We have identified a novel set of proteins that associate with the corepressor BCOR in a single 800-kDa complex that is recruited to a subset of BCL6 targets. The proteins in the BCOR complex include the PcG and PcG-associated proteins NSPC1, RING1, RNF2, and RYBP as well as components of an SCF ubiquitin ligase, SKP1, and FBXL10. Our biochemical and functional analyses indicate that BCOR recruits a unique combination of enzymatic activities to chromatin targets: a PcG E3 ubiquitin ligase for histone H2A, a demethylase for histone H3 K36, and an SCF E3 ubiquitin ligase. Corepressor recruitment by the POZ domain of BCL6 is required for the proliferation and survival of BCL6-positive lymphoma cells (61
). However, the relative contributions of the deacetylase and demethylation activities of the NCOR and SMRT complexes (32
) versus the histone ubiquitylation and demethylation activities of the BCOR complex to repression of individual BCL6 targets remain to be determined. Our ChIP results indicate that histone H2A ubiquitylation activity of the BCOR complex is present at the Cyclin D2
promoters, suggesting that BCOR likely contributes to the repression of these genes and perhaps to the enhanced proliferation and survival of BCL6-positive lymphoma cells.
Our finding that the BCOR complex contains PcG proteins which may contribute to BCL6-driven oncogenesis is consistent with a growing number of observations indicating a role for PcG proteins in stem cell identity, mammalian development, cell cycle regulation, and cancer (11
). PcG proteins are known to be part of a memory system that relies on epigenetic modification of chromatin to ensure faithful transmission of cell identities through cell division (10
). In addition to its potential roles in oncogenesis with BCL6 and MLL-AF9, BCOR is clearly an essential corepressor in multiple developmental pathways. OFCD patients, who have mutations in BCOR
, have severe craniofacial, digital, and cardiac defects. Peripheral blood lymphocytes from female patients show strongly biased inactivation of the X chromosome carrying the mutant BCOR allele (53
), indicating that BCOR is also required for normal hematopoiesis. The diverse phenotypes observed in OFCD patients and the widespread expression of BCOR suggest additional tissue-specific transcription factors recruit the BCOR complex and its associated PcG and SCF proteins.
The recruitment of BCOR complex PcG proteins to target genes by BCL6 in B cells suggests that BCL6 functions as a PcG-targeting factor. Interestingly, BCL6 is related to the Drosophila
POZ zinc finger transcription factor GAGA, which is thought to play a role in PcG and TrxG protein recruitment to DNA (6
). Typically, histone H3 K27 methylation, mediated by the ESC-E(Z) methyltransferase complex, creates a binding site for the chromo domain of the Pc protein in the PRC1 complex (13
). Although the BCOR complex, unlike other PRC1-like complexes (46
), does not copurify with a chromo domain-containing protein, the complex does contain protein domains with the potential to bind chromatin. CXXC domains like the one found in FBXL10 can bind unmethylated CpG sequences (9
). PHDs can bind nucleosomes (8
), and in two recent examples this binding was specific to H3 K4-trimethylated nucleosomes (67
). These domains of FBXL10 could stabilize the association of the BCL6-recruited complex with chromatin or may allow for BCL6-independent recruitment of the BCOR complex. Similarly, the BCOR-interacting protein AF9 (69
) interacts with the chromo domain protein MPc3 (Cbx8) (34
), which may facilitate chromatin contacts (7
). The BCOR complex may function analogously to the E2F6.com-1 complex recruited by the transcription factor E2F6 (56
). Like the BCOR complex, this complex includes RING1, RNF2, and a Psc homolog (MBLR) and also copurifies with the chromatin-binding domain protein (HP1γ), which may stabilize binding of E2F6.com-1 to chromatin trimethylated at K9 of histone H3 (56
The interaction of BCOR with PcG proteins as well as an F-box protein with a JmjC domain demethylase is particularly striking. JmjC domain demethylase enzymes are able to remove methyl groups from lysines that have been mono-, di-, or trimethylated (18
). The specificity of FBXL11 (JHDM1A) has been thoroughly characterized, and the protein has been found to demethylate mono- and dimethyl lysine 36 of histone H3 (74
). FBXL10 (JHDM1B) has been reported to also demethylate H3 K36 (74
), but its specificity remains to be fully established. The presence of methylated H3 K36 correlates with transcriptional elongation (44
), suppression of intragenic transcription (15
), and the defining of actively transcribed regions (2
). BCOR complexes containing the demethylase activity of the full-length FBXL10 may therefore additionally contribute to transcriptional regulation by altering H3 K36 methylation at target genes.
The F-box and the leucine-rich repeats at the C terminus of FBXL10 are presumed components of an SCF E3 ubiquitin ligase. Although we see a modest FBXL10-dependent effect on endogenous BCOR abundance (Fig. ), the direct target, nature (mono versus poly), and outcome (signaling versus degradation) of the ubiquitylation activity of FBXL10 are unknown. The presence of SKP1 at the BCL6 locus (Fig. ) indicates that SCF ubiquitin ligase components are found on chromatin with the BCOR complex. Future characterization of the SCF E3 ligase activity of FBXL10 will provide important insights into BCOR complex-dependent gene silencing.
Because deregulated expression of BCL6 plays an oncogenic role in non-Hodgkin's lymphomas and BCL6 can recruit BCOR to promoters involved in apoptosis and cell cycle regulation, we suggest that BCOR and its associated PcG and SCF proteins may also play a role in BCL6-associated lymphomas. Once a definitive functional role for BCOR is established, the enzymatic activities of the BCOR complex may be attractive therapeutic targets for non-Hodgkin's lymphoma.