Characterization of Bcor expression in mouse development.
ABSTRACT: Mutation of the gene encoding the transcriptional corepressor BCOR results in the X-linked disorder Oculofaciocardiodental syndrome (OFCD or MCOPS2). Female OFCD patients suffer from severe ocular, craniofacial, cardiac, and digital developmental defects and males do not survive through gestation. BCOR can mediate transcriptional repression by the oncoprotein BCL6 and has the ability to reduce transcriptional activation by AF9, a known mixed-lineage leukemia (MLL) fusion partner. The essential role of BCOR in development and its ability to modulate activity of known oncogenic proteins prompted us to determine the expression profile of Bcor during mouse development. Identification of independently transcribed exons in the 5' untranslated region of Bcor suggests that three independent promoters control the expression of Bcor in mice. Although Bcor is widely expressed in adult mouse tissues, analysis of known spliced isoforms in the coding region of Bcor reveals differential isoform usage. Whole mount in situ hybridization of mouse embryos shows that Bcor is strongly expressed in the extraembryonic tissue during gastrulation and expression significantly increases throughout the embryo after embryonic turning. During organogenesis and fetal stages Bcor is differentially expressed in multiple tissue lineages, with a notable presence in the developing nervous system. Strikingly, we observed that Bcor expression in the eye, brain, neural tube, and branchial arches correlates with tissues affected in OFCD patients.
Project description:Bcor (BCL6 corepressor) is a widely expressed gene that is mutated in patients with X-linked Oculofaciocardiodental (OFCD) syndrome. BCOR regulates gene expression in association with a complex of proteins capable of epigenetic modification of chromatin. These include Polycomb group (PcG) proteins, Skp-Cullin-F-box (SCF) ubiquitin ligase components and a Jumonji C (Jmjc) domain containing histone demethylase. To model OFCD in mice and dissect the role of Bcor in development we have characterized two loss of function Bcor alleles. We find that Bcor loss of function results in a strong parent-of-origin effect, most likely indicating a requirement for Bcor in extraembryonic development. Using Bcor loss of function embryonic stem (ES) cells and in vitro differentiation assays, we demonstrate that Bcor plays a role in the regulation of gene expression very early in the differentiation of ES cells into ectoderm, mesoderm and downstream hematopoietic lineages. Normal expression of affected genes (Oct3/4, Nanog, Fgf5, Bmp4, Brachyury and Flk1) is restored upon re-expression of Bcor. Consistent with these ES cell results, chimeric animals generated with the same loss of function Bcor alleles show a low contribution to B and T cells and erythrocytes and have kinked and shortened tails, consistent with reduced Brachyury expression. Together these results suggest that Bcor plays a role in differentiation of multiple tissue lineages during early embryonic development.
Project description:Oculofaciocardiodental (OFCD) and Lenz microphthalmia syndromes form part of a spectrum of X-linked microphthalmia disorders characterized by ocular, dental, cardiac and skeletal anomalies and mental retardation. The two syndromes are allelic, caused by mutations in the BCL-6 corepressor gene (BCOR). To extend the series of phenotypes associated with pathogenic mutations in BCOR, we sequenced the BCOR gene in patients with (1) OFCD syndrome, (2) putative X-linked ('Lenz') microphthalmia syndrome, (3) isolated ocular defects and (4) laterality phenotypes. We present a new cohort of females with OFCD syndrome and null mutations in BCOR, supporting the hypothesis that BCOR is the sole molecular cause of this syndrome. We identify for the first time mosaic BCOR mutations in two females with OFCD syndrome and one apparently asymptomatic female. We present a female diagnosed with isolated ocular defects and identify minor features of OFCD syndrome, suggesting that OFCD syndrome may be mild and underdiagnosed. We have sequenced a cohort of males diagnosed with putative X-linked microphthalmia and found a mutation, p.P85L, in a single case, suggesting that BCOR mutations are not a major cause of X-linked microphthalmia in males. The absence of BCOR mutations in a panel of patients with non-specific laterality defects suggests that mutations in BCOR are not a major cause of isolated heart and laterality defects. Phenotypic analysis of OFCD and Lenz microphthalmia syndromes shows that in addition to the standard diagnostic criteria of congenital cataract, microphthalmia and radiculomegaly, patients should be examined for skeletal defects, particularly radioulnar synostosis, and cardiac/laterality defects.
Project description:The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocardiodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) and Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmunoprecipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.
Project description:MLL is a target of chromosomal translocations in acute leukemias with poor prognosis. The common MLL fusion partner AF9 (MLLT3) can directly bind to AF4, DOT1L, BCOR, and CBX8. To delineate the relevance of BCOR and CBX8 binding to MLL-AF9 for leukemogenesis, here we determine protein structures of AF9 complexes with CBX8 and BCOR, and show that binding of all four partners to AF9 is mutually exclusive. Using the structural analyses, we identify point mutations that selectively disrupt AF9 interactions with BCOR and CBX8. In bone marrow stem/progenitor cells expressing point mutant CBX8 or point mutant MLL-AF9, we show that disruption of direct CBX8/MLL-AF9 binding does not impact in vitro cell proliferation, whereas loss of direct BCOR/MLL-AF9 binding causes partial differentiation and increased proliferation. Strikingly, loss of MLL-AF9/BCOR binding abrogated its leukemogenic potential in a mouse model. The MLL-AF9 mutant deficient for BCOR binding reduces the expression of the EYA1 phosphatase and the protein level of c-Myc. Reduction in BCOR binding to MLL-AF9 alters a MYC-driven gene expression program, as well as altering expression of SIX-regulated genes, likely contributing to the observed reduction in the leukemia-initiating cell population.
Project description:The BCL-6 co-repressor (BCOR) represses gene transcription by interacting with BCL-6 (Refs 1, 2). BCOR mutation is responsible for oculo-facio-cardio-dental (OFCD) syndrome, which is characterized by canine teeth with extremely long roots, congenital cataracts, craniofacial defects and congenital heart disease. Here we show that BCOR mutation increased the osteo-dentinogenic potential of mesenchymal stem cells (MSCs) isolated from a patient with OFCD, providing a molecular explanation for abnormal root growth. AP-2alpha was identified as a repressive target of BCOR, and BCOR mutation resulted in abnormal activation of AP-2alpha. Gain- and loss-of-function assays suggest that AP-2alpha is a key factor that mediates the increased osteo-dentinogenic capacity of MSCs. Moreover, we found that BCOR maintained tissue homeostasis and gene silencing through epigenetic mechanisms. BCOR mutation increased histone H3K4 and H3K36 methylation in MSCs, thereby reactivating transcription of silenced target genes. By studying a rare human genetic disease, we have unravelled an epigenetic mechanism for control of human adult stem cell function.
Project description:In this study, we report on 2 sisters from India with oculo-facio-cardio-dental (OFCD) syndrome caused by a novel heterozygous mutation c.3490C>T (p.R1164*) in the BCOR gene. OFCD syndrome is an X-linked inherited disorder which is lethal in males. Interestingly, both parents of the sisters were phenotypically normal, and DNA analysis from blood and buccal or saliva cells failed to detect the BCOR mutation found in their 2 daughters. To the best of our knowledge, for the first time, we provide indirect evidence of germline mosaicism for the BCOR mutation in one of the parents of the 2 sisters affected by OFCD syndrome. Although this condition is lethal in males, gonadal mosaicism could also be present in the father. The relevance of clinical diagnosis and mutation analysis required for genetic counseling is described in this family.
Project description:Oculo-facio-cardio-dental syndrome (OFCD) is a rare genetic disorder characterized by teeth with extremely long roots (radiculomegaly), and craniofacial, eye and cardiac abnormalities. The mutation of the transcriptional co-repressor BCOR has been identified as being responsible for oculo-facio-cardio-dental (OFCD) syndrome. Mesenchymal stem cells (MSCs) is isolated from the root apical papilla of an OFCD patient. Gene expression profiling is performed and compared between mutant MSCs and wild type MSCs. Total RNA were extracted from normal MSCs (MSCWT) and mutant MSCs (MSCO).
Project description:Oculo-facio-cardio-dental syndrome (OFCD) is a rare genetic disorder characterized by teeth with extremely long roots (radiculomegaly), and craniofacial, eye and cardiac abnormalities. The mutation of the transcriptional co-repressor BCOR has been identified as being responsible for oculo-facio-cardio-dental (OFCD) syndrome. Mesenchymal stem cells (MSCs) is isolated from the root apical papilla of an OFCD patient. Gene expression profiling is performed and compared between mutant MSCs and wild type MSCs. Overall design: Total RNA were extracted from normal MSCs (MSCWT) and mutant MSCs (MSCO).
Project description:BCL-6 encodes a POZ/zinc finger transcriptional repressor that is required for germinal center formation and may influence apoptosis. Aberrant expression of BCL-6 due to chromosomal translocations is implicated in certain subtypes of non-Hodgkin's lymphoma. The POZ domains of BCL-6 and several other POZ proteins interact with corepressors N-CoR and SMRT. Here we identify and characterize a novel corepressor BCoR (BCL-6 interacting corepressor), which is expressed ubiquitously in human tissues. BCoR can function as a corepressor when tethered to DNA and, when overexpressed, can potentiate BCL-6 repression. Specific class I and II histone deacetylases (HDACs) interact in vivo with BCoR, suggesting that BCoR may functionally link these two classes of HDACs. Strikingly, BCoR interacts selectively with the POZ domain of BCL-6 but not with eight other POZ proteins tested, including PLZF. Additionally, interactions between the BCL-6 POZ domain and SMRT, N-CoR, and BCoR are mutually exclusive. The specificity of the BCL-6/BCoR interaction suggests that BCoR may have a role in BCL-6-associated lymphomas.
Project description:With the advent of next-generation sequencing, an increasing number of novel gene fusions and other abnormalities have emerged recently in the spectrum of EWSR1-negative small blue round cell tumors (SBRCTs). In this regard, a subset of SBRCTs harboring either BCOR gene fusions (BCOR-CCNB3, BCOR-MAML3), BCOR internal tandem duplications (ITD), or YWHAE-NUTM2B share a transcriptional signature including high BCOR mRNA expression, as well as similar histologic features. Furthermore, other tumors such as clear cell sarcoma of kidney (CCSK) and primitive myxoid mesenchymal tumor of infancy also demonstrate BCOR ITDs and high BCOR gene expression. The molecular diagnosis of these various BCOR genetic alterations requires an elaborate methodology including custom BAC fluorescence in situ hybridization (FISH) probes and reverse transcription polymerase chain reaction assays. As these tumors show high level of BCOR overexpression regardless of the genetic mechanism involved, either conventional gene fusion or ITD, we sought to investigate the performance of an anti-BCOR monoclonal antibody clone C-10 (sc-514576) as an immunohistochemical marker for sarcomas with BCOR gene abnormalities. Thus we assessed the BCOR expression in a pathologically and genetically well-characterized cohort of 25 SBRCTs, spanning various BCOR-related fusions and ITDs and YWHAE-NUTM2B fusion. In addition, we included related pathologic entities such as 8 CCSKs and other sarcomas with BCOR gene fusions. As a control group we included 20 SBRCTs with various (non-BCOR) genetic abnormalities, 10 fusion-negative SBRCTs, 74 synovial sarcomas, 29 rhabdomyosarcomas, and other sarcoma types. In addition, we evaluated the same study group for SATB2 immunoreactivity, as these tumors also showed SATB2 mRNA upregulation. All SBRCTs with BCOR-MAML3 and BCOR-CCNB3 fusions, as well as most with BCOR ITD (93%), and all CCSKs showed strong and diffuse nuclear BCOR immunoreactivity. Furthermore, all SBRCTs with YWHAE-NUTM2B also were positive. SATB2 stain was also positive in tumors with YWHAE-NUTM2B, BCOR-MAML3, BCOR ITD (75%), BCOR-CCNB3 (71%), and a subset of CCSKs (33%). In conclusion, BCOR immunohistochemical stain is a highly sensitive marker for SBRCTs and CCSKs with BCOR abnormalities and YWHAE-rearrangements and can be used as a useful diagnostic marker in these various molecular subsets. SATB2 immunoreactivity is also present in the majority of this group of tumors.