Chromosome Inactivation Occurs in Female XX Animals

X-inactivation is a special form of imprinting found in animals. Females possess two X chromosomes, whereas males have one X chromosome plus a much shorter Y chromosome. Consequently, females have two copies of most genes carried on the X chromosomes, whereas males only have one. Evolution has developed a variety of mechanisms for gene dosage compensation in order to avoid different levels of gene expression in male and female.

In nematodes, such as C. elegans, the expression level of genes on both X chromosomes is halved. Conversely, in insects, such as Drosophila, the expression of genes on the single X chromosome in the male is doubled. In mammals, one of the pair of X chromosomes in each female cell is silenced (except for a few loci that are exempted and are referred to as "pseudo-autosomal" regions). In worms and insects, protein complexes that bind specifically to the X chromosomes are responsible for decreasing (worms) or increasing (insects) transcription from genes located on the X chromosomes. In female mammals, a mechanism involving non-coding RNA inactivates just one of the X chromosomes (see below).

In C. elegans, there is no Y chromosome and males have a single unpaired X chromosome (this situation is designated XO). Furthermore, XX animals are actually her-

X-inactivation The condensation and complete shutting down of gene expression of one of the two X-chromosomes in cells of female mammals maphrodites and possess both male and female sex organs. Dosage compensation relies on a protein, Sdc2, that is only expressed in XX animals. The Sdc2 protein binds to specific sites on the X chromosomes and the dosage compensation complex, consisting of half a dozen proteins, assembles on Sdc2 and decreases gene expression. The mechanism used by Drosophila is essentially a mirror image of that in C. elegans. In flies, a protein, Msl2, that is only expressed in XY animals binds to specific sites on the X chromosome. The dosage compensation complex assembles around Msl2 and increases gene expression. The dosage compensation complex of Drosophila includes two non-coding RNAs as well as several proteins.

In mammals, X-inactivation is controlled by methylation of the Xist gene, which is itself located on the X-chromosome. The Xist gene of the X-chromosome that remains active is inactivated by methylation. Once established, this methylation pattern is inherited at cell division; thus, the same one of the pair of X-chromosomes will remain active in the daughter cells. The expression of the Xist gene is in turn regulated by the antisense RNA, Tsix, which is transcribed from the Xist locus, but in the reverse direction. However, the timing of expression of Tsix RNA varies among different mammals and although Tsix appears to be involved in choosing which X chromosome to inactivate, its role is presently unclear.

Expression of the Xist gene causes the inactivation of the X chromosome that carries it. A long non-translated RNA is transcribed from the Xist gene. This Xist RNA coats the inactive X-chromosome. Starting from the Xist gene and proceeding along the X chromosome in both directions, the DNA is converted into heterochromatin, a condensed form of DNA that cannot be transcribed (Fig. 10.17). Highly condensed X chromosomes are visible in the cells of female mammals and are called Barr bodies, after Murray Barr, who discovered them in 1948. The presence or absence of Barr bodies has sometimes been used to check whether female Olympic athletes are genetic females.

If an active Xist gene is inserted into another chromosome, this is only partly inactivated. So another factor(s) is needed to explain X-inactivation. [The X chromosome has twice as many LINE-1 elements (see Ch. 4) per unit length than other chromosomes and it has been suggested that these may somehow promote the binding of Xist RNA. The converse theory argues that more LINE-1 elements have accumulated on the X chromosomes precisely because they are often inactivated!]

The mechanism of Xist-induced silencing only partly understood. After Xist RNA binds, it recruits proteins that are responsible for the actual transcriptional silencing and heterochromatin formation. Changes occur in the histones of the inactive X chromosome. First, histone 3 becomes methylated on Lys7 instead of on Lys4 as in active chromatin. Next, histone H4 loses most of its acetyl groups. Somewhat later, an unusual histone, macroH2A, an H2A variant with an extra C-terminal domain, is found solely on the inactive X chromosome. Finally, methylation of CpG islands occurs along the chromosome. Once silencing has been established, the Xist RNA is no longer required for its maintenance.

In those rare cases where three or more X chromosomes are present in female mammals, only one remains active. Moreover, mice with a single X chromosome (and no Y-chromosome) are healthy and fertile, implying that the second X chromosome is not even necessary. In marsupials, the X-chromosome from the father is always inactivated. In other mammals, the choice is random. Furthermore, which X chromosome is active varies in different cell lines. Consequently, female mammals consist of a genetic mosaic, in which different alleles of genes borne on the X-chromosome are expressed in different regions of tissues. This is illustrated by the variegated coat color seen in female mice that are heterozygous for a coat color mutation in an X-linked gene (Fig. 10.18).

Barr body Inactive and highly condensed X-chromosome as seen in the light microscope Xist gene A gene that causes the inactivation of the X-chromosome that carries it

Worms and flies use different mechanisms from mammals to regulate X chromosome expression.

The DNA of inactivated X chromosomes is highly condensed.

Xist RNA is involved in silencing X chromosomes.

FIGURE 10.17 X-inactivation Involves the Xist Gene and Xist RNA

B) Coating of onf x-chromosome by xut rna

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