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.

Conditional oncogenes provide a powerful tool for studying early events in transformation. Here we applied this approach to dissect the mechanism of leukemic transformation by MLL fusion proteins. MLL, located at 11q23, is rearranged in a variety of human acute lymphoid and myeloid leukemias. Our goals were to determine if MLL-induced growth transformation is reversible, to characterize the effect of MLL fusion protein expression on cell proliferation, differentiation, and survival in physiologically relevant cells, and finally to identify genes regulated by MLL fusion that are important for transformation.

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...