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Chromatin modifications are essential for directing transcription during embryonic development. Bromodomain-containing protein 2 (Brd2; also called RING3 and Fsrg1) is one of four BET (bromodomain and extra terminal domain) family members known to selectively bind acetylated histones H3 and H4. Brd2 associates with multiple subunits of the transcriptional apparatus including the mediator, TFIID and Swi/Snf multi-protein complexes. While molecular interactions of Brd2 are known, the functions of Brd2 in mammalian embryogenesis remain unknown. In developing a mouse model deficient in Brd2, we find that Brd2 is required for the completion of embryogenesis and proper neural tube closure during development. Embryos lacking Brd2 expression survive up to embryonic day 13.5, soon after mid-gestation, and display fully penetrant neurulation defects that largely result in exencephaly of the developing hindbrain. In this study, we find that highest expression of Brd2 is detected in the developing neural tube, correlating with the neural tube defects found in Brd2-null embryos. Additionally, embryos lacking Brd2 expression display altered gene expression programs, including the mis-expression of multiple genes known to guide neuronal development. Together these results implicate essential roles for Brd2 as a critical integrator of chromatin structure and transcription during mammalian embryogenesis and neurogenesis.
Bromodomains are conserved chromatin-targeting modules found in many eukaryotic transcriptional regulatory proteins and have been shown to bind specifically to acetylated histones H3 and H4 [1–5]. Brd2 belongs to the BET subfamily of bromodomain proteins that contain two tandem N-terminal bromodomains (B) and a single C-terminal extra-terminal (ET) domain . Epigenetic modifications of chromatin structure, such as histone acetylation and methylation, are known to have important consequences in the regulation of gene transcription . Therefore, understanding the roles of murine Brd2 in interpreting combinatorial histone modification of chromatin is a critical part of investigating the regulation of transcription during mammalian development.
Brd2 structure is conserved among plants, animals and fungi . The yeast orthologs of Brd2, TFIID-associated components bromodomain factors 1 and 2 (Bdf1 and Bdf2), have been shown to be required for anti-silencing functions at subtelomeric regions of the yeast genome and for correctly interpreting histone modifications [2, 8, 9]. In Drosophila, Brd2 is most closely related to female sterile homeotic 1 (fsh1), a trithorax-group gene required for proper gene expression and fly embryogenesis [10–12]. Three of the four mammalian BET proteins—Brd2, Brd3 and Brd4—are broadly expressed, while the fourth, Brdt, is selectively expressed in the germline [13, 14]. In the mouse, the ubiquitously expressed Brd2 has the highest levels of expression during embryogenesis as well as in the adult testis, ovary and brain [13–16]. Brd2 was initially identified as a nuclear kinase in human cells that is involved in guiding the expression of cell cycle genes through its binding to multiple E2Fs [17–19]. In addition, Brd2 has been shown to be associated with several multiprotein regulators of transcription, including the mediator, TFIID, and Swi/Snf complexes [16, 20]. These widespread interactions implicate Brd2 in targeting critical components of the transcriptional machinery to precisely modified regions of the eukaryotic genome.
While distinct interactions and expression patterns of the mammalian BET proteins have been described, little is known about the potential function of Brd2 in normal mammalian development. Disruption of the Brd2-related paralog, Brd4, in the mouse leads to early post-implantation lethality in vivo and an inability to maintain the inner cell mass in vitro . Recently, a single bromodomain of the testis-specific Brdt has been shown to be required for male germ cell differentiation . These studies suggest that although the basic structure of related BET family members is conserved, their expression patterns and functions in mammalian development are diverse. A number of studies in mammalian cells have implicated Brd2 function in the positive control of cell proliferation. Brd2 has been shown to bind several E2F cell cycle transcriptional activators and its exogenous expression was shown to help activate the cyclin A promoter [18, 19]. Moreover, specific over-expression of Brd2 in the lymphoid lineage was shown to result in B cell lymphoma and leukemia . Several studies have documented the nuclear accumulation of Brd2 during diverse proliferation events in cultured cells and in neural and reproductive tissues in vivo [16, 23, 24]. In an embryonic development study of the mouse, expression of Brd2 mRNA peaked between E8.5 and E12.5 and was prominently detected in the developing CNS . In humans, mutations in the promoter of the BRD2 gene have been linked to increased susceptibility to juvenile myoclonic epilepsy (JME), an adolescent-onset generalized epilepsy . BRD2 has also been genetically linked to photoparoxysmal response (PPR), a related seizure disorder in humans .
Given the current known biochemical functions of Brd2 and its potential role in neural development and disease, we disrupted the Brd2 gene in the mouse to investigate its biological function during mammalian development. Here, we show that Brd2-deficient embryos deviate from normal developmental programs at embryonic day 9.0 (E9.0), when they exhibit delayed development, later growth retardation and fail to survive after E13.5. Strikingly, as neural development progresses, Brd2-null embryos consistently manifest neural tube closure defects that most commonly appear as exencephaly of the hindbrain. Moreover, deregulation of transcription at E9.0 may underlie the developmental defects observed in the Brd2-null embryos before they become apparent. Together, these data indicate essential roles for Brd2 in regulating chromatin structure and transcription during mammalian development.
The ES cells RREO50, which carry a gene-trap construct in between the first and second coding exons of Brd2 (BayGenomics), were grown on mitotically inactive feeder layers until 90% confluent and were dissociated by trypsinization before injection. E3.5 blastocysts were derived from C57BL/6-Tyrc-Brd female mice and injected with 12–20 ES cells. The injected blastocysts were implanted into the uteri of day 2.5 pseudo-pregnant females, with eight to ten embryos implanted per uterine horn. The resulting male chimeras were mated with C57BL/6-Tyrc-Brd females to obtain F1 progeny. The strain carrying the germ line transmitted allele (named Brd2Gt1RFr and hereafter referred to as minus allele) was maintained on a mixed C57BL/6-129Ola background. Heterozygous animals were intercrossed to produce Brd2+/+, Brd2+/− and Brd2−/− embryos. A second gene-trapped ES cell line from EUCOMM (OTTMUSG00000017279) was used to derive a second disrupted Brd2 mouse line (named Brd2Gt2RFr and hereafter referred to as the lacZ allele). All breeding and procedures were carried out according to institutional regulations at Brown University Animal Facility and NIH Guide for the Use and Care of Laboratory Animals.
To differentiate wild-type (+) from mutant (−) alleles of Brd2, a 551bp amplicon of the mutant allele was PCR-amplified using Brd2For5-GTTCCCTGAGGTCAAGATGCTG and βGalRev7-ACCCCTTCCTCCTACATAGTTGGC and subsequently sequenced to identify the junction between the endogenous sequence and the insertion cassette. This analysis uncovered that 417 bp at the 5′-end of the gene trap was lost when it incorporated into the genome. However, the splice acceptor essential for the function of this disruption vector was retained. To identify the wild-type allele, a pair of primers, Brd2For1-GCTGAGCGGCGGCGGTTCCC proximal to Brd2For5 and Brd2IntRev93-CGGAACGCCGCCCCCCAACC downstream of the insertion cassette junction, were used to generate an amplification product of 106 bp. When resolved on 2% agarose-TAE gels stained with ethidium bromide, these two PCR products allowed us to identify genomic DNA of all three genotypes. Genomic DNA was isolated from either adult mouse-tail biopsies or yolk sacs of developing embryos using the DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer’s instructions.
Female mice of Brd2 heterozygous intercrosses were checked every morning for the presence of a copulation plug. The day on which the copulation plug was observed was designated as E0.5. Dissections took place on subsequent days of development. Females were sacrificed and the uterine horns were excised and placed in sterile PBS. Decidual swellings corresponding to embryos were dissected individually, and yolk sacs retained for PCR genotyping. Resultant embryos were imaged using a Zeiss Discovery V8 stereomicroscope equipped with an Axiocam MRc camera and Axiovert software. Immediately after imaging, embryos were dounced in Trizol (Invitrogen) to preserve RNA quality, and RNA was isolated using manufacturer’s protocols.
Total RNA from nine E9.0 embryos (3 Brd2+/+, 3 Brd2+/−, 3 Brd2−/−) was obtained as described above and further purified using micro RNeasy columns (Qiagen). RNA quality was checked using a Bioanalyzer, and concentration determined using a Nanodrop. 100 ng of each RNA sample were used in the Affymetrix Whole-transcript Sense Target Labeling Assay (Rev 3) followed by hybridization to a GeneChip® Mouse Gene 1.0 ST Array. Nine GeneChips were used to provide biological triplicates of each genotype. The Affymetrix Expression Console (v 1.1) was used to normalize data and determine signal intensity (RMA-Sketch). Analysis was performed in Microsoft Excel. Transcripts with two-fold or greater changes in Brd2−/− embryos are reported in Supplemental Table 1.
RNA was isolated from E9.5 embryos collected from timed heterozygous matings as described earlier. RNA concentrations were determined by Nanodrop (Thermo Scientific), and 0.5 to 1 μg of total RNA was used to prepare 20 μl of cDNA using the iScript Select kit (BioRad). Real-time PCR reactions were performed in triplicate using 1 μl of cDNA template, SYBR green PCR master-mix (ABI) and gene-specific primers for Brd2, Med26, Brachyury, NeuroD1, NeuroD4, Olig3, SlitRK6, and 18S rRNA (Invitrogen) in the ABI 7300 Real Time PCR System, according to manufacturers’ protocols. Primer sequences can be found in Supplemental Table 2. Relative mRNA expression levels were determined using ΔCt values and were normalized to 18S rRNA levels to correct for minor variations in starting RNA concentrations.
β-galactosidase staining of whole E13.0 embryos was performed using standard protocols. Freshly harvested embryos were fixed for 20 minutes at room temperature in 0.2% glutaraldehyde, washed three times in 0.1M phosphate buffer and incubated in 1 mg/ml X-gal (Invitrogen) overnight at room temperature. For immunostaining and TUNEL, freshly harvested embryos were fixed overnight at 4°C in 4% PFA, cryopreserved through serial incubation in 15% and 30% sucrose at 4°C, frozen in OCT blocks in liquid nitrogen and sectioned at 10 μm. For P-H3 antibody staining, slides were blocked for 1–2 hours in 10% goat serum and 10% BSA in PBT. Slides were then incubated at 1:100 dilution anti-P-H3 antibody (Cell Signaling, 9701S) in 10% goat serum, 10% BSA in PBT at 4°C overnight. The slides were washed three times in PBS at room temperature and incubated with a 1:100 dilution of goat anti-rabbit Alexa Fluor 594 (Molecular Probes, A11012) in 10% goat serum, 10% BSA in PBT for 2 hours at room temperature. For fluorescent analysis, the slides were washed three times in PBS, mounted with Vectashield plus DAPI (Vector Labs) and imaged on a Zeiss ImagerM1 fluorescence microscope using Axiovision software. For TUNEL staining, a Fluorescence In-Situ Cell Death Detection Kit (Roche) was utilized. Embryo sections were permeabilized for 6 minutes on ice in 0.1% Sodium Citrate, 0.1% Triton X-100 and rinsed twice in PBS. TUNEL substrate was mixed according to manufacturer’s protocol (Roche). Slides were covered, placed in dark, humidified chambers and incubated at 37°C for one hour, rinsed three times in PBS, mounted with Vectashield plus DAPI (Vector Labs) and imaged on a Zeiss ImagerM1 fluorescence microscope.
To ascertain the functions of Brd2 in mouse development, a heterozygous embryonic stem (ES) cell line (RRE050) with a gene trap vector insertion in between the first two coding exons (Figure 1A) of the mouse Brd2 gene was obtained from BayGenomics [27, 28]. The β-geo cassette of the gene-trap cassette contains a splice acceptor site which functions with the splice donor site of coding exon 1 of Brd2 to produce a truncated Brd2 transcript. This ES cell line was used to derive founder Brd2 heterozygous (Brd2+/−) mice. Initial genotyping of germ-line-transmitting founder Brd2+/− intercross progeny yielded viable wild type (Brd+/+) and heterozygous (Brd2+/−) progeny. However, intercrosses of heterozygous Brd2 males and females failed to produce any Brd2−/− mice at weaning age (Figure 1B). Thus, this disruption of Brd2 results in apparent embryonic or early postnatal lethality. Interestingly, the numbers of Brd2+/− progeny at weaning were lower than the expected Mendelian ratio of 2:1 (Figure 1B, p<0.0001). Therefore, having only a single functional copy of Brd2 may have some deleterious effects on embryonic development, resulting in partially penetrant haploinsufficiency of heterozygous offspring.
The lack of viable Brd2−/− offspring suggested that Brd2 is required to complete embryogenesis. To characterize the role of Brd2 during embryogenesis, timed matings of Brd2+/− males and females were used to recover embryos across multiple developmental time points. PCR genotyping of yolk sac derived genomic DNA reliably detected embryos of all three Brd2 genotypes in litters ranging from E8.5 to E13.5 from heterozygous intercrosses. To verify Brd2 disruption by the insertion of the gene trap vector, quantitative RT-PCR (qPCR) was used to detect and quantify transcripts of Brd2 and the β-galactosidase transgene in E9.5 in littermate embryos of all three Brd2 genotypes. Brd2 mRNA was undetectable in Brd2−/− embryos, while the relative Brd2 mRNA level in Brd2+/+ embryos was approximately two-fold higher than in Brd2+/− embryos (Figure 1C). Accordingly, Brd2−/− embryos were shown to have approximately two-fold higher expression of the β-galactosidase transgene relative to the Brd2+/− embryos. Brd2 mRNA levels were normalized to 18S rRNA levels to account for slight variation in total RNA yield from each individual embryo, and these experiments were repeated on multiple litters (data not shown). To assess the relative time of lethality, the progeny of multiple timed heterozygous intercrosses from E8.5 to E13.5 were recovered and genotyped. Of the 166 embryos recovered, 38 Brd2−/− embryos (23%) were identified between E8.5 and E11.5. In contrast, only 5 Brd2−/− embryos from a total of 59 recovered between E12 and E13 have been detected (8%; Table 1). At developmental stages E12 and later, uterine evidence of embryonic lethality was observed, which likely represents resorption of non-viable Brd2−/− embryos. These data indicate that only a fraction of the Brd2−/− embryos progress past E12, with a continuum of embryonic lethality occurring through several days (E8.5 to E11.5) during mid-gestation.
Phenotypic analysis of multiple embryonic litters derived from Brd2 heterozygous intercrosses revealed fully penetrant severe growth retardation and defects in neural tube closure for Brd2−/− embryos compared to matched wild type littermates. Representative whole embryo images of wild type and Brd2−/− littermates from E9.0 to E13.5 are shown (Figure 2). Comparison of wild type and Brd2−/− embryos reveals a reduction in the overall size of the Brd2−/− embryos compared to matched wild type littermates. Severe growth retardation and developmental delay are most obvious at E9.0, when some Brd2−/− embryos are significantly smaller than their wild type counterparts and have not undergone axial rotation (Figure 2A and 2C). This lag in development was observed in 60% of Brd2-null embryos recovered at this early time point. Since most Brd2−/− embryos fail to develop past E12 (Table 1), it is likely that this severe developmental delay has lethal consequences for most embryos that fail to express Brd2. As development proceeds, all of the Brd2−/− embryos display cranial defects in neural tube closure (Figure 2E-P). Most Brd2−/− embryos do complete axial rotation, and the most common neurodevelopmental defect observed is exencephaly in the developing hindbrain region (Figure 3). The neural folds in the rhombencephalon are large, thickened, and splayed open to the outside of the embryo (Figure 2, M-P and Figure 3). Additionally, multiple Brd2−/− embryos display a curved caudal neural tube with frequent openings within the neural region (Figure 4). In contrast, other aspects of development such as limb, heart and somite development appear relatively intact in the older Brd2−/− embryos (Figure 2, M-P). The neural tube defects of the Brd2−/− embryos are fully penetrant, as a Brd2−/− embryo with proper neurulation (n=43) has not been detected; however, the defects are pleiotropic in nature. In addition, several heterozygous Brd2+/− embryos have been identified with similar neural tube defects, although variable in nature (Supplemental Figure 1). Taken together, these data indicate that Brd2 function is necessary for proper neural tube closure during embryonic development and suggest that Brd2 may play an essential role in the regional specification of the developing rhombencephalon.
To address the localization of Brd2 function during mouse embryonic development, the expression of a β-galactosidase reporter gene in a second targeted Brd2 strain (Figure 1A), derived from an EUCOMM ES cell line, was used to examine the endogenous expression pattern of Brd2 in developing embryos. Brd2Gt2RFr mice demonstrated embryonic β-galactosidase activity in genotypically Brd2+/lacZ versus matched wild type Brd2+/+ control embryos. Much of the light blue β-galactosidase staining is readily detectable on the inside of the E13.0 embryos (Figure 5). Strikingly, β-galactosidase staining is most readily detected in the developing brain and spinal cord, precisely in the neural tube that fails to close properly in the Brd2−/− embryos. This neural-specific expression pattern is consistent with an RNA in situ pattern reported in a previous study of Brd2 expression during mouse embryogenesis . The heightened and localized expression of Brd2, together with the developmental defects in null mutants reported here, suggests a critical role in the development of the mouse CNS.
A number of diverse cellular processes are known to underlie proper neural tube closure in rodents and humans [29, 30]. To begin to assess the cellular etiology of the neural tube defects associated with loss of Brd2, phosphorylated histone H3 (P-H3) and terminal deoxynucleotidyl transferase (TUNEL) staining were performed to detect proliferation and apoptosis, respectively. Cell proliferation in the thickened neural folds of the Brd2−/− embryo is evidenced by the nuclear staining of P-H3 on the ridges of the neural folds. The extent and localization of staining in the mutant, whose neural folds are spread apart, is similar, although not identical, to the wild type control, where the neural folds are bending towards each other (Figure 6A and B). Similarly, little difference in apoptosis is observed between the Brd2+/+ and Brd2−/− embryos at this E11.5 time point (Fig. 6C and D). Together, these data indicate that there are no significant defects in cell proliferation and apoptosis in this Brd2−/− embryo at this time point; however, such cellular defects may arise in more severely affected embryos that are more difficult to assay or at different time points in development.
To assess global gene transcription in the Brd2−/− embryos, the expression profiles of Brd2+/+, Brd2+/− and Brd2−/− embryos were compared at E9.5 by microarray analysis. The transcript levels of a number of genes were found to differ, with two-fold or greater enrichment found in 46 genes and two-fold or greater reduction seen in only 10 transcripts (Supplemental Table 1). A subset of the reduced transcripts in the Brd2−/− embryos encode several key neuronal regulators such as the neurogenic differentiation factors 1 and 4 (NeuroD1 and NeuroD4) and the oligodendrocyte transcription factor 3 (Olig3; Table 2). The ephrin receptor A3 (EphA3) and the integral membrane protein SLITRK6, which shares homology with the neurotrophin receptor family, were also found to be expressed at two to three fold lower levels in the Brd2−/− embryos by expression array (Table 2). The reduced expression of these genes was confirmed by quantitative RT-PCR of RNA extracted from additional Brd2+/+ and Brd2−/− littermates, demonstrating a three fold reduction in NeuroD4 and SLITRK6 and a four fold reduction in NeuroD1 and Olig3 in the Brd2−/− embryos compared to Brd2+/+ littermates (Figure 7A). To confirm that these expression changes were not due simply to differences in embryonic stage of development, additional quantitative RT-PCR was performed on Brd2+/+ and Brd2−/− embryos with 37–39 somite pairs. Similar fold reductions between Brd2+/+ and Brd2−/− embryos were observed (Figure 7B). All gene expression levels were normalized to 18S rRNA.
Spina bifida is one of the most common birth defects worldwide, whereas juvenile myoclonic epilepsy (JME) is much less common; however, both may have links to Brd2 deregulation. Spina bifida involves a posterior opening of the spinal cord. Brd2 may play an indirect or direct role in this neural development defect. The curly tail mouse has been an extensively studied model of spina bifida, and recent progress has implicated the reduced expression of the transcription factor encoding gene Grainy-head-like-3 (Grhl3) as being responsible for the opening of the posterior neuropore in this mutant [31–33]. As spina bifida is only partially penetrant in the curly tail strain, a number of curly tail modifier genes have been mapped in the mouse genome. Strikingly, one of these curly tail modifiers, Mct1, has been mapped to the HLA region of mouse chromosome 17, in close proximity to the Brd2 gene . Based on the neural tube defects of the Brd2-null embryos presented here, Brd2 and Grhl3 may collectively coordinate precise transcriptional events required for proper neural tube closure.
Mutations in the promoter of the human BRD2 gene have been linked to increased susceptibility to juvenile myoclonic epilepsy (JME), an adolescent-onset generalized epilepsy . In addition, BRD2 has also been genetically linked to photoparoxysmal response (PPR), a related seizure disorder in humans . It is worth noting that differences between null mutations in the mouse that elicit striking neurodevelopmental defects (this study) and more subtle regulatory mutations of the human BRD2 promoter may have profoundly different consequences. The human mutations are predicted to alter the relative levels of BRD2 expression in certain individuals and are not predicted to alter the full-length protein product . This hypothesis posits a threshold model of BRD2 expression and susceptibility to JME. Given the striking neural tube closure defects of the Brd2-null embryos, it is possible that subtle changes in BRD2 expression may result in viable offspring with neurodevelopmental changes consistent with an increased susceptibility to seizures.
Given Brd2’s diverse molecular interactions and its relevance to human neural developmental defects, a functional investigation of Brd2 in mammalian development was warranted. Using reverse genetics to establish a Brd2-null mouse line, we demonstrate that the disruption of the Brd2 gene causes embryonic lethality. Brd2-null embryos deviate from normal developmental programs at embryonic day 9.0 (E9.0) where they exhibit developmental delay and generalized growth retardation. As development progresses, Brd2−/− embryos consistently manifest neural tube closure defects that most commonly appear as exencephaly of the hindbrain. This observation correlates with a high expression of Brd2 in the developing CNS.
Consistent with the notion of Brd2’s involvement in cell proliferation, we find an overall reduction in the growth potential of the Brd2−/− embryos compared to Brd2-containing embryos (Figure 2). However, in contrast to Brd4-null embryos which fail much earlier in development, Brd2-null embryos have traversed many cell division cycles to reach these time points . Here, we observed similar neural epithelial proliferation in a Brd2−/− embryo compared to a matched wild type control and conclude that significant differences in proliferation are not apparent (Figure 6). However, we cannot rule out the possibility that subtle differences in proliferation may accumulate over time and detract from normal neuronal development. Defects in coordinated specification and proliferation of early neural tissue may be associated with the neural tube defects of the Brd2-null embryos [29, 30, 36]. Expression of Brd2 mRNA peaks between E8.5 and E12.5, and is prominently detected in the developing CNS . Thus, at approximately E8.5 when neuronal proliferation is initiated, Brd2 may be required to promote neurogenesis by regulating the gene expression networks required to drive the expansion of newly born neuronal cell types . The nuclear accumulation of Brd2 in multiple proliferating neuronal cell types is consistent with this notion . Thus, the inability of neural folds to fuse might reflect the inability of the Brd2-null embryos to produce enough neuronal precursor cells during early CNS development. Alternatively, Brd2−/− embryos may be unable to execute the correct amount of neuronal apoptosis, as Brd2 has been previously shown to be induced during apoptosis in PC12 cells and in neurons; this function may be important also during early neurogenesis . Thus, in the absence of Brd2, a subtle loss in the balance of proliferation and apoptosis may help establish an unmanageable expansion of neural precursor cells, which may result in an inability to correctly fuse the neural folds. Additionally, Brd2 function may be required outside of the developing neural ectoderm. This result would be similar to neural tube closure defects in Twist knockout mice in which head mesenchyme or neural crest derivatives are the root of neural tube closure defects [38, 39]. Future studies will aim to distinguish these diverse, yet related, possibilities.
Other models which provide insight into Brd2 function include the Drosophila Brd2 ortholog, fsh1, and murine Gcn5 mutants. A recent report indicates that fsh1 mutants undergo homeosis of the head and tail region that may be similar in nature to the neuronal defects of the Brd2-null mouse embryos . In this regard, genes that are known to be critical regulators of midbrain-hindbrain specification and regionalization during early neuronal development may also be targets of Brd2 function [41, 42]. In a recent report by Bu et al., cranial neural tube closure defects, similar to that observed in the Brd2-null embryos, are described in homozygous Gcn5 mutants, which contain a single point mutation in the catalytic core of the histone acetyltransferase (HAT) domain . Disruption of a related HAT, p300, leads to similar hindbrain exencepahly as in the Brd2-null embryos . In addition to HATs, disruptions of de novo DNA methyltransferases Dnmt3b and Dnmt1o in the mouse result in neural tube closure defects [45, 46]. As histone acetylation and DNA methylation are functionally linked in epigenetic regulation, it is possible that Brd2 might play a central role in stabilizing methylation marks on the developing mammalian genome required for proper neurulation. Thus, phenotypic variation in the Brd2-null mutants may reflect mosaic methylation patterns between individual embryos . Future studies using conditional alleles of Brd2 will focus on the molecular mechanism of Brd2 in regulating the specification and regionalization of the developing mouse brain.
Supplemental Figure 1. Whole mount analysis of Brd2+/+, Brd2+/−, and Brd2−/− littermates at E9.5 and E10.5 show graded effects of Brd2 copy number. Note the minor growth retardation in the heterozygotes compared to wild-type and Brd2-null littermates, as well as the less severe delay in neural tube closure as compared to the Brd2-null mice. The intermediate phenotypes seen in the Brd2+/− mice suggest partial haploinsufficiency. All images at each time point are shown at identical magnification for size comparison.
The authors would like to thank John Coleman, Gary Wessel, Angus Wilson and Mike Marr for critical input throughout these studies and for insightful comments on the manuscript. We thank John Wallingford, Mark Zervas, Stephen Brown and Nellwyn Hagan for input and expertise in assaying neuronal development. We thank Bill Skarnes, BayGenomics and EUCOMM for generously providing the gene-trapped ES cell lines used in our study. We thank Mandy Pereira and Erin Paul in the transgenic mouse core facility at Brown University for establishing the Brd2-deficient mouse lines. We thank Dr. Christoph Schorl and the Genomics and Proteomics Core Facility at Brown University for expertise with microarray analysis and qPCR. This research was supported in part by NIH/NCRR COBRE Award # P20RR015578 and Ellison Medical Foundation awards to R.N.F.
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