SUMMARY Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt's lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.

The E2 protein encoded by human papillomaviruses (HPVs) inhibits expression of the viral E6 oncoprotein, which, in turn, regulates p53 target gene transcription. To identify cellular proteins involved in E2-mediated transcriptional repression, we isolated an E2 complex from human cells conditionally expressing HPV-11 E2. Surprisingly, the double bromodomain-containing protein Brd4, which is implicated in cell cycle control and viral genome segregation, was found associated with E2 and conferred on E2 the ability to inhibit AP-1-dependent HPV chromatin transcription in an E2-binding site- [Keywords: HPV; E2; AP-1; Brd4; chromatin transcription; gene silencing] Supplemental material is available at http://www.genesdev.org.

Transcription factor TFIID is a multiprotein complex composed of a TATA-box-binding subunit, TBP, and several tightly associated factors (TAFs). Human TFIID-promoter interactions are restricted to the TATA-box region on most core promoters but extend over a large promoter region downstream of the TATA box and the transcription start site on the Ad2ML promoter. TFIID downstream interactions are thought to be functionally relevant because they can be induced by transcriptional activators, which in some cases requires TFIIA, result in stabilization of the TFIID-promoter complex, and correlate with increased recruitment of the remaining general transcription factors. Here we examine the topological organization of human TFIID complexes bound to the Ad2ML promoter. Our data provide insight into the relative disposition of DNA and several TFIID subunits, as well as evidence for DNA wrapping by TFIID, and suggest a direct role of TFIIA in the stable positioning of promoter DNA relative to TAFs.

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

Bromodomain-containing protein 4 (Brd4) contains two tandem bromodomains (BD1 and BD2) that bind preferentially to acetylated lysine residues found in histones and nonhistone proteins. This molecular recognition allows Brd4 to associate with acetylated chromatin throughout the cell cycle and regulates transcription at targeted loci. Recruitment of positive transcription elongation factor b, and possibly the general initiation cofactor Mediator as well, plays an important role in Brd4-regulated transcription. Selective contacts with acetyl-lysines in nucleosomal histones and chromatin-binding factors likely provide a molecular switch modulating the steps from chromatin targeting to transcriptional regulation, thus further expanding the ‘acetylation code’ for combinatorial regulation in eukaryotes.

SUMMARY Post-translational modification can modulate protein conformation and alter binding partner recruitment within promoter regulatory regions. Here, we find that bromodomain-containing protein 4 (BRD4), a transcription cofactor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV) to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NFκB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and also by relocalization of NFκB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is also critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and cofactor recruitment.

Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5 end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5 ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5 end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3-end addition to the template. These new findings support the interpretation that the 5 end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.The two major species of RNA that accumulate during hepatitis delta virus (HDV) replication are the unit-length genome and its exact complement, the antigenome (Fig. 1, panels 1 and 2) (2). These 1,679-nucleotide (nt) RNAs are detected in both circular and linear forms. In addition, there are relatively smaller amounts of an 800-nt polyadenylated RNA of antigenomic polarity (Fig. 1, panel 3). This putative mRNA contains the open reading frame for the small delta protein, a 195-amino-acid species that is essential for HDV genome replication (10). All other proteins needed for HDV genome replication are provided by the host cell. This means that somehow a host RNA polymerase that is normally DNA dependent has to be redirected to act on HDV RNA as template.One approach to clarify the mechanism of HDV genome replication with its unique RNA-directed RNA synthesis and processing has been to adopt the rationale that a detailed characterization of the mRNA species will provide valuable insights. Previous studies include (i) Northern analyses (2), (ii) primer extension (9), (iii) ribonuclease protection assays (27), and (iv) 5Ј rapid amplification of cDNA ends (5Ј-RACE) (6). By primer extension, a 5Ј end was first mapped to position 1631 Ϯ 1 of the 1,679-nt RNA sequence (9). A more recent study using 5Ј-RACE indi...

Brd4 is a chromatin adaptor containing tandem bromodomains binding to acetylated histone H3 and H4. Although Brd4 has been implicated in the transcriptional control of papillomavirus-encoded E2 protein, it is unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Using DNase I footprinting performed with in vitro reconstituted human papillomavirus (HPV) chromatin and nucleosome-free DNA templates, we found that Brd4 facilitates E2 binding to its cognate sequences in chromatin depending on bromodomains and the E2-interacting region of Brd4. Moreover, the coactivator and corepressor function of Brd4 requires at least one intact bromodomain and is mediated by its direct association with E2 proteins encoded by cancer-inducing high risk HPV-16 and HPV-18, wart-causing low risk HPV-11, and bovine papillomavirus type 1, in part through enhancing the protein stability of E2 that is normally degraded via the ubiquitindependent proteasome pathway. Our findings indicate that a chromatin adaptor can bridge and enhance the binding of a sequence-specific transcription factor to chromatin and further promote the stability of a labile transcription factor via direct protein-protein interaction. E2 encoded by human papillomaviruses (HPVs)2 is a multifunctional protein regulating viral DNA replication, genome segregation, transcription, cell cycle control, and senescence (1). Its primary function relies on the sequence-specific recognition of a 12-nucleotide palindrome, ACCN 6 GGT, located at the upstream regulatory region of HPVs and animal papillomaviruses, such as bovine papillomavirus type 1 (BPV-1). The sequence context and the location of E2-binding sites (E2BSs) as well as the nature of E2 proteins all contribute to E2 activity in viral gene regulation. The transcriptional activity of E2 appears to be commonly shared by different types of E2 proteins encoded, for example, by cervical cancer-inducing HPV types 16 (HPV-16) and 18 (HPV-18), genital wart-associated HPV type 11 (HPV-11), and BPV-1. In general, HPV-16 E2 (16E2) exhibits strongest transcriptional activity, followed by BPV-1 E2 (BE2), HPV-18 E2 (18E2), and HPV-11 E2 (11E2), and correlates well with their corresponding binding affinities to E2BSs derived from the promoter-proximal regions of naturally occurring HPV-11, HPV-16, and HPV-18 sequences (2). Because two of the four E2BSs in genital HPVs are flanked by an upstream Sp1-binding site and the downstream TATA box of the E6 early promoter, HPV E2 typically functions as a transcriptional repressor by excluding Sp1 and TFIID/TBP from binding to their cognate sequences and thus prevents the assembly of a transcriptional preinitiation complex (3-7). HPV E2 also exhibits transactivation activity functioning in a heterologous promoter context where multimerized E2BSs are situated away from the TATA box (8), as seen in several natural BPV-1 promoter regions (9, 10). Like many other cellular transcription factors, E2...