Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pho is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, we purified Pho protein complexes from Drosophila embryos and found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt. Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono-and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. Our results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). We propose that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state.[Keywords: Polycomb group; PRE; Pho/dYY1; MBT repeat; histone methylation] Supplemental material is available at http://www.genesdev.org. The regulation of gene expression by Polycomb group (PcG) and trithorax group (trxG) proteins represents a paradigm for understanding the establishment and maintenance of heritable transcriptional states during development. PcG and trxG genes were first genetically identified as regulators that are required for the long-term maintenance of HOX gene expression patterns in Drosophila. PcG proteins keep HOX genes silenced in cells in which they must stay inactive, whereas trxG proteins maintain the active state of these genes in appropriate cells (for review, see Ringrose and Paro 2004). This regulatory relationship is conserved in vertebrates, where PcG and trxG proteins also regulate HOX gene expression. In addition, mammalian PcG and trxG proteins have also been implicated in X-chromosome inactivation, hematopoietic development, control of cell proliferation, and oncogenic processes.Drosophila HOX genes are among the best-studied target genes of the PcG/trxG system. Different studies have led to the identification of specific cis-regulatory sequences in HOX genes that are called Polycomb response elements (PREs) and are required for silencing by PcG proteins. PREs are typically several hundred base pairs in length, and they function as potent transcriptional silencer elements in the context of HOX reporter genes as well as in a variety of other reporter gene assays (e.g., Chan et al. 1994;Zink and Paro 1995;Sengupta et al. 2004). This ope...

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state.

Transcriptional on and off states of HOX genes and other developmental control genes are maintained by antagonistic regulators encoded by trithorax group (trxG) and Polycomb group (PcG) genes. The trxG proteins Ash1 and hTRX and the PcG repressor E(z) are histone methyltransferases (HMTases) that methylate distinct lysine residues in the N-terminal tail of histone H3. trxG proteins are generally thought to function as activators of HOX genes, but how histone methylation by Ash1 and Trx promotes HOX gene transcription is not clear. Here, we show that in ash1 and trx mutants expression of HOX genes is lost within their normal expression domains, but we find that, contrary to expectation, this expression is restored in ash1 and trx mutants that also lack PcG gene function. Moreover, such trxG PcG double mutants show severe misexpression of HOX genes and, hence, ectopic activation of HOX genes caused by the removal of PcG gene function also occurs in the absence of ash1 and trx function. Together, these results suggest that the Ash1 and Trx HMTases are not 'coactivators' required for transcriptional activation of HOX genes, but function specifically as antirepressors. We propose that histone methylation by Ash1 and Trx is required continuously throughout development to prevent inappropriate PcG silencing of HOX genes in cells in which they must stay transcriptionally active.

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcγR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DRspecific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.Introduction mAbs are becoming increasingly utilized in the treatment of lymphoid disorders (1, 2). In particular, mAb directed to cell-surface antigens on malignant B cells has proven the most clinically effective, with the anti-CD20 mAb, rituximab, being the first to be approved by the US FDA for the treatment of cancer. Rituximab has substantially improved outcome for patients with many different types of non-Hodgkin lymphoma and has now been administered to over 1 million patients in the decade since its approval. Despite such success, treatment is not curative and there is intense preclinical and clinical investigation of many other engineered mAbs directed to both CD20 and a host of other cell-surface antigens (2).

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