BCOR Expression Research Articles

Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones.

BCOR Is Involved in Myeloid Cell Growth Control by Regulating Hox Genes

AbstractAbstract 3445BCOR interacts with Polycomb group (PcG) proteins to form a PRC1-like transcriptional repressive complex. BCOR is an essential developmental regulator: hemizygous mutation of BCOR in males causes gestational lethality, and heterozygous mutation in females causes the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD). Studies in humans, cell lines and mice indicate that BCOR plays key roles in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. Recently, recurrent somatic BCOR mutations have been reported in retinoblastoma, medulloblastoma and acute myelogenous leukaemia (AML) suggesting BCOR functions as a tumor suppressor protein. The goal of the current study was to begin elucidating the function of BCOR in normal hematopoiesis and in hematopoietic transformation. To examine this in the mouse we transduced bone marrow (BM) from mice harboring a conditional Bcor deletion allele (BcorFl/Y) with lentivirus expressing CRE. FACS analysis showed that with CRE, 83% of Bcor-/Y (Bcor KO) BM cells kept in IL3, IL6 and stem cell factor (SCF) were CD34+ compared to 95% of non-induced BcorFl/Y (Bcor WT) BM cells. Moreover, following G-CSF treatment, Bcor KO BM cells showed a much higher incidence of cell differentiation (40.5%) compared to Bcor WT cells (5.4%) as shown by CD11b/Gr-1 FACS analysis. Surprisingly, Bcor KO BM cells also showed significantly higher proliferation rates in liquid culture and methycellulose colony assays. To identify BCOR downstream target genes, we compared the gene expression profiles between Bcor KO and WT BM cells. Real-time-PCR of individual Hox genes confirmed the upregulation of HoxA5, HoxA7 and HoxA9 in Bcor KO BM cells, which are known to increase self-renewal of immature hematopoietic cells. In addition, ChIP analysis showed specific binding of BCOR to HoxA7 promoter. In conclusion, our results from mouse experiments suggest that BCOR is involved in maintaining the CD34+ stem cell state and loss of BCOR leads to enhanced myeloid cell differentiation and proliferation possibly by regulating Hox genes. In addition, we examined BCOR expression levels in a large panel of human leukemic cell lines; high level BCOR mRNA and protein were detected in most cell lines. Notably, 2 cell lines, U937 and HL60, had low levels of BCOR mRNA and no detectable BCOR protein. Treatment of both cell lines with the differentiation inducers ATRA, Vitamin D3 or DMSO lead to a mark induction in BCOR expression both at the mRNA and protein level, suggesting that BCOR may be involved in myeloid cell differentiation. Further investigation should shed more light on the roles BCOR that plays in normal hematopoiesis and whether BCOR loss later in hematopoiesis could result in a differentiation block. Disclosures:No relevant conflicts of interest to declare.

Function analysis of mesenchymal Bcor in tooth development by using RNA interference

Teeth, an excellent model for studying organogenesis, develop from a series of epithelial-mesenchymal interactions that are mediated by a complex molecular network. Bcor (BCL-6 interacting corepressor) has recently been discovered, but little is known about its function in tooth development. Mutations in BCOR affect humans with oculofaciocardiodental syndrome, which is an X-linked dominant disorder with presumed male lethality and which comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. In this study, the Bcor expression pattern has been intensively investigated during mouse molar development. Bcor is expressed in both dental epithelium and the mesenchyme at E11.5. To understand the function of Bcor, knockdown of Bcor has been examined by using lentivirus-mediated RNA interference. Silencing of Bcor expression in dental mesenchymal cells at E14.5 causes dentinogenesis defects and retardation of tooth root development. Thus, our results suggest that Bcor expressed in the mesenchyme plays crucial roles during early tooth development. The function of Bcor expressed in the epithelium remains to be elucidated.

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.

Characterization of Bcor expression in mouse development

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.