The Mll gene is a member of the mammalian trithorax group, involved with the antagonistic Polycomb group in epigenetic regulation of homeotic genes. MLL contains a highly conserved SET domain also found in various chromatin proteins. In this study, we report that mice in which this domain was deleted by homologous recombination in ES cells (⌬SET) exhibit skeletal defects and altered transcription of particular Hox genes during development. Chromatin immunoprecipitation and bisulfite sequencing analysis on developing embryo tissues demonstrate that this change in gene expression is associated with a dramatic reduction in histone H3 Lysine 4 monomethylation and DNA methylation defects at the same Hox loci. These results establish in vivo that the major function of Mll is to act at the chromatin level to sustain the expression of selected target Hox genes during embryonic development. These observations provide previously undescribed evidence for the in vivo relationship and SET domain dependence between histone methylation and DNA methylation on MLL target genes during embryonic development.histone methyltransferase ͉ MLL-SET domain ͉ homeotic transformations T he control of cell identity during development is specified, in large part, by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of the embryo (1). The trithorax and polycomb groups (trx-G and PcG) were identified for their role in faithfully maintaining the transcriptional states of these key developmental regulators, providing an epigenetic mechanism of cellular memory (2-4).The gene expression maintenance function of the trxG and PcG proteins is highly conserved. Mixed lineage leukemia (Mll), a human homolog of Drosophila trithorax and a member of the trxG family, was identified first for its involvement in chromosomal translocations associated with lymphoid and myeloid acute leukemia in infants and adults (5, 6). Mll encodes a 3,969-aa nuclear protein with multiple domains, including three AT-hook motifs, a DNA methyltransferase homology domain (DNMT) in the aminoterminal half of the protein, a central zinc finger (PHD) region, and a highly conserved 130-aa carboxyl-terminal SET domain. The MLL protein was shown to be proteolytically processed into two portions (MLL N and MLL C ) with antagonistic transcriptional effector properties, that reassociate and stabilize each other (7-9). The MLL protein is critical for proper regulation of the Hox genes during embryonic development (10). In Mll null mutant mice (MllϪ͞Ϫ), Hox gene expression is correctly initiated but is not sustained as the function of Mll becomes necessary (11), leading to embryonic lethality.It is strongly believed that maintenance of the transcriptional status of target genes by PcG and trxG proteins is achieved through chromatin modifications (12). The structure similarity between some trxG͞PcG and suppressors or enhancers of position effect variegation (PEV) further substantiates this point. One of the most remarkable shared domains within th...

BackgroundThe INK4/ARF locus encodes three tumor suppressor genes (p15Ink4b, Arf and p16Ink4a) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized.Principal FindingsHere we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence.ConclusionsWe identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus.