Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes.

Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are cotranscriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their cotranscriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing cotranscriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is important for nucleosome eviction.

The multifunctional protein nucleophosmin (NPM1) is a human linker histone H1 chaperone.

Linker histone H1 plays an essential role in chromatin organization. Proper deposition of linker histone H1 as well as its removal is essential for chromatin dynamics and function. Linker histone chaperones perform this important task during chromatin assembly and other DNA-templated phenomena in the cell. Our in vitro data show that the multifunctional histone chaperone NPM1 interacts with linker histone H1 through its first acidic stretch (residues 120-132). Association of NPM1 with linker histone H1 was also observed in cells in culture. NPM1 exhibited remarkable linker histone H1 chaperone activity, as it was able to efficiently deposit histone H1 onto dinucleosomal templates. Overexpression of NPM1 reduced the histone H1 occupancy on the chromatinized template of HIV-1 LTR in TZM-bl cells, which led to enhanced Tat-mediated transactivation. These data identify NPM1 as an important member of the linker histone chaperone family in humans.

Histone chaperone as coactivator of chromatin transcription: role of acetylation.

Histone chaperones are a group of histone-interacting proteins, involved in several important cellular functions. These chaperones are essential to facilitate ordered assembly of nucleosomes, both in replication dependent and independent manner. Replication independent function of histone chaperone is necessary for histone eviction during transcriptional initiation and elongation. In this chapter we have discussed a method to evaluate the role of histone chaperone NPM1 (the only known chaperone to get acetylated with functional consequence) in the transcriptional activation which is acetylation dependent.

Methods to study histone chaperone function in nucleosome assembly and chromatin transcription.

Histone chaperones are histone interacting proteins that are involved in various stages of histone metabolism in the cell such as histone storage, transport, nucleosome assembly and disassembly. Histone assembly and disassembly are essential processes in certain DNA-templated phenomena such as replication, repair and transcription in eukaryotes. Since the first histone chaperone Nucleoplasmin was discovered in Xenopus, a plethora of histone chaperones have been identified, characterized and their functional significance elucidated in the last 35 years or so. Some of the histone chaperone containing complexes such as FACT have been described to play a significant role in nucleosome disassembly during transcription elongation. We have reported earlier that human Nucleophosmin (NPM1), a histone chaperone belonging to the Nucleoplasmin family, is a co-activator of transcription. In this chapter, we describe several methods that are used to study the histone chaperone activity of proteins and their role in transcription.

Differential expression of TLR-2 and TLR-4 in the epithelial cells in oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory condition of the mucosa mediated by a complex signalling network between the keratinocytes and the sub-epithelial lymphocytes. Since OLP occurs in constantly renewing epithelium continuously exposed to commensals, we hypothesised that the epithelial cell microflora interactions may mediate the persistent inflammation. By virtue of their ability to respond to most oral commensal microorganisms, the toll like receptor-2 (TLR-2) and TLR-4 are the most widely investigated receptors in oral diseases. The overall objective of this study was to investigate the role of TLR-2 and TLR-4 in OLP. DESIGN: Systemically healthy OLP and control subjects were recruited after obtaining the institutional review board approval. Expression of TLR-2 and TLR-4 proteins and transcripts in the tissue epithelium and in the epithelial cells isolated from saliva were determined by immunohistochemistry and quantitative real-time polymerase chain reaction respectively. RESULTS: The tissue epithelium and the salivary epithelial cells expressed reduced TLR-2 and increased TLR-4 proteins and transcripts in OLP. The salivary epithelial cells from OLP subjects secreted elevated IL-12. However, upon stimulation with bacterial lipopolysaccharide the epithelial cells from OLP exhibited a mixed Th1 (IL-12) and Th2 (IL-4) response. Presence of dexamethasone significantly reduced inflammatory cytokines in the in vitro stimulated cultures of salivary epithelial cells from OLP subjects. CONCLUSION: Collectively, our data support a critical role for the host-microbial interactions in the OLP pathogenesis. The potential use of exfoliated oral epithelial cells in saliva for functional analysis exponentially increases its value as biological specimen for clinical research.

Acetylated NPM1 localizes in the nucleoplasm and regulates transcriptional activation of genes implicated in oral cancer manifestation.

Nucleophosmin (NPM1) is a multifunctional protein involved in the regulation of centrosome duplication, ribosome biogenesis, genomic stability, histone chaperone function, and transcription. Overexpression of NPM1 is associated with cancers of diverse histological origins. Here, we have found that p300-mediated acetylation of NPM1 modulates its subcellular localization and augments its oncogenic potential. Acetylated NPM1 is predominantly localized in the nucleoplasm, where it associates with transcriptionally active RNA polymerase II. Deacetylation of NPM1 is brought about by human SIRT1 and reduces its transcriptional activation potential. Remarkably, increased levels of acetylated NPM1 were found in grade II and III oral squamous cell carcinoma (OSCC) patient samples. Small interfering RNA (siRNA)-mediated knockdown of NPM1 in an OSCC cell line, followed by microarray analysis and chromatin immunoprecipitation experiments, revealed that some of the genes involved in oral cancer malignancy are regulated by NPM1 and have acetylated NPM1 localized at their promoters. Either suppression of p300 by siRNA or mutation of acetylatable lysine residues of NPM1 resulted in reduced occupancy of acetylated NPM1 on the target gene promoter concomitant with its decreased transcript levels. These observations suggest that acetylated NPM1 transcriptionally regulates genes involved in cell survival and proliferation during carcinogenesis.

Curcumin, a novel p300/CREB-binding protein-specific inhibitor of acetyltransferase, represses the acetylation of histone/nonhistone proteins and histone acetyltransferase-dependent chromatin transcription.

Acetylation of histones and non-histone proteins is an important post-translational modification involved in the regulation of gene expression in eukaryotes and all viral DNA that integrates into the human genome (e.g. the human immunodeficiency virus). Dysfunction of histone acetyltransferases (HATs) is often associated with the manifestation of several diseases. In this respect, HATs are the new potential targets for the design of therapeutics. In this study, we report that curcumin (diferuloylmethane), a major curcumanoid in the spice turmeric, is a specific inhibitor of the p300/CREB-binding protein (CBP) HAT activity but not of p300/CBP-associated factor, in vitro and in vivo. Furthermore, curcumin could also inhibit the p300-mediated acetylation of p53 in vivo. It specifically represses the p300/CBP HAT activity-dependent transcriptional activation from chromatin but not a DNA template. It is significant that curcumin could inhibit the acetylation of HIV-Tat protein in vitro by p300 as well as proliferation of the virus, as revealed by the repression in syncytia formation upon curcumin treatment in SupT1 cells. Thus, non-toxic curcumin, which targets p300/CBP, may serve as a lead compound in combinatorial HIV therapeutics.