The bromodomain-containing gene BRD2 is regulated at transcription, splicing, and translation levels

Abstract

The human BRD2 gene has been linked and associated with a form of common epilepsy and electroencephalographic abnormalities. Disruption of Brd2 in the mouse revealed that it is essential for embryonic neural development and that viable Brd2(+/-) heterozygotes show both decreased GABAergic neuron counts and increased susceptibility to seizures. To understand the molecular mechanisms by which mis-expression of BRD2 might contribute to epilepsy, we examined its regulation at multiple levels. We discovered that BRD2 expresses distinct tissue-specific transcripts that originate from different promoters and have strikingly different lengths of 5' untranslated regions (5'UTR). We also experimentally confirmed the presence of a highly conserved, alternatively spliced exon, inclusion of which would result in a premature termination of translation. Downstream of this alternative exon is a polymorphic microsatellite (GT-repeats). Manipulation of the number of the GT-repeats revealed that the length of the GT-repeats affects the ratio of the two alternative splicing products. In vitro translation and expression in cultured cells revealed that among the four different mRNAs (long and short 5'UTR combined with regular and alternative splicing), only the regularly spliced mRNA with the short 5'UTR yields full-length protein. In situ hybridization and immunohistochemical studies showed that although Brd2 mRNA is expressed in both the hippocampus and cerebellum, Brd2 protein only can be detected in the cerebellar Purkinje cells and not in hippocampal cells. These multiple levels of regulation would likely affect the production of functional BRD2 protein during neural development and hence, its role in the etiology of seizure susceptibility.

Figure 1

Molecular organization of the BRD2 gene and its multiple transcripts. A. A cartoon showing the presence of the exons, introns, and promoters of the human BRD2 gene. P_I, P_II, P_III, P_IV represent multiple upstream transcription initiation sites; P_S represents the major transcription initiation site which produces the shorter transcript; 1 represents the first coding exon included in the long transcript; 1s represents the part of exon 1 included in the short transcript; 2a (yellow bar) represents the alternatively spliced exon; green bars represent the 5’ untranslated region; blue bars represent coding exons; purple bars represent the 3’ untranslated region. B. Cartoon depiction of the four corresponding mRNAs that result from the use of different transcription start sites and the alternatively spliced exon 2a (the color coding of various genomic regions is the same as that in Figure 1A); the small flags indicate the Flag-tag sequences that were inserted in the expression constructs used in the transfection experiments; probes 1 and 2 indicate the region of sequence used as probes in the Northern blot hybridization analysis in Fig 2C and 2D. C. A cartoon of the full-length BRD2 protein; the two ovals represent the two bromodomains; the blue rectangle represents the ET domain; the two small rectangles represent additional conserved regions; the region used for generating the antibody is indicated by the blue bar and arrow; the flag indicates the position of the inserted Flag tag.

Figure 2

Expression of the multiple transcripts in selected mouse tissues and in human brain. A. A cartoon depicting alignment of DNA sequences in the genomic region between the genomic DNA sequence of exon 1 and cDNA sequences in the EST database using NCBI’s BLASTN, showing that most cDNA clones start from the middle of exon 1. B. A Cartoon depicting sequence alignment between the 3.5 kb genomic sequence upstream of the TATA Box in exon 1 and cDNA sequences in the EST database, indicating multiple transcription initiation sites of BRD2 in a broad 3 kb region. The predicted promoters are labeled P_I to P_IV. C. Northern hybridization analysis of Brd2 mRNAs in selected mouse tissues using probe 1 (indicated in figure 1) to detect the longer transcripts with long 5’ UTR. D. Northern hybridization analysis of Brd2 mRNAs in selected mouse tissues using probe 2 (indicated in figure 1) to detect both the longer and shorter transcripts (with long and shorter 5’ UTR). T, Testis; B, Brain; Sk, Skeletal muscle; Lu, Lung; O, Ovary; Mg, Mammary gland; K, Kidney; H, Heart; Li, Liver; In, Intestine; U, Uterus; Sp, Spleen. E. Northern hybridization of human brain mRNA using human BRD2 cDNA as a probe, showing the long and short transcripts. Cb, Cerebellum; Cc, Cerebral Cortex; Md, Medulla; Sc, Spinal Cord; Oc, Occipital cortex; Fc, Frontal cortex; Tc, Temporal cortex; Pt, Putamen.

Figure 3

The conserved region in intron 2 of BRD2 contains an alternatively spliced exon. A. A cartoon portraying the ~180 bp region in intron 2 of BRD2 that is highly conserved between human, dog, mouse, rat, and opossum, adapted from the ECR Browser. The yellow bar indicates the alternative exon 2a (92 bp) in intron 2. The green column near exon 3 is a microsatellite (GT repeats). A vertical axis cut-off of 75% identity is utilized to visualize only the significant alignments. Note that the level of conservation in intron 2 is higher than in the two adjacent coding exons. B. RT-PCR of human brain RNA using primers located in exon 2 and exon 3 of human BRD2. The upper weaker band contains a 92 bp conserved region in intron 2 (exon 2a), indicating the existence of mRNAs containing the alternatively spliced exon. C. RT-PCR of RNA from constructs containing different length of the GT repeats using primers across exon 2 to exon 3. It shows that the proportion of the alternatively spliced transcript (top band) increases relative to the regularly spliced transcript (lower band) with the shortening of the GT repeats.

Figure 4

Translation of the BRD2 mRNAs. A. In vitro translation of BRD2 mRNAs using ³⁵S methionine labeling. L, BRD2 transcript with long 5’ UTR. S, BRD2 transcript with short 5’ UTR. 2a, exon 2a containing-alternatively spliced. The arrow indicates the full-length BRD2 protein. Luc, Luciferase positive control. (--), negative control. B. Expression of BRD2 protein from Flag-tagged BRD2 cDNA constructs transfected into HEK293T cells as detected by immunoblot analysis with anti-Flag antibody.

Figure 5

Expression of Brd2 mRNAs and protein in selected regions of the adult mouse brain. A. in situ hybridization in cerebellum; B. in situ hybridization in hippocampus; the pictures shown in panels A and B were obtained with probe 2 (indicated in Fig. 1) which recognizes both the long and short transcripts ; C. immunostaining in cerebellum; D. immunostaining in hippocampus. PC, Purkinje cells; GC, granule cells; DG, dentate gyrus. CA1, CA2, CA3, sub-regions of hippocampus.