Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.

Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by highresolution, genome-wide copy number analysis coupled with gene-expression profiling. Of 272 recurrent chromosomal aberrations that were associated with gene-expression alterations, 30 were used differentially by the DLBCL subtypes (P < 0.006). An amplicon on chromosome 19 was detected in 26% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs. A highly up-regulated gene in this amplicon was SPIB, which encodes an ETS family transcription factor. Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines but not to GCB DLBCL, PMBL, or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL. Deletion of the INK4a/ARF tumor suppressor locus and trisomy 3 also occurred almost exclusively in ABC DLBCLs and was associated with inferior outcome within this subtype. FOXP1 emerged as a potential oncogene in ABC DLBCL that was up-regulated by trisomy 3 and by more focal high-level amplifications. In GCB DLBCL, amplification of the oncogenic mir-17-92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent, but these events did not occur in ABC DLBCL. Together, these data provide genetic evidence that the DLBCL subtypes are distinct diseases that use different oncogenic pathways.gene-expression profiling ͉ oncogenes ͉ tumor suppressor genes ͉ comparative genomic hybridization

Gene activation and repression regulated by acetylation and deacetylation represent a paradigm for the function of histone modifications. We provide evidence that, in contrast, histone H2B monoubiquitylation and its deubiquitylation are both involved in gene activation. Substitution of the H2B ubiquitylation site at Lys 123 (K123) lowered transcription of certain genes regulated by the acetylation complex SAGA. Gene-associated H2B ubiquitylation was transient, increasing early during activation, and then decreasing coincident with significant RNA accumulation. We show that Ubp8, a component of the SAGA acetylation complex, is required for SAGA-mediated deubiquitylation of histone H2B in vitro. Loss of Ubp8 in vivo increased both gene-associated and overall cellular levels of ubiquitylated H2B. Deletion of Ubp8 lowered transcription of SAGA-regulated genes, and the severity of this defect was exacerbated by codeletion of the Gcn5 acetyltransferase within SAGA. In addition, disruption of either ubiquitylation or Ubp8-mediated deubiquitylation of H2B resulted in altered levels of gene-associated H3 Lys 4 methylation and Lys 36 methylation, which have both been linked to transcription. These results suggest that the histone H2B ubiquitylation state is dynamic during transcription, and that the sequence of histone modifications helps to control transcription.

The transcription factor IRF4 is required during an immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells 1-3 . Multiple myeloma, a malignancy of plasma cells, has a complex molecular etiology with several subgroups defined by gene expression profiling and recurrent chromosomal translocations 4,5 . Moreover, the malignant clone can sustain multiple oncogenic lesions, accumulating genetic damage as the disease progresses 6,7 . Current therapies for myeloma can extend survival but are not curative 8,9 . Hence, new therapeutic strategies are needed that target molecular pathways shared by all subtypes of myeloma. Using a loss-offunction, RNA-interference-based genetic screen we show here that IRF4 inhibition was toxic to myeloma cell lines, regardless of transforming oncogenic mechanism. Gene expression profiling and genome-wide chromatin immunoprecipitation analysis uncovered an extensive network of IRF4 target genes and identified MYC as a direct target of IRF4 in activated B cells and myeloma. Unexpectedly, IRF4 was itself a direct target of MYC transactivation, generating an autoregulatory circuit in myeloma cells. Though IRF4 is not genetically altered in most myelomas, they are nonetheless addicted to an aberrant IRF4 regulatory network that fuses the gene expression programs of normal plasma cells and activated B cells.Recently, we developed a genetic method to identify therapeutic targets in cancer in which small hairpin RNAs (shRNAs) that mediate RNA interference are screened for their ability to block cancer cell proliferation and/or survival 10 . Here we report the results of such an "Achilles heel" screen in multiple myeloma (Supplementary Table 3). We used myeloma cell lines from three molecular subtypes: KMS12 (CCND1 translocation), H929 (FGFR3/MMSET * Corresponding author: Louis M. Staudt, MD, PhD 9000 Rockville Pike Building 10, Room 4N114 Bethesda, MD 20892 301-402-1892 Fax: 301-496-9956 e-mail: lstaudt@mail.nih.gov Reprints and permissions information can be found at www.nature.com/reprints. Author contributions were as follows: Experimental design/discussion, A.L.S., V.N.N., J.E., L.M.S.; preparation and performance of experiments, A.L.S, N.C.T.E, L.L., V.N.N., S.D., X.Y., H.Z., Y.Z., B.C.; data analysis/interpretation, A.L.S., N.C.T.E., W.X., G.W., J.P., J.E., L.M.S.; manuscript preparation, A.L.S., L.M.S. New datasets have been deposited under GEO accessions GSE8958, GSE9067 (gene expression), and GSE9367 (ChIP-CHIP).Full Methods are available in the Supplementary Materials in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of this paper at www.nature.com/nature.The authors declare no competing financial interests. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2009 July 10. Published in final edited form as:Nature. (Fig.1a). Lymphoma cell lines were largely unaffected by IRF4 knockdown, with the exception of OCILy3, an activated B cell-like...