Understanding the role of BRD8 in human carcinogenesis

1.1 Splicing variants of BRD8 and their expression in human organs

According to the Ensembl database in EMBL-EBI, there are 11 protein-coding transcripts of human BRD8. Two major structural forms, isoform 1 or Q9H0E9-2 (951 amino acids) and isoform 2 or Q9H0E9-1 (1235 amino acids), are encoded by transcript variants ENST00000230901/NM_006696 and ENST00000254900/NM_139199, respectively. BRD8 isoform 2 differs from isoform 1 in the C-terminal region and lacks 73 amino acids in the central region. Importantly, both isoforms have bromodomain 1 (BD1), but isoform 2 has another bromodomain (BD2) at the C-terminus (Figure 1A). Immunoblotting of BRD8 seems to detect bands above the calculated molecular weight: isoforms 1 (102,839 Da) and 2 (135,336 Da) appear between 150 and 250 kDa and close to 250 kDa, respectively.¹¹

RNA sequencing data from the GTEx project (https://gtexportal.org/) revealed the expression patterns of BRD8 variants across human tissues. A variant ENST00000254900, corresponding to isoform 2, is expressed abundantly and exclusively in the testis (Figure 1B). On the other hand, ENST00000230901, corresponding to isoform 1, is broadly expressed in all tissues. This variant (ENST00000230901) is also expressed in colonic mucosa and tumors.¹¹ Thus, understanding the splicing mechanism and the role of the testis-specific variant could help to determine its phenotype(s), including the “preweaning lethality” in Brd8 KO mice shown by the International Mouse Phenotyping Consortium (https://www.mousephenotype.org).

1.2 Bromodomains of BRD8

As described above, BRD8 isoform 2 has two bromodomains, BD1 between codons 705 and 813 and BD2 between codons 1101 and 1209 at the C-terminus. The amino acid sequences of BD1 (109 a.a.) and BD2 (109 a.a.) are 54.1% identical (ClustalW: https://www.genome.jp/tools-bin/clustalw). The BD1 of human BRD8 has been reported to interact at least with histone H4¹¹ and H2AZ.¹² In addition, the BD1 bound to acetylated K73/76 of Twist family bHLH transcription factor 1 (TWIST1), while BD2 bound to acetylated K5/8 of histone H4.¹³ Regarding the interaction with histone H2AZ, BRD8 binds to both the unacetylated and acetylated forms. Unexpectedly, acetylated H2AZ has a relatively lower binding affinity for BRD8 than the unacetylated H2AZ, implying a recognition preference of the bromodomain.¹²

It is noteworthy that the double bromodomain-containing yeast Bdf1 (BD1, 149–256 a.a.; BD2, 314–423 a.a.), a part of the SWR chromatin remodeling complex, is thought to be homologous to human BRD8 isoform 2.^14-16 The amino acid sequences of BD1 and BD2 between human BRD8 isoform 2 and yeast Bdf1 are 25.0% and 23.9% identical, respectively (ClustalW). Bdf1 was first identified as a protein to interact with histone H4 by yeast two-hybrid screening.¹⁷ Subsequently, Matangkasombut and Buratowski showed that point mutations in the bromodomains of Bdf1 disrupted histone binding in vitro, which leads to defects in its transcription.¹⁸ Bdf1 contains an additional extra terminal domain, and thus may belong to the BET family (https://www.yeastgenome.org/). Of note, mutant Bdf1 proteins with a substitution in the ZA-loop of BD1 (Y187A) or BD2 (Y354A) showed reduced histone H4 binding. In addition, its binding to the core histones, H2A, H2B, H3, and H4 was impaired by substitutions of P194/P361 or M195/M362 in the ZA loop, or N230/N397 in the BC loop (of BD1 and BD2). The Y187/Y354, P194/P361, M195/M362, and N230/N397 correspond to Y744/Y1140, P751/P1147, M752/M1148, and N787/N1183 of BD1 and BD2 in human BRD8 (Q9H0E9-1, NP_631938), respectively (Figure 1A).^{19, 20}

The bromodomain also acts as a binding module for the nonhistone proteins.^{11, 13, 21} BRD8 has been known to bind MRG-binding protein (MRGBP)^{11, 21} and TWIST1¹³ through BD1. Although it is likely that BRD8 interacts directly with EP400 and EP400 N-terminal like (EP400NL), it remains unclear whether the bromodomain(s) mediates interactions with these proteins.²²

1.3 BRD8 is a coactivator for nuclear hormone receptors

Enhancement of the transcriptional activity of the TR was the first reported function of BRD8.¹⁰ In addition, BRD8 acts as a co-activator for other nuclear receptors including androgen receptor (AR),^{10, 23, 24} progesterone receptor,²⁴ estrogen receptor,²⁴ glucocorticoid receptor,²³ and retinoid X receptor (RXR)/peroxisome proliferator-activated receptor γ (PPARγ).^{25, 26} The BRD8 protein contains multiple short sequence motifs “LXXLL” (amino acids 271–275 and 340–344 in BRD8 isoform 1) that is necessary for binding nuclear receptors.²⁷ Consistent with these data, a variant of BRD8 lacking the N-terminal LXXLL motif (p120β) lost ligand-inducible enhancement by TR, PPARγ, and RXR, while the variant retained the ability of AR transcriptional activity.²³ This may imply that the mechanism by which BRD8 functions as an AR coactivator may differ from other nuclear hormone receptors. BRD8 has also been reported to play an important role in the regulation of PPARγ target genes during adipogenesis through the recruitment of chromatin-modifying complex including EP400 and TIP60.²⁵

1.4 BRD8 is part of the multiprotein complex of NuA4/Tip60

Previous proteomic analysis revealed that BRD8 is a member of the human NuA4/TIP60 histone acetyltransferase complex, suggesting that BRD8 is involved in transcriptional regulation in concert with this complex (Figure 2).²⁸ NuA4/TIP60 is an evolutionarily conserved and multifunctional histone acetyltransferase complex that to date consists of at least 20 subunits (Table 1).^{15, 16, 22, 28-35} The catalytic subunit TIP60 (KAT5: lysine acetyltransferase 5) acetylates nonhistone proteins as well as histones H4, H2A, and H2A variants.^36-39 EP400, originally identified as an interacting partner of the adenovirus E1A protein,⁴⁰ is a functional subunit with ATP-dependent chromatin remodeling activity, allowing the replacement of conventional histone H2A with variant histone H2AZ.³⁴ This complex is well-known to be recruited to DNA damage sites and protect the cells against damage.

Unexpectedly, yeast NuA4 likely lacks Bdf1, the yeast counterpart of human BRD8.¹⁶ However, deletion of yeast Bdf1 was lethal in combination with a mutant allele of Esa1, the yeast counterpart of human TIP60, suggesting that the presence of human BRD8 in the NuA4/TIP60 complex may reflect the functional interaction between Bdf1 and Esa1 in yeast.¹⁸ Consistent with their observation in yeast, BRD8 facilitated acetylation of histone H4K16 where it is catalyzed by TIP60 at DNA damage sites.^{41, 42} In addition, BRD8 mediated the incorporation of H2AZ by EP400 and the subsequent induction of PPARγ target genes during adipogenesis.²⁵ Taken together, BRD8 may play an important role in transcription and DNA repair as a subunit of the NuA4/TIP60 complex.

1.5 BRD8 interacts with EP400NL to form a nuclear complex distinct from the NuA4/TIP60 complex

We recently reported that BRD8 regulates the expression of multiple subunits of the prereplicative complex, such as minichromosome maintenance genes (MCMs) and origin recognition complex genes (ORCs), independently of TIP60.¹¹ This can be explained by the formation of an independent functional complex. Indeed, a recent interactome analysis indicated that BRD8 interacts directly with EP400NL to form a tetrameric protein complex named TINTIN (Trimer independent of NuA4 for transcription interactions with nucleosomes) (Figure 2, Table 1).²² The EP400NL gene is located adjacent to EP400 and encodes a 51.8 kDa protein that shares a similar N-terminal sequence with the large EP400 protein (343.5 kDa). Another group also showed that EP400NL induced PD-L1 expression in the presence of serum- and γ-interferon-stimulation, suggesting a possible link between this complex and tumor immune evasion.³⁵ Interestingly, BRG1, a core ATPase subunit of the human SWI/SNF complex, was found in the EP400NL-associated complex instead of EP400, suggesting that this complex also has chromatin remodeling activity.³⁵ Although the components of the EP400NL-associated complex are not fully understood, this complex can control gene expression in the absence of histone acetyltransferase activity.³⁵ Collectively, BRD8 may govern gene expression in concert with different complexes.

1.6 Alterations of BRD8 in cancer tissues

It has been reported that a mutation in BRD8 was identified in one of four microphthalmia-associated transcription factor (MITF) family translocation renal cell cancers by whole exome sequencing, and the patient showed a partial response to ipilimumab, an immune checkpoint inhibitor.⁴³ However, BRD8 mutations are not frequently observed in urinary tract cancer (45 of 805 patients in the COSMIC database) or bladder urothelial cancer (13 of 411 patients in The Cancer Genome Atlas [TCGA] PanCancer Atlas dataset).

A rearrangement of BRD8 with the PHD finger protein 1 (PHF1) gene has been reported in low-grade endometrial stromal sarcoma (ESS).⁴⁴ This rearrangement is expected to lead to an in-frame fusion between exon 16 of BRD8 (NM_006696) and exon 2 of PHF1 (NM_002636), whose transcript preserves the entire coding sequence of PHF1 but lacks the bromodomain from BRD8 (Figure 3A). PHF1 encodes a polycomb-like protein that mediates recruitment of polycomb repressive complex 2 (PRC2) to chromatin, resulting in transcriptional repression of key developmental genes through H3K27 methylation. It is of note that rearrangements of PHF1 with members of the NuA4/TIP60 complex have also been identified in soft tissue sarcomas. Partners of the fusion genes include JAZF1,⁴⁵ EPC1,⁴⁵ MEAF6,⁴⁶ EPC2,⁴⁷ MBTD1,⁴⁸ ING3,⁴⁹ and EP400 (Figure 3B).⁵⁰ A recent study has shown that the EPC1-PHF1 fusion protein forms a megacomplex in which the NuA4/TIP60 and PRC2 complexes are bound.⁵¹ This aberrant complex leads to mislocalized acetylation by TIP60, resulting in the dysregulation of gene expression, particularly in the increased expression of PRC2 target genes.

In addition to the BRD8-PHF1 fusion, its reciprocal fusion form (exon 1 of PHF1 and exon 9 of BRD8) was also found in ossifying low-grade ESS.⁵² Notably, the predicted protein retains the C-terminal region of BRD8 containing the bromodomain. In addition, the expression of this C-terminal BRD8 was probably governed by the PHF1 promoter and was increased in tumor cells compared to the control endometrial stromal cells. The significant oncogenic function of these fusion proteins is still unknown, but given the characteristics of its partners, it might impair DNA damage response, cell cycle, and DNA replication through epigenetic dysregulation.^{11, 12, 41}

Although genetic alterations in BRD8 are rare, increased expression of BRD8 has been found in several tumors including colorectal cancer and glioblastoma.^{11, 12} According to TCGA data, BRD8 transcripts are highly expressed in cholangiocarcinoma (CHOL), thymoma (THYM), and pheochromocytoma and paraganglioma (PCPG) compared to their matched normal tissues (GEPIA, http://gepia.cancer-pku.cn/). Posttranscriptional modification is also involved in the regulation of BRD8 expression. As the expression of BRD8 was enhanced by the treatment with MG132, a proteasome inhibitor, BRD8 protein is thought to undergo proteasomal degradation through its ubiquitination. Indeed, a total of 31 ubiquitination sites were identified inside and outside of the bromodomain.¹¹ In colorectal cancer cells, BRD8 accumulates through the inhibition of ubiquitin-dependent protein degradation by the interaction with MRGBP. In addition, the isoform-specific regulation by microRNA has been reported. BRD8 isoform 2 is specifically upregulated through the loss of microRNA-185 in prostate cancer, which leads to the activation of AR-mediated transcription.^{23, 53}

1.7 Dysregulation of BRD8 is associated with tumorigenesis

The first report linking BRD8 to carcinogenesis identified that accumulation of BRD8 was associated with resistance to spindle poisons in metastatic colon cancer cell lines and advanced rat colon carcinomas.⁵⁴ Although the growth-promoting effect of BRD8 isoform 2 in prostate cancer may be mediated through activation of AR-mediated transcription,^{23, 53} the molecular mechanisms by which BRD8 is associated with cancer progression were largely unknown. Recently, integrated analyses of genome-wide mapping of BRD8-binding sites and BRD8-regulated genes revealed direct BRD8 target genes in human lung⁵⁵ and colorectal cancer cells.¹¹ In lung epithelial cells, BRD8 affected genes essential for the cell cycle, antimicrobial proteins, and chemokines, suggesting an involvement of BRD8 in cell proliferation and immune responses in the mucosal barrier in the airway epithelium.⁵⁵ In colorectal cancer cells, genes regulated by BRD8 were significantly associated with DNA replication and the cell cycle.¹¹ In glioblastoma, BRD8 blocked the binding of WT p53 to chromatin, thereby inhibiting p53-mediated transactivation.¹² In line with the data, knockdown of BRD8 increased the expression of p53 target genes such as p21, Puma, and TIGAR in colorectal cancer, which induced apoptosis and cell cycle arrest.⁴¹ However, the involvement of BRD8 in the regulation of p53 expression remains controversial.¹¹ Furthermore, BRD8 mediated the interaction between TWIST1 and the TIP60 complex, and recruited the TIP60 complex to the TWIST1 target loci, thereby promoting epithelial–mesenchymal transition, proliferation, and metastasis of breast cancer.¹³ Given that the bromodomain of BRD8 is responsible for the recruitment, interaction, and gene expression, it may be a promising therapeutic target for patients with glioblastoma and colorectal, prostate, and breast cancer.