ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of > or =29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.Supplemental material is available at http://www.genesdev.org.Received September 16, 2008; revised version accepted November 4, 2008. Chromosomal translocations involving the mixed-lineage leukemia gene (MLL) are a frequent occurrence in human acute leukemias of both children and adults (Eguchi et al. 2005). In over half of all infant acute leukemias, the MLL protein fuses to one of >50 identified partner genes, resulting in a MLL fusion protein that acts as a potent oncogene (Krivtsov and Armstrong 2007). While extensive gene expression signatures have been determined for primary human leukemia samples (Armstrong et al. 2002;Yeoh et al. 2002;Ferrando et al. 2003;Ross et al. 2003;Rozovskaia et al. 2003;Haferlach et al. 2005), the direct genomic targets of MLL fusion proteins remain unknown. This information is essential to determine how MLL fusion proteins impose oncogenic transcriptional programs and to identify targets for therapeutic intervention in human disease.Distinct chromatin-modifying complexes and histone modifications are associated with distinct phases of transcription (Li et al. 2007). The trithorax group proteins, including MLL, catalyze histone H3-Lys-4 trimethyl (H3K4me3) modifications at the start sites of transcriptionally engaged genes (Ruthenburg et al. 2007). These H3K4me3-modified regions are largely constrained to the transcription start site regions of genes that are transcriptionally initiated, but not necessarily fully transcribed (Bernstein et al. 2006;Barski et al. 2007;Guenther et al. 2007). As a gene becomes fully transcribed, elongating RNA Polymerase II (Pol II) molecules proceed through gene coding regions along with associated elongation factors including DOT1, which catalyzes dimethylation of histone H3-Lys-79 (H3K79me2) (Li et al. 2007). Physical interactions between the most common MLL partner proteins and transcriptional elongation components suggest that defects in H3K4 and H3K79 methylation might be a key factor in MLL leukemogenesis In order to define the portion of gene regulatory circuitry that is controlled directly by MLL fusion proteins in human leukemia, we determined the binding patterns of an MLL fusion protein and chromatin modifications across the entire human genome. We performed this mapping in leukemic cells harboring the MLL-AF4 fusion gene, because this rearran...

Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, we examined ASH1L, the mammalian homolog of the Drosophila melanogaster Trithorax group (TrxG) protein Ash1. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. Here we demonstrate that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines we found that their recruitments occur independently of each other. Collectively, our data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes.

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The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and͞or transgenic f lies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI͞SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI͞SNF complex is recruited to ALL-1͞trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1͞SNR1.The ALL-1 gene is involved in human acute leukemia, in particular infant and secondary leukemia, through chromosome translocations or partial tandem duplications (1-3). The chromosome translocations result in expression of chimeric proteins composed of the N-terminal Ϸ1,300 residues of ALL-1 linked to a C-terminal polypeptide encoded by any (Ͼ25) of the partner genes. The gene duplications produce altered (longer) ALL-1 proteins. ALL-1 is the human homolog of Drosophila trx (4). The latter is a member of the trithorax group (trx-G) gene family that together with the Polycomb group (Pc-G) genes act to control expression of the Antennapedia and bithorax homeotic gene complexes determining segment identify in Drosophila (ref. 5 and reviewed in refs. 6 and 7). Expression or silencing of the homeotic genes (HOM) is established by the gap and pair-rule genes at the cellular blastoderm stage. This determined state is maintained during subsequent development by the collective action of the trx-G and Pc-G genes, which function as transcriptional activators and repressors, respectively. trx-G͞Pc-G genes act through specific response elements present within their target genes (8,9). This activity is evidenced by the physical localization of the protein products of several trx-G͞Pc-G genes, including trx and Pc, to multiple sites (presumed to be the response elements in target genes) on salivary gland polytene chromosomes of third star larvae (refs. 10-13 and references therein).Converging lines of evidence suggest that trx-G and Pc-G genes act by establishing open and repressive chromatin states, respectively. A striking feature is the similarity...

The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.C hromosome band 11q23 is a region of recurrent rearrangements in human acute leukemias. These rearrangements, usually in the form of reciprocal chromosome translocations, affect 5-10% of children and adults with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The most common translocations are t(4;11) and t(9;11), accounting for 40% and 27%, respectively, of all 11q23 rearrangements. There is a strong association between leukemia phenotype and particular rearrangements. Thus, t(4;11) occurs nearly exclusively in ALL, and 85% of cases with t(9;11) are AMLs (1, 2). Essentially all 11q23 abnormalities involve the ALL-1 gene (also termed MLL, HRX, or HTRX), which rearranges with Ͼ30 partner genes to produce fusion proteins composed of ALL-1 N terminus and the C terminus of the partner protein (3, 4). A second and less frequent type of ALL-1 rearrangement does not involve partner genes but rather partial duplications of ALL-1 N-terminal segments (5). ALL-1-associated leukemias show some unusual and intriguing features (reviewed in refs. 6 and 7). First, they predominate infant acute leukemias, amounting to 80% of infants with ALL and 65% of those with AML. Second, they account for the majority of therapyrelated (secondary) leukemias, developing in 5-15% of primary cancer patients treated with drugs, such as etoposide (VP16), that inhibit DNA topoisomerase II. Third, in infant leukemia and in therapy-related leukemia the disease arises after a brief latency. In fact, studies of monozygotic twins and newborns with leukemia and analysis of neonatal blood spots from children who were diagnosed with leukemia indicate that in most or all infant leukemias ALL-1 rearrangements occur in utero. The short latency suggests that ALL-1 fusion proteins induce leukemia with few, if any, additional mutations. Fourth, prognosis of patients with 11q23 abnormalities is ...

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox͞homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an Ϸ10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesionmediated signaling.