MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.

Much of the genome is transcribed into long noncoding RNAs (ncRNAs). Previous data suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex (BX-C) prevent silencing of Ultrabithorax (Ubx) and recruit activating proteins of the trithorax group (trxG) to their maintenance elements (MEs). We found that, surprisingly, Ubx and several bxd ncRNAs are expressed in nonoverlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. Our data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. We propose that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription.

Summary Propagation of gene expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysine 4 or 27 are present during transcription, but surprisingly are replaced by non-methylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste that are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones, and thus may act as epigenetic marks.

MLL (mixed-lineage leukemia) is a histone H3 Lys-4 specific methyltransferase that is a positive regulator of Hox expression. MLL rearrangements and amplification are common in acute lymphoid and myeloid leukemias and myelodysplastic disorders and are associated with abnormal up-regulation of Hox gene expression. Although MLL is expressed throughout hematopoiesis, Hox gene expression is sharply down-regulated during differentiation, suggesting that either the activity of MLL or its association with target promoters must be regulated. Here we show that MLL associates with actively transcribed genes but does not remain bound after transcriptional down-regulation. Surprisingly, MLL is associated not only with promoter regions but also is distributed across the entire coding regions of genes. MLL interacts with RNA polymerase II (pol II) and colocalizes with RNA pol II at a subset of actively transcribed target in vivo. Loss of function Mll results in defects in RNA pol II distribution. Together the results suggest that an intimate association between MLL and RNA pol II occurs at MLL target genes in vivo that is required for normal initiation and͞or transcriptional elongation.histone methyltransferase ͉ Hox genes ͉ transcription P roper expression of the clustered HOX genes is essential for normal embryonic development. In addition, overexpression of select HOX genes such as HOXA9 and the HOX cofactor MEIS1 has been implicated in human myelodysplastic disorders as well as acute lymphoid and myeloid leukemias. HOXA9 and MEIS1 are normally expressed only in early hematopoietic lineages, but during later stages of differentiation, expression is down-regulated to undetectable levels (1). The mixed-lineage leukemia protein MLL, which is homologous to Drosophila trithorax, is one important regulator of HOXA9 expression. MLL-knockout mice show severe hematopoietic defects associated with defects in Hox gene (including Hoxa9) expression (2-5). Conversely, MLL rearrangements are commonly associated with lymphoid and myeloid leukemias (6-8). Translocations involving MLL delete the sequences most conserved with D. trithorax and replace them with an in frame fusion to 1 of Ͼ40 different translocation partners (9, 10). MLL fusion proteins enforce persistent expression of HOXA9 and MEIS1, which appears to be critical for leukemogenesis (11). By itself, overexpression of HOXA9 induces stem cell expansion and is associated with poor-prognosis acute myeloid leukemia (12, 13). However, when coexpressed with MEIS1, HOXA9 is acutely transforming (14).Recently, we and others found that the C-terminal SET domain of MLL protein is a histone methyltransferase that is specific for histone H3 Lys-4 (15, 16). MLL binds directly to Hox gene promoters and promotes transcriptional activation by methylating histone H3 on Lys-4 (15, 16) and also by recruiting MOF, a histone H4 Lys-16 specific acetyltransferase (17). Although an H3 Lys-4 demethylase has recently been identified (18), in general Lys-4 methylation is a long-lasting mark for sustained...