Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX),which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z)decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.

The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm [del(9p)], unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The a-and J31-interferon genes have been assigned to this chromosome region (9p21-22). We now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5'-methylthioadenosine phosphorylase (EC 2.4.2.28), an enzyme of purine metabolism.

Skin provides an attractive organ system for exploring coordinated regulation of keratinocyte (KC) proliferation, differentiation, senescence, and apoptosis. Our main objective was to determine whether various types of cell cycle arrest confer resistance to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report, simultaneous expression of cyclin-dependent kinase inhibitors (p15, p16, p21, and p27), a marker of early differentiation (keratin 1), mediators of apoptosis (caspases 3 and 8), and NF-B were analyzed in three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express p16; 3) Abruptly induced growth arrest, confluency, and senescence pathways are associated with resistance to apoptosis; 4) The death-defying phenotype of KCs does not require early differentiation; 5) NF-B is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the promoter), dysfunctional NF-B, and diminished capacity to respond to antiproliferative treatments, and they remain highly sensitive to apoptosis with cleavage of caspases 3 and 8. These data indicate that KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-B but not early differentiation.

The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. T he MLL (mixed-lineage leukemia) gene located on chromosome band 11q23 is involved in many chromosomal translocations associated with acute leukemia (1-5). MLL is involved in translocations with Ͼ40 different genes, and breakpoints in MLL fall in an 8.3-kb breakpoint cluster region (refs. 6 and 7; see Fig. 6). The 430-kDa MLL protein is cleaved specifically into amino and carboxyl terminal peptides, which associate with each other (8-10). Domains of MLL include AT hooks, repression and activation domains, plant homeodomain (PHD) fingers, and a SET [Su(var)3-9, enhancer of zeste, and trithorax] domain, which was shown recently to have histone methyltransferase activity (10, 11). Recent murine models of MLL leukemia, including one from our lab, have confirmed that the aminoterminal portion of MLL fused in-frame to the partner gene is critical for leukemogenesis (12, 13). These fusions retain the AT hooks and the repression domain of MLL but lose the PHD, activation, and SET domains. The MLL repression domain initially was defined by using a reporter gene system (14) and was shown to be critical in the context of an MLL fusion for bone marrow transformation in vitro (15). Recently, this region of MLL also was shown to bind nonmethylated CpG DNA in vitro (16). Only by understanding how the MLL protein, including the repression domain in the amino-terminal portion of MLL, normally performs its regulatory functions can one infer how the MLL fusion proteins lead to hematopoietic cell transformation and leukemia development. In this respect, it will be important to characterize more extensively the interaction between the MLL repression domain and corepressors, and to assess the significance of these interactions.It was shown recently that DNA methyltransferase 1 (DNMT1) binds to histone deacetylase 1 (HDAC1), and this activity maps immediately adjacent to the region of sequence similarity between DNMT1 and MLL (17). The region of similarity, the cysteine-rich CXXC domain, is highly conserved among a small group of proteins, including DNMT1, MLL, and methyl-CpG-binding protein 1 (MBD1͞PCM1) (5,14,(18)(19)(20). Two regions of MLL, the CXXC domain (RD1) and the adjacent region (RD2), behave independently as transcriptional repressors in a reporter gene system (14). Although it is unknown how HDAC1 mediates the repression function of DNMT1, several possibilities exist. HDACs are believed to repress transcription by recruiting repressor complexes (21) or by removing acetyl groups from core histone tails in chromatin; hypoace...