the others (reviewed by Kelley and Kuroda, 1995). This observation suggests that these gene products must interact Dosage compensation is a regulatory process that and form a complex. Preliminary direct evidence for such insures that males and females have equal amounts of an interaction is provided by the observation that MSL-1 X-chromosome gene products. In Drosophila, this is and MSL-2 can be co-precipitated with antisera against achieved by a 2-fold enhancement of X-linked gene either protein (Kelley et al., 1995). Binding of MLE and transcription in males, relative to females. The the MSL proteins to the X chromosome in males is enhancement of transcription is mediated by the correlated with the appearance of histone 4 acetylated at activity of a group of regulatory genes characterized Lys16 (H4Ac16) on the same chromosome (Turner et al., by the male-specific lethality of their loss-of-function 1992) and at the same sites (Bone et al., 1994). alleles. The products of these genes form a complexWe have used the phenotype of male-specific lethality that is preferentially associated with numerous sites on to screen the X chromosome of Drosophila melanogaster the X chromosome in somatic cells of males but not of for ethyl methane sulfonate (EMS)-induced mutations, females. Binding of the dosage compensation complex identifying additional genes that may be involved in the is correlated with a significant increase in the presence regulatory process of dosage compensation. We isolated of a specific histone isoform, histone 4 acetylated at one such mutation (mof) and observed that dying mutant Lys16, on this chromosome. Experimental results and males lack the X-associated isoform of H4Ac16. MOF sequence analysis suggest that an additional gene, exhibits the signature motif for the acetyl coenzyme males-absent on the first (mof), encodes a putative acetyl A binding site found in numerous and diverse acetyl transferase that plays a direct role in the specific histone transferases, and the mof mutation is a single amino acid acetylation associated with dosage compensation. The substitution in the most conserved residue of this motif. predicted amino acid sequence of MOF exhibits a This provides evidence that MOF is the histone acetyl significant level of similarity to several other proteins, transferase (HAT) responsible for the particular histone including the human HIV-1 Tat interactive protein acetylation involved in the male-specific hypertranscripTip60, the human monocytic leukemia zinc finger tion of X-linked genes. protein MOZ and the yeast silencing proteins SAS3 and SAS2.

Fragile X syndrome carriers have FMR1 alleles, called premutations, with an intermediate number of 5' untranslated CGG repeats between patients (>200 repeats) and normal individuals (<60 repeats). A novel neurodegenerative disease has recently been appreciated in some premutation carriers. As no neurodegeneration is seen in fragile X patients, who do not express FMR1, we hypothesize that lengthened rCGG repeats of the premutation transcript may lead to neurodegeneration. Here, using Drosophila melanogaster, we show that 90 rCGG repeats alone are sufficient to cause neurodegeneration. This phenotype is neuron specific and rCGG repeat dosage sensitive. Although devoid of mutant protein, this neurodegeneration exhibits neuronal inclusion bodies that are Hsp70 and ubiquitin positive. Overexpression of Hsp70 could suppress the neurodegeneration. These results demonstrate that neurodegenerative phenotype associated with fragile X premutation is indeed caused by the lengthened rCGG repeats and provide the first in vivo experimental demonstration of RNA-mediated neurodegeneration.

Posttranslational acetylation of core histone amino termini has long been associated with transcriptionally active chromatin. Recent reports have demonstrated histone acetyltransferase activity in a small group of conserved transcriptional regulators directly linked to gene activation. In addition, the presence of a putative acetyltransferase domain has been discovered in a group of proteins known as the MYST family (for its founding members MOZ, YBF2͞ SAS3, SAS2, and Tip60). Members of this family are implicated in acute myeloid leukemia (MOZ), transcriptional silencing in yeast (SAS2 and YBF2͞SAS3), HIV Tat interaction in humans (Tip60), and dosage compensation in Drosophila (MOF). In this report, we express a yeast ORF with homology to MYST family members and show it possesses histone acetyltransferase activity. Unlike the other MYST family members in Saccharomyces cerevisiae this gene is essential for growth.

In many multicellular organisms, males have one X chromosome and females have two. Dosage compensation refers to a regulatory mechanism that insures the equalization of X-linked gene products in males and females. The mechanism has been studied at the molecular level in model organisms belonging to three distantly related taxa; in these organisms, equalization is achieved by shutting down one of the two X chromosomes in the somatic cells of females, by decreasing the level of transcription of the two doses of X-linked genes in females relative to males, or by increasing the level of transcription of the single dose of X-linked genes in males. The study of dosage compensation in these different forms has revealed the existence of an amazing number of interacting chromatin remodeling mechanisms that affect the function of entire chromosomes.

We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasiamutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant ⌬hMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.In eukaryotic cells, DNA damage activates signal transduction pathways that rapidly affect downstream processes such as gene transcription, cell cycle progression, and DNA replication (13,25). All of these processes involve alterations in chromatin structure. Posttranslational covalent modifications of histones have emerged as key regulatory events in DNA damage response. A widespread modification is acetylation catalyzed by histone acetyltransferases and reversed by deacetylases (3, 13, 50). Reversible acetylation of four lysines (K) at positions 5, 8, 12, and 16 in the amino-terminal tail of histone H4 occurs in vivo in all eukaryotes (3). The hyperacetylation of histone H4 could lead to the unfolding of the nucleosomal fiber (50), and the acetylation of histone H4 at K16 occurs on the hyperactive male X chromosome of Drosophila polytene chromosomes (51). Ikura et al. (19) noted that Tip60 (Tat-interacting protein), which acetylates histones H2A, H3, and H4, plays a role in DNA repair. More recently, Kusch et al. (28) demonstrated that the Drosophila Tip60 acetylates nucleosomal phosphoH2Av and exchanges it with an unmodified H2Av. Bird et al. (5) reported that the acetylation of histone H4 by Esa1 (essential SAS2-related acetyltransferase) is required for DNA repair in yeast and suggested that a similar modification may function in mammalian cells.ATM (ataxia-telangiectasia-mutated protein) is crucial for the initiation of signaling pathways in mammalian cells following exposure to ionizing radiation (IR) and other DNA-damaging agents (36, 46), and cells deficient in ATM function also have defective telomere chromatin (47). Bakkenist and Kastan (4) have suggested that chromatin structural perturbations induced by DNA double-strand breaks (DSBs) serve as a trigger for ATM activation. Recent studies indicate that the MRN (Mre11, Rad50, and Nbs1) complex as well as TRF2 either influences activation of ATM (9, 29, 52) or serves as a modulator/amp...

In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.Dosage compensation is a regulatory mechanism to ensure that the level of expression of genes on the single X chromosome of Drosophila males equals the level attained from the two X chromosomes in females. This equalization, achieved by a twofold increase in the rate of X-linked gene transcription in males relative to females, has been observed for a wide variety of genes with promoters of different strengths, in many cell types, and at different developmental stages. For this reason, the study of dosage compensation may provide valuable insights into the mechanisms that regulate levels of transcription.Five genes involved in dosage compensation have been identified based on the male-specific lethality of their loss-of-function alleles (26). The products of these genes, collectively referred to as MSL proteins, colocalize to the male X chromosome, a chromosome that is also highly enriched with histone H4 acetylated at lysine 16 (6, 41). Since in all eukaryotes acetylation of the histones has been correlated directly with the establishment and regulation of transcription (reviewed in reference 28), it is likely that the MSL complex mediates its effect, at least in part, through histone acetylation. Indeed, the most recent MSL to be discovered is MOF (for "males absent on the first"), a protein with homology to acetyltransferases of the MYST family (8,18,33).Another protein component of the complex is MLE (for "maleless"), an ATP-dependent RNA or DNA helicase (25). Unlike the other members of the MSL complex, MLE can be dissociated from the X chromosome by treatment with RNase, suggesting that the complex may interact with either nascent or some other form of RNA (34). This speculation has been reinforced, if not validated, by the recent discovery of two genes, roX1 and roX2 (for "RNA on the X 1 and 2") that encode RNAs with no apparent open reading frames (1, 27). These RNAs are found only in males, and their presence depends on the MSL complex, with which they are seen to colocalize on the X chromosome (15,20).In this paper, we report the initial functional characterization of ...

We describe a stable, multisubunit human histone acetyltransferase complex (hMSL) that contains homologs of the Drosophila dosage compensation proteins MOF, MSL1, MSL2, and MSL3. This complex shows strong specificity for histone H4 lysine 16 in chromatin in vitro, and RNA interference-mediated knockdown experiments reveal that it is responsible for the majority of H4 acetylation at lysine 16 in the cell. We also find that hMOF is a component of additional complexes, forming associations with host cell factor 1 and a protein distantly related to MSL1 (hMSL1v1). We find two versions of hMSL3 in the hMSL complex that differ by the presence of the chromodomain. Lastly, we find that reduction in the levels of hMSLs and acetylation of H4 at lysine 16 are correlated with reduced transcription of some genes and with a G 2 /M cell cycle arrest. This is of particular interest given the recent correlation of global loss of acetylation of lysine 16 in histone H4 with tumorigenesis.The dynamic regulation of chromatin structure can be brought about by a complex series of posttranslational modifications to histones, particularly on the amino-terminal histone tails (33,48). Among the lysines that can be acetylated, lysine 16 of histone H4 appears to be uniquely targeted in a number of organisms. Studies of telomeric silencing in Saccharomyces cerevisiae have shown that lysine 16-specific acetyltransferase and deacetylase activities determine the boundaries of silenced chromatin (32, 59). Furthermore, chromatin immunoprecipitation and site-directed mutagenesis in budding yeast clearly indicate that acetylation of H4 lysine 16 has an independent specific function in relation to gene transcription when compared to other histone acetylation sites (16,34). In Drosophila, lysine 16 acetylation is targeted to the hypertranscribed male X chromosome in the process of dosage compensation (8,35). Biochemical analysis of bulk histones in mammalian cells has indicated that most of the monoacetylated H4 is acetylated at lysine 16, as is most of the di-and triacetylated forms, leading to the suggestion that this is the first acetylation mark on H4 or possibly the last to be taken off (61, 69). One rationale for a special role for lysine 16 acetylation is that it is the only known site of acetylation in the basic patch of H4, a region from amino acids 16 to 20 that is implicated in the formation of higher-order chromatin structure (17). Recently, loss of acetylation at lysine 16 of histone H4 has been identified as a common hallmark of human cancer (22).In Drosophila, dosage compensation-the equalization of X-linked gene products in males and females-is achieved by enhancing the transcriptional level of X-linked genes in males.Five genes involved in this process were identified due to male-specific lethality when mutated (4,5,26). These genes are male-specific lethal 1 (msl1), msl2, msl3, maleless (mle), and males-absent on the first (mof). All five gene products form the MSL complex, which harbors histone acetyltransferase (HAT) activity with...

SUMMARYDosage compensation in Drosophila increases the transcription of genes on the single X chromosome in males to equal that of both X chromosomes in females. Site-specific histone acetylation by the malespecific lethal (MSL) complex is thought to play a fundamental role in the increased transcriptional output of the male X. Nucleation and sequence-independent spreading of the complex to active genes serves as a model for understanding the targeting and function of epigenetic chromatin-modifying complexes. Interestingly, two noncoding RNAs are key for MSL assembly and spreading to active genes along the length of the X chromosome.