No abstract

Dosage compensation in Drosophila occurs by an increase in transcription of genes on the X chromosome in males. This elevated expression requires the function of at least four loci, known collectively as the male-specific lethal (ms/) genes. The proteins encoded by two of these genes, maleless (m/e) and male-specific lethal-1 (ms/-/), are found associated with the X chromosome in males, suggesting that they act as positive regulators of dosage compensation. A specific acetylated isoform of histone H4, H4Acl6, is also detected predominantly on the male X chromosome. We have found that MLE and MSL-1 bind to the X chromosome in an identical pattern and that the pattern of H4Acl6 on the X is largely coincident with that of MLE/MSL-1. We fail to detect H4AcI6 on the X chromosome in homozygous msl males, correlating with the lack of dosage compensation in these mutants. Conversely, in Sxl mutants, we detect H4Acl6 on the female X chromosomes, coincident with an inappropriate increase in X chromosome transcription. These data suggest that synthesis or localization of H4Acl6 is controlled by the dosage compensation regulatory hierarchy. Dosage compensation may involve H4Acl6 function, potentially through interaction with the products of the msl genes.

In female mammalian cells, dosage compensation for X-linked genes is achieved by the transcriptional silencing, early in development, of many genes on just one of the two X chromosomes. Several properties distinguish the inactive X (Xi) from its active counterpart (Xa). These include expression of Xist, a gene located in the X-inactivation center (Xic), late replication, differential methylation of selected CpG islands and underacetylation of histone H4. The relationship between these properties and transcriptional silencing remains unclear. Female mouse embryonic stem (ES) cells have two active X chromosomes, one of which is inactivated as cells differentiate in culture. We describe here the use of these cells in studying the sequence of events leading to X-inactivation. By immunofluorescent labeling of metaphase chromosome spreads from ES cells with antibodies to acetylated H4, we show that an underacetylated X chromosome appears only after 4 days of differentiation, and only in female cells. The frequency of cells with an underacetylated X reaches a maximum by Day 6. In undifferentiated cells, H4 in centric heterochromatin is acetylated to the same extent as that in euchromatin but has become relatively underacetylated, as in adult cells, by Day 4 of differentiation (i.e. , when deacetylation of Xi is first seen). The overall deacetylation of Xi follows Xist expression and the first appearance of a single, late-replicating X, both of which occur on Day 2. It also follows the silencing of X-linked genes. Levels of mRNA from four such genes, Hprt, G6pd, Rps4, and Pgk-1, had all fallen by approximately 50% (relative to the autosomal gene Aprt) by Days 2-4. The results show that properties that characterize Xi are put in place in a set order over several days. H4 deacetylation occupies a defined place within this sequence, suggesting that it is an intrinsic part of the X-inactivation process. The stage at which a completely deacetylated Xi is first seen suggests that deacetylation may be necessary for the maintenance of silencing but is not required for its initiation. Nor is it required for, or an immediate consequence of, late replication. However, we note that selective deacetylation of H4 on specific genes would not be detected by the microscopical approach we have used and that such selective deacetylation may still be part of the silencing process.

Antibodies to histone deacetylases (HDACs) have been used to immuno-isolate deacetylase complexes from HeLa cell extracts. Complexes shown to contain HDAC1, HDAC3, HDAC6, and HDAC1؉2 as their catalytic subunits have been used in an antibody-based assay that detects deacetylation of whole histones at defined lysines. The class II deacetylase HDAC6 was inactive in this assay, but the three class I enzymes deacetylated all histone lysines tested, although with varying efficiency. In comparison to HDAC1, HDAC3 preferentially deacetylated lysines 5 and 12 of H4 and lysine 5 of H2A. H4 tails in purified mononucleosomes were refractory to deacetylation by both HDAC1 and HDAC3, unless ATP was added to the reaction mix. Surprisingly, ATP also consistently enhanced cleavage of free, non-nucleosomal histones, but not small peptides, by both enzyme complexes. We found no evidence that ATP operates by phosphorylation of components of the HDAC complex, but have shown that HDACs 1, 2, and 3 all co-immunoprecipitate with the ATP-dependent chaperone protein Hsp70. Another common ATP-dependent chaperone, Hsp90, was absent from all HDAC complexes tested, whereas Hsp60 associated with HDAC1 only. We suggest that Hsp chaperone proteins enhance the deacetylase activity of HDAC complexes by ATP-dependent manipulation of protein substrates.

L.P.O'Neill and A.M.Keohane contributed equally to this workWe have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P 1 . This domain includes the promoter P 0 , which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation.