Abstract
Brd2 belongs to the BET family of epigenetic transcriptional co-regulators that act as adaptor-scaffolds for the assembly of chromatin-modifying complexes and other factors at target gene promoters. Brd2 is a protooncogene and candidate gene for juvenile myoclonic epilepsy in humans, a homeobox gene regulator in Drosophila, and a maternal-zygotic factor and cell death modulator that is necessary for normal development of the vertebrate central nervous system (CNS). As two copies of Brd2 exist in zebrafish, we use antisense morpholino knockdown to probe the role of paralog Brd2b, as a comparative study to Brd2a, the ortholog of human Brd2. A deficiency in either paralog results in excess cell death and dysmorphology of the CNS, whereas only Brd2b deficiency leads to loss of circulation and occlusion of the pronephric duct. Co-knockdown of both paralogs suppresses single morphant defects, while co-injection of morpholinos with paralogous RNA enhances them, suggesting novel genetic interaction with functional antagonism. Brd2 diversification includes paralog-specific RNA variants, a distinct localization of maternal factors, and shared and unique spatiotemporal expression, providing unique insight into the evolution and potential functions of this gene.
Highlights
Brd2 is a member of the bromodomain-extra terminal domain (BET) family of proteins, which act as epigenetic transcriptional co-regulators and are major interpreters of the acetyl-lysine-histone code [1,2]
To verify that brd2b-S was an actual in vivo transcript rather than a cDNA library cloning artifact, and to empirically confirm the presence of predicted exons of the longer transcript, as found in the present databases, we designed primer pairs spanning diagnostic exon–exon junctions in the two cDNAs, and conducted RT-PCR on RNA isolated from stage I–IV oocytes and from 4, 24, and 48 h post fertilization embryos (Figure 1B, exon 5/6, exon 9/10, exon 11/12 primers from brd2b-L, and exon5/intron5 primers from brd2b-S; Figure 1C, key of primer pairs and their predicted products; gels showing RT-PCR products obtained). brd2b-L exons are present in all tested oocyte stages and embryos (Figure 1C, blue arrowheads, lanes labeled 3, 4, and 5, all sample gels)
Primers derived exclusively from within intron 1 show no product (Figure 1C, lane labeled 9 for each sample gel), ruling out genomic DNA contamination as a source template. These data corroborate our previous analyses by Northern blot, where we detected brd2b RNA variants of corresponding sizes, with longer RNAs (4 and 6 kb) observed in ovaries and embryos of all stages, and a short mRNA of about 1.8 kb only in embryos starting between 8 and 10 hpf and persisting through 48 hpf [25]
Summary
Brd is a member of the bromodomain-extra terminal domain (BET) family of proteins, which act as epigenetic transcriptional co-regulators and are major interpreters of the acetyl-lysine-histone code [1,2]. BET proteins recognize and bind acetylated histones via their N-terminal bromodomains and recruit other proteins via their C-terminal ET domains [3]. They function as adaptor-scaffolds at target gene promoters, providing a surface for the ordered assembly and regulation of various chromatin-modifying factors and bridging transcription factors at distal elements with the basal transcriptional machinery [3,4]. As master regulators of multiple target genes, BET proteins likely facilitate crosstalk among different regulatory pathways and biological processes, and act as critical relay stations for the integration of multiple signals [6,7]. The study of BET protein Brd across species in the context of embryonic development illustrates this point
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