BCOR (BCL6 corepressor) is a transcriptional co-repressor that was identified based on its ability to interact with the POZ domain of the oncoprotein BCL6 (Huynh et al., 2000
). Chromosomal translocations in the promoter and 5′ untranslated region of the human BCL6 gene are a common genomic alteration in non-Hodgkin’s B cell lymphomas (Baron et al., 1993
; Dalla-Favera et al., 1999
; Kerckaert et al., 1993
; Miki et al., 1994
; Ye et al., 1993
). These translocations result in aberrant expression of BCL6 (Chen et al., 1998
; Ye et al., 1995
). Mice engineered to model one of these translocations develop B cell lymphomas, demonstrating that BCL6 is a bona fide oncogene (Cattoretti et al., 2005
). BCOR potentiates BCL6 mediated transcriptional repression of reporter constructs in transiently transfected cells (Huynh et al., 2000
). In germinal center B cells, BCOR is found with BCL6 at several known BCL6 target genes, including regulators of cell proliferation and apoptosis (Gearhart et al., 2006
). BCOR co-purifies with an 800 kDa complex comprised of Polycomb group transcriptional repressor proteins and SCF ubiquitin E3 ligase components (Gearhart et al., 2006
). Epigenetic modification of BCL6 target gene chromatin by the BCOR repression complex is likely to play a role in mediating their repression.
In addition to its role in B-cells, BCOR aids in the control of gene expression in multiple tissues and organ systems during development and into adulthood as mutations in human BCOR
result in X-linked Oculofaciocardiodental syndrome (OFCD) (Ng et al., 2004
). OFCD is the primary subtype of OMIM #300166 microphthalmia, syndromic 2 (MCOPS2) and is characterized by ocular, dental, cardiac and digital anomalies in heterozygous females (Ng et al., 2004
; Schulze et al., 1999
). Males with OFCD do not survive due to presumed embryonic lethality (Ng et al., 2004
). Since BCOR
lies on the X chromosome in both mice and humans, random X-inactivation results in mosaic expression of the mutant allele contributing to varying disease severity in females (Ng et al., 2004
; Schulze et al., 1999
). Additionally, peripheral leukocytes of female patients show preferential survival of cells in which the mutant allele of BCOR
lies on the inactivated X chromosome, indicating a strong requirement for BCOR
in hematopoiesis (Hedera and Gorski, 2003
; Ng et al., 2004
). Another form of MCOPS2, which is distinct from OFCD, occurs in males with a single missense mutation (p.P85L) in the fourth coding exon of BCOR
(Horn et al., 2005
; Ng et al., 2004
). In the described family, this syndrome is inherited in an X-linked recessive pattern and comprises microphthalmia/anophthalmia, mental retardation, and skeletal and other anomalies (Ng et al., 2002
). RNAi knock-down of Bcor
in zebrafish results in eye, skeletal and nervous system abnormalities consistent with those found in MCOPS2 patients (Ng et al., 2004
). BCOR has also been shown to interact with and repress transcriptional activation of the MLL fusion protein AF9 (Srinivasan et al., 2003
), a known regulator of Hox gene expression (Collins et al., 2002
) and skeletal development, further implicating BCOR as a key developmental regulator. The pleiotropic effects induced by the loss of functional BCOR in humans and zebrafish (Ng et al., 2004
) clearly illustrate the essential role of BCOR
during embryogenesis and emphasizes the importance of determining the spatial and temporal expression of BCOR
during development. Herein, we provide a detailed analysis of Bcor
mRNA expression during mouse development and in the adult mouse.
In adult mice and humans, Bcor
is widely expressed (Huynh et al., 2000
; Nagase et al., 2000
). However, these studies are limited in scope, relying on RNA dot blot analysis of human tissues and reverse transcription PCR (RT-PCR) on a limited number of mouse tissues. Expanding upon these results, we have dissected the pattern of Bcor
expression by Northern blot analysis, more extensive RT-PCR, and whole mount and section in situ hybridization experiments. To determine the expression pattern of Bcor
in adult mouse tissues, we harvested total RNA from 14 different organs and conducted Northern blot analysis on 4 organs and RT-PCR on all 14 organs using a probe that recognizes all mRNA isoforms (). Three transcripts migrating at approximately 7 kb are found in all four organs analyzed however the stoichiometry of the different transcripts is not identical (). Bcor
mRNA is detected by RT-PCR in all tissues tested (). The ubiquitous expression of Bcor
in adult mouse tissue is consistent with expression of BCOR
in human adult tissue (Huynh et al., 2000
Figure 1 Three independent promoters drive widespread expression of Bcor mRNA in embryonic and adult mouse tissue (A-F). Northern blot analysis of adult mouse organs reveals three major Bcor transcripts. Gapdh was used as a loading control (A). Promoter 1, 2 and (more ...)
The identification of three Bcor
transcripts by Northern blot analysis prompted us to consider that the Bcor
gene may utilize multiple promoters, alternative splicing and/or polyadenylation sites. Although alternative splicing in the coding region has been reported ( and (Srinivasan et al., 2003
)) this can only affect transcript size by up to 156 bp. In silico analysis of spliced expressed sequence tags (EST) databases supported use of alternative promoters and polyadenylation sites at the mouse Bcor
genomic locus. Two alternative polyadenylation sites separated by 550 bp are present. CpG islands are also found in close proximity upstream of exons 1a, 1b and 1c, further supporting the presence of three independent promoters (). To determine whether all three promoters suggested by the EST database are actively used in most tissues, we generated an exon 2 specific reverse primer and three forward primers in putative Bcor
exons 1a, 1b and 1c that correspond with sequences specifically driven by putative promoters P1, P2 and P3 (). RT-PCR and sequencing of the products demonstrates that Bcor
uses all three putative independent promoters ( and data not shown). In the panel of adult mouse tissues tested, each promoter appears to be used at similar levels in all tissues tested, with the exception of whole blood, which appears to preferentially use promoter 3 relative to other tissues. Interestingly, amplification from promoter 2 results in two specific amplicons () due to the use of an alternative splice donor sites in exon 1b (data not shown). We also examined promoter use during embryonic development (E11.5 - E18.5). Although all promoters are used at these stages the distal splice donor of exon 1b is not used at E15.5 and E16.5 ().
Splicing bypassing exon 5 and/or alternative splice acceptor usage at exon 8 results in the previously identified Bcor
isoforms a-d () (Srinivasan et al., 2003
). Only isoforms a and b contain sequences required for the interaction with the transcriptional regulator AF9 (Ng et al., 2004
; Srinivasan et al., 2003
). To determine which isoforms are differentially expressed in the embryo and adult mouse tissues, we conducted RT-PCR using primers that span exon 4 to exon 10 on samples used in . Bcor
isoforms a, c and d can be amplified from the samples tested (). Isoform c shows the most ubiquitous expression in the sample set and isoform a shows the most tissue-specific expression. Relative to the other tissues, isoform a is more strongly represented in the brain and testis. Isoform b is only barely detected in ovary, eye, spleen and kidney at this level of amplification but not at embryonic stages.
The clinical presentation of OFCD in female patients and presumed embryonic lethality in males suggested that Bcor might have a unique expression pattern during embryogenesis that might give insight into future studies on Bcor function. To determine the spatial and temporal expression of Bcor during gastrulation and early organogenesis we conducted whole mount and section in situ hybridization on embryonic day 7.5 - 15.5 CD-1 outbred mouse embyros. Using the same probe sequence as was used for northern analysis we generated digoxigenin labeled RNA antisense and sense (- control) probes that would detect all known Bcor transcripts. These probes were used in all subsequent whole mount and section in situ expression images.
Whole mount in situ hybridization on embryonic day 7.5 and 8.5 shows that Bcor is strongly expressed in the extraembryonic tissue during gastrulation (). Anti-sense probe specificity is validated by absence of signal in embryo hybridized with the sense strand control (). show that expression of Bcor is restricted to the ectoplacental cone and trophectoderm but absent from extraembryonic visceral and parietal endoderm. Consistent with this, trophoblastic stem cells express Bcor (data not shown). Although Bcor is weakly expressed in the neuroectoderm of the embryo proper (), the level is significantly lower in comparison to extraembryonic tissues ().
Figure 2 Bcor mRNA is strongly expressed in extraembryonic tissue during early embryogenesis (A-G). Whole mount in situ hybridization of mouse embryos at day 7.5 and 8.5 shows strong expression of Bcor in the extraembryonic tissue (A and C). Sense strand control (more ...)
After completion of embryonic turning at approximately E9.0, Bcor transcript levels in the embryo proper increase dramatically. Whole mount in situ hybridization of E9.0 embryos () reveals the initial increase in Bcor expression throughout the embryo proper, led by striking increases in the ectodermal tissue of the tail bud. By E9.5 Bcor transcripts are differentially expressed showing strong expression in the limb buds and branchial arches (; sense control is 3F), while maintaining less robust expression throughout the rest of the embryo. Strong expression of Bcor also extends from the tail bud (shown in detail in ) down the ridge of the closing neural tube along the dorsal side of the embryo. Strong expression of Bcor in tissues that will give rise to future craniofacial structures correlates well with craniofacial abnormalities present in female OFCD patients.
Figure 3 Bcor mRNA expression increases throughout the embryo proper post embryonic turning. Whole mount in situ hybridization shows Bcor expression starting in the tail bud (TB) at embryonic day 9 (A and B) and abundant expression in the tail bud, branchial arches (more ...)
After E9.5, Bcor is differentially expressed in multiple tissue and organ systems during the fetal period of mouse development. Section in situ hybridization of a mid-body cross section at E11.5 reveals strong expression of Bcor in the neural tube and flanking dorsal root ganglion (, sense control in 4B). Bcor is also expressed in the notochord and cervical sclerotome and myotome in addition to more subtle and general expression in the forelimb region (). Bcor expression can also be found surrounding the esophagus, right and left bronchi and connected lung buds and in the sympathetic chains (). A cross section through the head of an E13.5 fetal mouse displays significant expression in the eye, neural tube, the olfactory epithelium and the teeth primordium (, sense control in 4F). Bcor expression in the eye is interestingly found in the retina, lens tissues and in the boundary of the eyelid ().
Figure 4 Early fetal stage mouse embryos show widespread but differential expression of Bcor mRNA (A-H). Section in situ hybridization on horizontal sections of an E11.5 embryo shows Bcor expression in multiple tissues including the right and left forelimb buds (more ...)
At E14.5, a sagittal section near the midline of a fetus reveals expression of Bcor in the trigeminal (V) ganglion, the neopallial cortex, the corpus striatum and the olfactory epithelium ( sense control in 5B). In more lateral sagittal sections through an E15.5 fetus, Bcor expression is retained in the eye but is localized to the retina and the dorsal side of the lens (, sense control in 5F). Bcor is also expressed in the lung and gut epithelium and muscle tissue in the fetus e.g. in the temporalis and masseter muscles ().
Figure 5 Later fetal stage mouse embryos continue to express Bcor mRNA in a widespread yet tissue specific manner (A-H). Section in situ hybridization on a sagittal section of an E14.5 embyro (A-D) shows Bcor expression in trigeminal ganglion (TG), neopallial (more ...)
Here we have characterized the unique expression pattern of the transcriptional corepressor Bcor
. We have shown that Bcor
is widely expressed in adult tissue, is expressed in ES cells and displays a widespread but specific expression during embryonic development. There is very strong expression of Bcor
in extraembryonic tissue. Expression of Bcor
in the developing eye, tooth primordium, limb buds, branchial arches and multiple nervous system tissues also distinctly correlates with many of the tissues adversely affected in OFCD and LM patients (Ng et al., 2004
; Schulze et al., 1999
). This analysis provides important information for the analysis of future mouse models of OFCD.