xist that is critical for silencing activity, suggesting that Tsix modulation of Xa choice could involve regulation of of xist RNA's silencing activity by affecting its interactions with trans-acting factors.

Xce and its effects

Xce is the X choosing element locus which lies down stream of Xist, beyong Tsix.

X-chromosomes bearing stronger Xce alleles are chosen more frequently as Xa and show markedly lower levels of Xist RNA while producing higher levels of Tsix transcripts.

There is a correlation of Xce strength with methylation levels at DXPas34, the CpG island located at the major Tsix promoter, and methyation appears to follow X-inactivation.

Imprinted X-inactivation

Figure 5. Xist and Tsix expression during early female mouse development.

Figure 3. Xist and Tsix gene organization.

In contrast to mouse embryonic tissue, in mouse extraembryonic tissue, Xa is always maternally inherited Xm and Xi is paternally inherited, Xi. Interruptions in this pattern are lethal in mice when the imprinted pattern is overridden by alterred levels of Xist or Tsix. In humans, variants from imprinted patterns are not lethal and even XXX females are possible.

The imprint controlling X-inactivation is located on the Xm, designated Xa-fate, and is established during oocyte growth between prophase between meiosis I and meiosis II.

Silencing and alterations in chromatin structure

Figure 6. The three steps of the silencing process: initiation, establishment, and maintenance.

Figure 7. Chromatin modifications on the Xi occur sequentially during the differentiaiton of female ES cells.

MacroH2A is recruited to the Xi by Xist.

MacroH2A followed by exclusion of H2ABBD from Xi. (formation of Barr body).

Xist/XIST espression is not necessary to continue Xi after establishment.

DNA methylation appears to be extremely important sor the stability and maintenance of gene silencing on Xi.

Late replication is not necessary for continued silencing.

Genes that escape X-inactivation lack chromatin modifications characteristic of Xi.

X-linked genes escaping silencing do not have H3 methylated Lys-9 but methylation at Lys-4.

Escapees do not show late replication timing or methylation of CpG islands in their promoter regions.

Escaping genes are found to be clustered and flanked by DNA boundary elements.

Xist RNA consists of functional modules

Figure 8. the repeat structure of Xist/XIST RNA.

Silencing function ascribed to A repeat.

Figure 9. Functional domains of Xist RNA