Inactivation of an X-chromosome in female cells is part of the dosage compensation mechanism necessary to allow equivalent expression of X-linked genes in female and male cells, which have two and one X-chromosomes, respectively. The suggestion that X-inactivation might occur was first presented as an hypothesis by Mary Lyon (see Lyon Hypothesis). The result of the inactivation process is the appearance of the Barr body. In eutherian (placental) mammals, the initial choice between inactivation of maternal or paternal X-chromosomes is random (see Random X-Inactivation), but once established in a repressed state, the same X-chromosome is inactivated after every cell division. This is an excellent example of establishing and maintaining a chromosomally based state of determination.
Abnormalities in the process of X-chromosome inactivation have allowed the genetic definition of the X-inactivation center required for inactivation to occur (1). The X-inactivation center is actually on the inactive X-chromosome and contains the gene encoding Xist RNA. This site actually remains actively transcribed, whereas the rest of the X-chromosome is silenced. The Xist RNA is the key regulator of inactivation (2, 3). Remarkably the gene does not encode messenger RNA, but it produces instead a long, untranslated RNA that remains associated with the inactive X-chromosome (4, 5). Through some unknown mechanism, the Xist RNA has a causal role in directing the heterochromatinization of the inactive X-chromosome. The process of heterochromatinization leads to numerous differences between active and inactive X-chromosomes including DNA methylation, histone acetylation status, and replication timing (see Facultative Heterochromatin, Euchromatin).
DNA methylation is characteristic of many inactive promoters and genes. Consistent with this observation, many genes in the inactive X-chromosome are heavily methylated in contrast to the active X-chromosome (6). However, the kinetics with which particular sites within the X-linked genes become methylated during the differentiation of embryonic female somatic cells do not always correlate with the timing of transcriptional inactivation (7). Although it remains to be determined if a subset of key sites around regulatory elements is always methylated before transcription is repressed, it seems probable that other mechanisms must supplement any influence of DNA methylation on transcription.
Heterochromatin normally replicates late during S-phase. Replication timing has been proposed as a determinant of transcriptional activity. Genes that replicate late during S-phase might do so under conditions of limiting transcription factors, or they might be assembled into a repressive chromatin structure using components translated only late in S-phase. The active X-chromosome normally replicates early in S-phase, whereas the inactive X-chromosome replicates late (8). However, female lymphoma cell lines have been isolated in which the opposite occurs (9). Thus the inactive X-chromosome does not have to replicate late in S-phase to be transcriptionally quiescent.
The formation of local repressive chromatin structures in which key genetic regulatory elements are rendered inaccessible to transcription factors by inclusion within positioned nucleosomes is an important mechanism for transcriptional repression. Promoters in the inactive X-chromosome are incorporated into positioned nucleosomes, whereas promoters in the active X-chromosome are free of such structures and have transcription factors bound to them (10). Thus specific local chromatin structures clearly have a role in regulating differential gene activity between the two X-chromosomes. Nevertheless, a causal relationship between chromatin structure and transcription has yet to be established. Nucleosomes on the active X-chromosome contain predominantly acetylated histones, whereas those on the inactive X-chromosome are not acetylated (11). Thus establishing and maintaining specific chromatin structures containing modified histones is an excellent candidate mechanism for establishing and maintaining differential expression of genes between the two X-chromosomes. Differential methylation and replication timing may stabilize these different states of gene activity.
