Moon Kyoo Jang

Selective Recognition of Acetylated Histones by Bromodomain Proteins Visualized in Living Cells

Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, we show that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacted with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone was essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persisted on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions.

The Bromodomain Protein Brd4 Stimulates G1 Gene Transcription and Promotes Progression to S Phase

Brd4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. Brd4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of Brd4 in cell growth and transcription using a small hairpin (sh) RNA approach. The Brd4 shRNA vector stably knocked down Brd4 protein expression by ∼90% in NIH3T3 cells and mouse embryonic fibroblasts. Brd4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, Brd4 knockdown cells were arrested at G1, whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G1 in control cells, many of these G1 genes were not up-regulated in Brd4 knockdown cells. Reintroduction of Brd4 rescued expression of these G1 genes in Brd4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that Brd4 was recruited to the promoters of these G1 genes during G0-G1 progression. Furthermore, Brd4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. Brd4 recruitment was low to absent at genes not affected by Brd4 shRNA. The results indicate that Brd4 stimulates G1 gene expression by binding to multiple G1 gene promoters in a cell cycle-dependent manner.

Cutting Edge: Autoantigen Ro52 Is an Interferon Inducible E3 Ligase That Ubiquitinates IRF-8 and Enhances Cytokine Expression in Macrophages

IFN regulatory factor (IRF)-8 is a transcription factor important for the development and function of macrophages. It plays a critical role in the induction of cytokine genes, including IL-12p40. Immunopurification and mass spectrometry analysis found that IRF-8 interacted with Ro52 in murine macrophages upon IFN-γ and TLR stimulation. Ro52 is an IFN-inducible protein of the tripartite motif (TRIM) family and is an autoantigen present in patients with Sjögren’s syndrome and systemic lupus erythematosus. Ro52 has a RING motif and is capable of ubiquitinating itself. We show that IRF-8 is ubiquitinated by Ro52 both in vivo and in vitro. Ectopic expression of Ro52 enhanced IL-12p40 expression in IFN-γ/TLR-stimulated macrophages in an IRF-8-dependent manner. Together, Ro52 is an E3 ligase for IRF-8 that acts in a non-degradation pathway of ubiquitination, and contributes to the elicitation of innate immunity in macrophages.

Structural Basis for Acetylated Histone H4 Recognition by the Human BRD2 Bromodomain.

Recognition of acetylated chromatin by the bromodomains and extra-terminal domain (BET) family proteins is a hallmark for transcriptional activation and anchoring viral genomes to mitotic chromosomes of the host. One of the BET family proteins BRD2 interacts with acetylated chromatin during mitosis and leads to transcriptional activation in culture cells. Here, we report the crystal structures of the N-terminal bromodomain of human BRD2 (BRD2-BD1; residues 74–194) in complex with each of three different Lys-12-acetylated H4 peptides. The BRD2-BD1 recognizes the H4 tail acetylated at Lys-12 (H4K12ac), whereas the side chain of hypoacetylated Lys-8 of H4 binds at the cavity of the dimer interface of BRD2-BD1. From binding studies, we identified the BRD2-BD1 residues that are responsible for recognition of the Lys-12-acetylated H4 tail. In addition, mutation to Lys-8 in the Lys-12-acetylated H4 tail decreased the binding to BRD2-BD1, implicating the critical role of Lys-8 in the Lys-12-acetylated H4 tail for the recognition by BRD2-BD1. Our findings provide a structural basis for deciphering the histone code by the BET bromodomain through the binding with a long segment of the histone H4 tail, which presumably prevents erasure of the histone code during the cell cycle.

Crystal Structure of the Human BRD2 Bromodomain INSIGHTS INTO DIMERIZATION AND RECOGNITION OF ACETYLATED HISTONE H4

The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators. One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74-194) of human BRD2. Strikingly, the BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail.

Papillomavirus Genomes Associate with BRD4 to Replicate at Fragile Sites in the Host Genome

It has long been recognized that oncogenic viruses often integrate close to common fragile sites. The papillomavirus E2 protein, in complex with BRD4, tethers the viral genome to host chromatin to ensure persistent replication. Here, we map these targets to a number of large regions of the human genome and name them Persistent E2 and BRD4-Broad Localized Enrichments of Chromatin or PEB-BLOCs. PEB-BLOCs frequently contain deletions, have increased rates of asynchronous DNA replication, and are associated with many known common fragile sites. Cell specific fragile sites were mapped in human C-33 cervical cells by FANCD2 ChIP-chip, confirming the association with PEB-BLOCs. HPV-infected cells amplify viral DNA in nuclear replication foci and we show that these form adjacent to PEB-BLOCs. We propose that HPV replication, which hijacks host DNA damage responses, occurs adjacent to highly susceptible fragile sites, greatly increasing the chances of integration here, as is found in HPV-associated cancers.

Phosphorylation of NF-κB by calmodulin-dependent kinase IV activates anti-apoptotic gene expression

We previously presented that calmodulin-dependent kinase IV (CaMKIV) mutually interacts with NF-kappa B and phosphorylates it directly, inducing the increased transcriptional regulation dependent on NF-kappa B target genes [J. Biol. Chem. 276 (2001) 20005]. Here, we show that Ser(535) residue is phosphorylated by CaMKIV. S535A mutant of p65 was specifically defective in transactivation of NF-kappa B target gene expression induced by CaMKIV. While coexpression of active CaMKIV with wild-type p65 led to a recovery from etoposide-induced apoptosis and an increase of Bcl-2 protein in cells, cells expressing S535A mutant did not. Taken together these results suggest that phosphorylated NF-kappa B p65 on Ser(535) by CaMKIV increases NF-kappa B target gene expression, including anti-apoptotic gene, hence leading to inhibition of apoptosis.

Brd4 is displaced from HPV replication factories as they expand and amplify viral DNA.

Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.

Papillomavirus E2 Proteins and the Host Brd4 Protein Associate with Transcriptionally Active Cellular Chromatin

ABSTRACT The interaction of papillomavirus E2 proteins with cellular Brd4 protein is important for transcriptional regulation of viral genes and partitioning of viral genomes. Bovine papillomavirus type 1 (BPV-1) E2 binds cellular chromatin in complex with Brd4 in both mitotic and interphase cells. To identify specific sites of E2 interaction on cellular chromatin, a genome-wide chromatin immunoprecipitation-on-chip analysis was carried out using human promoter sequences. Both E2 and Brd4 were found bound to most transcriptionally active promoters in C33A cells. These promoters were also bound by RNA polymerase II and were modified by histone H3 acetylation and K4 trimethylation, all indicators of active transcription. E2 binding strongly correlated with Brd4 and RNA polymerase II occupancy and H3K4me3 modification at all human promoters, indicating that E2 bound to active promoters. E2 binding did not correlate with the presence of consensus E2 binding sites in the promoters. Furthermore, the mRNA levels of E2-bound cellular genes were not significantly changed by E2 expression. Thus, the papillomavirus E2 proteins bind to transcriptionally active cellular genes but do not change their activity. We propose that this may be a way for the virus to ensure that the viral genome is retained in transcriptionally active regions of the nucleus to escape silencing. Therefore, E2-mediated tethering of viral genomes to host chromatin has multiple roles: to partition the viral genome to daughter cells, to ensure that the genomes are retained in the nucleus, and to make certain that the genomes are retained in functionally active nuclear domains.

The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional.

IntroductionWe previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research.MethodsIn this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression, proliferation, behaviour and lifespan in the presence and absence of Y-27632.ResultsWe demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely, keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification.ConclusionsROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research, as well as for regenerative and personalized medicine.