The molecular mechanism of X chromosome inactivation

Published on August 31, 2016   38 min

Other Talks in the Series: Epigenetics

0:00
The molecular mechanism of X chromosome inactivation, presented by Professor Neil Brockdorff of the Department of Biochemistry, University of Oxford. I'll begin my talk with a short introduction followed by a discussion of critical steps in the X inactivation process.
0:16
X chromosome inactivation is an important form of epigenetic regulation that evolved in mammals to equalize the levels of expression of genes on the X chromosome, in XX females relative to XY males. Briefly, during early development of a female embryo, each individual cell triggers inactivation of one of the two X chromosomes that are present. The process is normally random. So in an individual cell, there's an equal probability of the X chromosome inherited from the mother or the father being selected as the inactive X. Once X inactivation has occurred, cells remember which of the two X chromosomes was inactivated through all subsequent cell generations. As a consequence, female mammals or chimeras comprised of a mosaic or patchwork of cell populations with either one or the other X chromosome being inactive. A classical illustration of X chromosome inactivation is seen in the coat of the calico cat, which you can see in this image. A gene that gives rise to orange coat color lies on the X chromosome. And calico cats are heterozygous for this gene. The wild type allele encodes black coat color. So when X inactivation occurs in early development, cells either inactivate the chromosome with the wild type gene, giving rise to orange coat color, or the chromosome with the orange gene, giving rise to black coat color. These individual cells then expand into a clonal patch cells during further development, giving rise to these areas with either orange or black coat color.
1:42
X chromosome inactivation results in the transcriptional silencing of genes located on that chromosome. The underlying basis for this process is a change in the state of chromatin, referring to DNA and its associated histone and non-histone proteins. In simplified terms, chromatin can exist either in an open conformation, termed U-chromatin, or a compacted conformation, termed heterochromatin. The inactive X is an example of the latter. This point is illustrated in the image of an interphase nucleus from a female somatic cell, stained with DAPI, a blue-fluorescent dye that binds the DNA. The intense DAPI focus, indicated with the arrow, is the compacted heterochromatic inactive X chromosome. This structure was first observed by Barr and Bertram in 1953 and is sometimes referred to as "The Barr body".
2:32
Although the majority of genes on the X chromosome is silenced by X inactivation, a small proportion of genes escape the X inactivation signal. The number of escaped genes varies between different species. In humans, there are around 100 X-linked genes that escape silencing. However in mouse, there are only a handful of such genes. Analysis of the three-dimensional position of gene loci in the interphase nucleus, which is determined using fluorescence microscopy, indicates the escapees, escaped genes, loop-out of the compacted inactive X chromosome territory as is illustrated in the schematic. This organization likely reflects engagement of the escaped loci with what are termed transcription factories located elsewhere in the interphase nucleus. In some instances, the genes that escape X inactivation have a direct functional homologue on the Y chromosome, which explains why there's no requirement to equalize their expression levels in females relative to males. In other instances, there's no functional homologue and it's assumed that cells can tolerate lower levels of expression of those specific genes in males compared to females.
3:39
So how do female cells inactivate one of their two X chromosomes? An important clue towards understanding this came from classical studies in the 1960s and 1970s in which it was showing that imbalance chromosome translocations involving the X chromosome in an autosome, only one of the two translocation products is able to undergo X inactivation. In the example shown here, the balanced translocation T(X;16)H, the 16H translocation product on the normal X chromosome can undergo X inactivation. However, the X16 product is never inactivated. Through comparison of several different X autosome translocations, it was deduced that there is a critical region that must be present on the X chromosome or X chromosome translocation product in order for it to undergo X inactivation. This region was termed the X inactivation center or Xic. A key point to get across here is that the Xic functions in cis, that is, it needs to be present on an X chromosome or part of X chromosome in order for that chromosome to be inactivated.
4:44
Delineation of the critical Xic region, enabled studies aimed at defining the gene or genes present at this locus. The key breakthrough came in the early 1990s with the identification of an unusual gene that's located within the Xic region. This gene was termed the X inactive specific transcript or Xist. The Xist gene produces a large RNA, around 17 kilobases in length that is processed similarly to other messenger RNAs that is it's capped, spliced, and polyadenylated. However, unlike most mRNAs, Xist does not have the capacity to encode a protein, that is, it's a non-coding RNA. As its name suggests, Xist is expressed only from the inactive X chromosome allele. Its expression immediately precedes the onset of X inactivation in ovary development which fits with having a role in initiating the process. Expression of Xist RNA by XX cells continues throughout development and in adult animals. A key feature of Xist RNA is that it's not exported into the cytoplasm unlike other messenger RNAs. Instead, it is retained within the nucleus, localizing to a domain or a territory that approximately corresponds to the chromosomes from which it's transcribed. This property is illustrated in the image here in which Xist RNA has been detected with a green fluorescent probe and can be seen to overlie the Barr body in the panel on the right. Localization of Xist RNA over the chromosome for which it is transcribed suggests the mechanism by which it could function in cis and as such provides a possible explanation for Xic function as defined in classical studies. Proof that Xist is indeed the Xic gene came from genetic analysis. First gene knockouts demonstrated that Xist is required for X chromosome inactivation and also for the viability of female embryos. Subsequently, using Xist transgenes, it was shown that Xist is sufficient to inactivate the X chromosome or for that matter, any other chromosome from which it is expressed.
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The molecular mechanism of X chromosome inactivation

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