Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
The chromatin-targeting protein Brd2 is required for neural tube closure and embryogenesis
Introduction
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], [2], [3], [4], [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 [6]. Epigenetic modifications of chromatin structure, such as histone acetylation and methylation, are known to have important consequences in the regulation of gene transcription [7]. 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 [6]. 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], [11], [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], [14], [15], [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], [18], [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 [21]. Recently, a single bromodomain of the testis-specific Brdt has been shown to be required for male germ cell differentiation [22]. 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 [23]. 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 [24]. 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 [25]. BRD2 has also been genetically linked to photoparoxysmal response (PPR), a related seizure disorder in humans [26].
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.
Section snippets
ES cells and generation of Brd2-null mice
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
Gene-trap-mediated disruptions of the mouse Brd2 gene
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 (Fig. 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
Discussion
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
Acknowledgements
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
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These authors contributed equally to this work.