A cluster of six microRNAs (miRNAs), miR-17-92, is processed from the transcript of C13orf25, a gene amplified in some lymphomas and solid tumors. We find that levels of the miRNAs in the cluster do not vary entirely in parallel with each other or with the primary RNA in B-cell lines or normal cells, suggesting that processing or stability of the miRNAs is differentially regulated. Using luciferase reporter assays, we identified the region required for maximum promoter activity. Additional deletions and mutations indicated that the promoter is regulated by the collaborative activity of several transcription factors, most of which individually have only a moderate effect; mutation of a cluster of putative SP1-binding sites, however, reduces promoter activity by 70%. MYC is known to regulate C13orf25; surprisingly, mutation of a putative promoter MYCbinding site enhanced promoter activity. We found that the inhibitory MYC family member MXI1 bound to this region. The chromatin structure of a >22.5-kb region encompassing the gene contains peaks of activating histone marks, suggesting the presence of enhancers, and we confirmed that at least two regions have enhancer activity. Because MicroRNAs (miRNAs) are single-stranded RNAs of ϳ22 nucleotides that negatively regulate expression of their target genes posttranscriptionally. 1-4 Various miRNAs are overexpressed or underexpressed in different types of cancer in humans and may function as either tumor suppressors or oncogenes. 5-13 A cluster of six miRNAs (miR-17-92, comprising miR-17, miR18a, miR-20a, miR-19a, miR-19b-1, and miR-92a-1) is processed from the transcript of C13orf25 (also known as MIR17HG or MIRHG1), a gene amplified in some lymphomas and solid tumors 5,14 -17 and overexpressed in a large fraction of lymphomas. 5 Overexpression of miR-17-92 accelerates lymphomagenesis in mouse models. 5,8 The miRNAs in the cluster can target transcripts of genes, such as E2F1, PTEN, and BIM, which are important in cell proliferation and apoptosis. 18 -22 The transcriptional regulation of the miR-17-92 cluster, however, is poorly understood.C13orf25 is activated by MYC and E2F transcription factors, and MYC was first shown to bind to a region containing a conserved CATGTG sequence in the first intron of the gene locus. 8 By chromatin immunoprecipitation (ChIP) in HeLa cells, endogenous E2F1, E2F2, and E2F3 were found to directly bind the promoter of the gene and regulate its transcription. 21 The primary transcript initiates from a consensus initiator sequence downstream of a nonconsensus TATA box. 23 This TATA box is flanked by a TP53 binding site that medi-

BCL6 encodes a transcriptional repressor that is essential for the germinal center (GC) reaction and important in lymphomagenesis. Although its promoter has been well studied, little is known concerning its possible regulation by more distal elements. To gain such information, we mapped critical histone modifications associated with active transcription within BCL6 as well as far upstream sequences at nucleosomal resolution in B-cell lines and in normal naive and GC B cells. Promoter-associated and intronic CpG islands (CGIs) in BCL6 showed a reciprocal pattern of histone modifications. Gene expression correlated with a paradoxical loss from the intronic CGI of histone H3 lysine-4 trimethylation, normally associated with transcription, suggesting that the intronic CGI may interfere with transcription. In an ∼110-kb region extending 150-260 kb upstream of BCL6, highly active histone modifications were present only in normal GC B cells and a GC B-cell line; this region overlaps with an alternative breakpoint region for chromosomal translocations and contains a GC-specific noncoding RNA gene. By chromosome conformation capture, we determined that the BCL6 promoter interacts with this distant upstream region. It is likely that transcriptional enhancers in this region activate BCL6 and overcome strong autorepression in GC B cells.chromatin structure | epigenetic | DNA looping | transcriptional regulation | ChIP-on-chip

BCL6 gene transcribes two main mRNA isoforms, variant 1 and variant 2, which have distinct transcription start sites. However, these two isoforms encode identical BCL6 protein. Recently, a third variant was sequenced from a human hippocampus cDNA library (DB465062). In this study, we cloned and sequenced a novel BCL6 spliced isoform (BCL6S) which lacks exon 7 that encodes the first two zinc fingers of BCL6. We found a splicing isoform, BCL6S, that excludes exon 7 but retains the last four of the six zinc fingers (ZFs) coding region of the BCL6 gene. BCL6S mRNA and protein were expressed in human cell lines and tissues that expressed BCL6, but accounted for a minor portion of BCL6 transcripts or protein. BCL6S protein was also detectable in BCL6 positive cells. BCL6S could form homodimers or heterodimers with BCL6 and could bind to classical BCL6 binding sites. Luciferase reporter assays demonstrated that BCL6S could effectively repress typical BCL6 target genes. BCL6S is a compact repressor that may have a functional role in normal and neoplastic germinal center B cells. Keywords BCL6; zinc finger; alternative splicing; transcription repressor; germinal centerThe BCL6 proto-oncogene encodes a BTB/POZ zinc finger (ZF) transcriptional repressor, locating on chromosome 3q27 [1] with 10 exons over 26 kilobases (kb) region. The protein contains an amino-terminal POZ motif encoded by sequence starting at exon 3 and the six C2H2-type ZFs at the carboxy-terminal is encoded by exons 7 to 10.The BCL6 protein is a nuclear phosphoprotein that is the master regulator in germinal center (GC) formation and GC B-cell differentiation and survival. Deregulation of BCL6 has been implicated in the pathogenesis of lymphomas especially diffuse large B cell lymphoma (DLBCL). The BCL6 amino-terminal POZ domain interacts with the co-repressors N-COR, SMRT and BCOR, and the six carboxy-terminal ZF motifs recognize specific DNA sequences. The PEST domain, the second repression domain (RDII) in the middle of BCL6, interacts with the corepressor MTA3 [2] and the function of BCL6 may be modified by phosphorylation or acetylation of this domain [3]. In addition to direct DNA binding to conserved cis-elements of Correspondence to: Wing C Chan. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. BCL6 transcribes two main isoforms, variant 1 and variant 2, which have distinct transcription start sites and are regulated by two discrete promoters. Recently, a third variant was sequenced from a human hippocampus cDNA library (DB465062). These observations indicate BCL6 gene can be transcribed from altern...

BCL6, a proto-oncogene frequently mutated and translocated in B-cell lymphoma, encodes a transcriptional repressor essential for the germinal center (GC) reaction. BCL6 mRNA and protein have higher expression in GC-B cells than in pre- and post-GC-B cells despite strong autorepression. Hypothesizing that histone modifications epigenetically regulate BCL6 during B-cell differentiation, we mapped a number of histone modifications associated with active transcription far upstream to the promoters of BCL6 and ~100 other genes in pre-GC-B, GC-B and plasma cell lines at nucleosomal resolution using the ChIP-chip assay. We identified numerous regions of histone H3 acetylation and H3 lysine-4 mono- and trimethylation (H3K4me1 and -3) within and upstream of BCL6; most striking was a vast region of H3K4me1 extending 140-260 kb upstream of BCL6 seen in a GC-B-cell line, but not in pre- or post-GC-B-cells. We also observed high expression of an intergenic transcript within this far upstream region in GC-B-cell lines but not in numerous other lines, suggesting distant regulatory regions, active only in GC-B-cells, account for high BCL6 expression. Many other genes also showed evidence for stage-specific distal regulatory regions. Defining a histone code for B cell master regulators will elucidate transcriptional networks central to both the immune response and cancer. The work is supported by UNMC Eppley Cancer Institute.

BCL6 encodes a BTB/POZ zinc finger (ZF) transcriptional repressor which is essential for normal germinal center (GC) reactions. Its dysregulated expression contributes to the pathogenesis of B cell non-Hodgkin lymphoma (NHL). The BCL6 gene consists of ten exons, BCL6 translation start site is located in exon 3, and the six-ZF DNA binding domain is encoded by exons 7 to 10. BCL6S, was cloned from the cDNA of a BCL-6 positive cell line, DHL-16. DNA sequencing showed that BCL6S was a normal splicing isoform that excludes the entire exon 7 of the BCL6 gene and encodes a 650 amino acid (aa) protein. BCL6S was detected by RT-PCR in all human cell lines and tissues expressing BCL6, but was not detected in the mouse B cell lymphoma cell line A20. BCL6S accounts for 1/8 to 1/10 of total BCL6 transcripts. Luciferase reporter assays demonstrated that BCL6S could repress typical BCL6 target genes ( BCL6, CD23b, Blimp1, MIP1α ) as effectively as the full length BCL6. BCL6S retains the last four ZFs of BCL6 and localizes in the cell nucleus. Protein-protein interaction assays confirmed that BCL6 and BCL6S could form homodimers and heterodimers. DNA binding assays showed no affinity differences between BCL6 and BCL6S; however, a single mutation at the N-terminus of the POZ domain (L19 to H) not only disrupted BCL6S dimerization but also significantly decreased the DNA binding affinity, indicating that BCL6 dimerization may stabilize the ZF-DNA binding. In conclusion, we have identified a novel BCL6 splicing isoform. BCL6S, a compact repressor as potent as BCL6. POZ dimerization plays an important role in stabilization of BCL6 DNA binding whereas the first two ZFs are not absolutely required. The biological role of BCL6S in GC B cells needs to be further investigated.

Chromosomal translocations at 3q27 involving BCL6 occur in two different regions. Rearrangements with breaks within the major breakpoint region (MBR), comprising the first exon and part of the first intron of BCL6, are among the most common genetic abnormalities in of B-cell non-Hodgkin lymphoma, whereas breaks within an alternative breakpoint region (ABR), located between 245 and 285 kb 5′ to BCL6, have also been reported in follicular lymphoma grade and a small group (6.4%) of diffuse large B-cell lymphomas (DLBCLs). As a result of the MBR translocation, BCL6 expression is deregulated by promoter substitution with either immunoglobulin (Ig) genes or non-Ig genes as partners. A role for deregulated BCL6 expression in the pathogenesis of DLBCL has previously been confirmed in a mouse model. However, the biological role of the more distant ABR region is still not known. Using real-time PCR, we identified in the ABR region an evolutionarily conserved DNase I hypersensitive site (named Far5) which contains a conserved composite binding site for transcription factors PU.1 and IRF4, both of which play important roles in B-cell differentation. Further studies demonstrated that chromatin in the Far5 region, 190kb upstream of BCL6 promoter, has an open configuration in DHL6, Granta 519 and U266 cell lines. Far5 DNA showed enhancer activity by a luciferase reporter assay. PU.1 binds to Far5 in vivo (DHL16 cell line) by a chromatin immunoprecipitation (ChIP) assay, and PU.1 binds in vitro to the conserved PU.1/IRF composite site in Far5 synergistically with either IRF4 or IRF8. ChIP-on-chip assays showed Far5 histone H3K4 monomethylation, a chromatin modification associated with gene enhancers and other regulatory elements. In addition, we identified a transcript upstream of the Far5 region that is specifically expressed in germinal center (GC) B cells, but not at other stages of B-cell differentation. These results indicated that Far5 may play a role in selective expression of intergenic transcripts in GC B-cells. In other genes, intergenic transcription plays a role in looping between distal regulatory regions and the promoter. Our data showed that ABR region is constitutively active in GC B-cells and may play an important role in increasing BCL6 transcription when naïve B cells differentiate into GC B-cells. Further investigation of interactions between the ABR and the BCL6 promoter will uncover the regulatory function of the BCL6 ABR in B-cell differentiation and B-cell lymphomas.