OFCD syndrome and extraembryonic defects are revealed by conditional mutation of the Polycomb-group repressive complex 1.1 (PRC1.1) gene BCOR.

BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9-10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.

Polycomb Repressive Complex 1.1 Component, BCOR, Promotes Syncytiotrophoblast Differentiation in Mice and Humans.

Early defects in placenta development are thought to underlie a range of adverse pregnancy conditions including miscarriage, fetal growth abnormalities, preeclampsia, and stillbirth. Differentiating trophoblast stem cells undergo a choreographed allocation of syncytiotrophoblast and extravillous trophoblast cells in response to signaling cues from the developing fetus and the uterine environment. The expression and activity of transcription factors and chromatin modifying enzymes change during differentiation to appropriately reshape the chromatin landscape in each cell type. We have previously found in mice that extraembryonic loss of BCOR, a conserved component of the epigenetic silencing complex Polycomb Repressive Complex 1.1 (PRC1.1), leads to a reduced labyrinth and expanded trophoblast giant cell population in the placenta. Molecular analysis of wild-type and BCOR loss-of-function male and female placentas by RNA-seq identified gene expression changes as early as E6.5. We found that BCOR is required to down regulate stem cell genes and repress factors that promote alternate lineages which leads to reduced levels of syncytiotrophoblasts. ChIP-seq experiments identified a number of directly bound functional targets including Pdgfa and Wnt7b . In humans, BCOR is mutated in X-linked syndromes involving fetal growth restriction and females with a heterozygous null mutation in BCOR can experience recurrent miscarriages. To establish a direct role for BCOR in human placental development, we used CRISPR/Cas9 to knockout BCOR in male (CT29) and female (CT30) human trophoblast stem cells. Mutant cell lines retained capacity for induced differentiation into syncytiotrophoblast and extravillous trophoblasts and exhibited minimal changes in gene expression. However, in 3D cell culture using trophoblast organoid media, BCOR knockout lines had significantly altered gene expression including homologs of stem cell genes upregulated in Bcor knockout mice. CUT&RUN experiments in self-renewing and 3D cell culture identified genes directly bound by BCOR. Single cell profiling of wild type, knockout, and a P85L pathogenic knock-in BCOR mutation showed a reduced capacity to differentiate into syncytiotrophoblasts after four days of differentiation. Together, these results suggest that BCOR is a conserved regulator of trophoblast development that represses stem cell genes during differentiation and maintains lineage fidelity by repressing genes that promote alternate cell fates.

Oculofaciocardiodental and Lenz microphthalmia syndromes result from distinct classes of mutations in BCOR.

Lenz microphthalmia is inherited in an X-linked recessive pattern and comprises microphthalmia, mental retardation, and skeletal and other anomalies. Two loci associated with this syndrome, MAA (microphthalmia with associated anomalies) and MAA2, are situated respectively at Xq27-q28 (refs. 1,2) and Xp11.4-p21.2 (ref. 3). We identified a substitution, nt 254C-->T; P85L, in BCOR (encoding BCL-6-interacting corepressor, BCOR) in affected males from the family with Lenz syndrome previously used to identify the MAA2 locus. Oculofaciocardiodental syndrome (OFCD; OMIM 300166) is inherited in an X-linked dominant pattern with presumed male lethality and comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. Given their phenotypic overlap, we proposed that OFCD and MAA2-associated Lenz microphthalmia were allelic, and we found different frameshift, deletion and nonsense mutations in BCOR in seven families affected with OFCD. Like wild-type BCOR, BCOR P85L and an OFCD-mutant form of BCOR can interact with BCL-6 and efficiently repress transcription. This indicates that these syndromes are likely to result from defects in alternative functions of BCOR, such as interactions with transcriptional partners other than BCL-6. We cloned the zebrafish (Danio rerio) ortholog of BCOR and found that knock-down of this ortholog caused developmental perturbations of the eye, skeleton and central nervous system consistent with the human syndromes, confirming that BCOR is a key transcriptional regulator during early embryogenesis.

Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets.

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.

EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis.

The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

Structure of a BCOR corepressor peptide in complex with the BCL6 BTB domain dimer.

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.

ETO protein of t(8;21) AML is a corepressor for Bcl-6 B-cell lymphoma oncoprotein.

The multiplicity of transcription factors involved in hematologic malignancies suggests a complicated scenario in which many different molecular mechanisms lead to malignant transformation. We hypothesized that some of these proteins might physically and functionally interact and thus mechanistically link different diseases. The ETO protein of t(8;21) acute myeloid leukemia (AML) is an excellent candidate as a common factor because it is normally expressed in human hematopoietic cells, it binds to histone deacetylases (HDACs), and it interacts with the PLZF protein of t(11;17) acute promyelocytic leukemia. To determine whether ETO functionally links a broader range of disease entities, we asked whether ETO forms a complex with the Bcl-6 oncoprotein of B-cell lymphomas. We found that ETO and Bcl-6 are coexpressed in normal and malignant lymphoid tissue, where they interact and colocalize in nuclear speckles. ETO binds to the fourth zinc finger of Bcl-6, enhances Bcl-6 repression of artificial and endogenous genes in an HDAC-dependent manner, and forms a complex with Bcl-6 on the promoters of its endogenous target genes in B-cell lymphoma cells. Therefore, ETO is a bona fide corepressor that links the transcriptional pathogenesis of acute leukemias and B-cell lymphomas and offers a compelling target for transcriptional therapy of hematologic malignancies.