14-3-3 Mediates Histone Cross-Talk during Transcription Elongation in Drosophila

Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation

Squamocin modulates histone H3 phosphorylation levels and induces G1 phase arrest and apoptosis in cancer cells

<p>Abstract</p> <p>Background</p> <p>Histone modifications in tumorigenesis are increasingly recognized as important epigenetic factors leading to cancer. Increased phosphorylation levels of histone H3 as a result of aurora B and pMSK1 overexpression were observed in various tumors. We selected <it>aurora B </it>and <it>MSK1 </it>as representatives for testing various compounds and drugs, and found that squamocin, a bis-tetrahydrofuran annonaceous acetogenin, exerted a potent effect on histone H3 phosphorylation.</p> <p>Methods</p> <p>GBM8401, Huh-7, and SW620 cells were incubated with 15, 30, and 60 μM squamocin for 24 h. The expressions of mRNA and proteins were analyzed by qRT-PCR and Western blotting, respectively. The cell viability was determined by an MTT assay. Cell cycle distribution and apoptotic cells were analyzed by flow cytometry.</p> <p>Results</p> <p>Our results showed that squamocin inhibited the proliferation of GBM8401, Huh-7, and SW620 cells, arrested the cell cycle at the G₁phase, and activated both intrinsic and extrinsic pathways to apoptosis. In addition, we demonstrated that squamocin had the ability to modulate the phosphorylation levels of H3S10 (H3S10p) and H3S28 (H3S28p) in association with the downregulation of aurora B and pMSK1 expressions.</p> <p>Conclusions</p> <p>This study is the first to show that squamocin affects epigenetic alterations by modulating histone H3 phosphorylation at S10 and S28, providing a novel view of the antitumor mechanism of squamocin.</p

An Automated Phenotype-Driven Approach (GeneForce) for Refining Metabolic and Regulatory Models

Integrated constraint-based metabolic and regulatory models can accurately predict cellular growth phenotypes arising from genetic and environmental perturbations. Challenges in constructing such models involve the limited availability of information about transcription factor—gene target interactions and computational methods to quickly refine models based on additional datasets. In this study, we developed an algorithm, GeneForce, to identify incorrect regulatory rules and gene-protein-reaction associations in integrated metabolic and regulatory models. We applied the algorithm to refine integrated models of Escherichia coli and Salmonella typhimurium, and experimentally validated some of the algorithm's suggested refinements. The adjusted E. coli model showed improved accuracy (∼80.0%) for predicting growth phenotypes for 50,557 cases (knockout mutants tested for growth in different environmental conditions). In addition to identifying needed model corrections, the algorithm was used to identify native E. coli genes that, if over-expressed, would allow E. coli to grow in new environments. We envision that this approach will enable the rapid development and assessment of genome-scale metabolic and regulatory network models for less characterized organisms, as such models can be constructed from genome annotations and cis-regulatory network predictions

Recruitment and Activation of RSK2 by HIV-1 Tat

The transcriptional activity of the integrated HIV provirus is dependent on the chromatin organization of the viral promoter and the transactivator Tat. Tat recruits the cellular pTEFb complex and interacts with several chromatin-modifying enzymes, including the histone acetyltransferases p300 and PCAF. Here, we examined the interaction of Tat with activation-dependent histone kinases, including the p90 ribosomal S6 kinase 2 (RSK2). Dominant-negative RSK2 and treatment with a small-molecule inhibitor of RSK2 kinase activity inhibited the transcriptional activity of Tat, indicating that RSK2 is important for Tat function. Reconstitution of RSK2 in cells from subjects with a genetic defect in RSK2 expression (Coffin-Lowry syndrome) enhanced Tat transactivation. Tat interacted with RSK2 and activated RSK2 kinase activity in cells. Both properties were lost in a mutant Tat protein (F38A) that is deficient in HIV transactivation. Our data identify a novel reciprocal regulation of Tat and RSK2 function, which might serve to induce early changes in the chromatin organization of the HIV LTR

Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark

Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements

Stability of histone modifications across mammalian genomes: implications for 'epigenetic' marking.

The combination of chromatin immunoprecipitation (ChIP) with microarray analysis (ChIP-chip) or high-throughput sequencing (ChIP-seq and ChIP-SAGE) has provided maps of a wide variety of site-specific histone modifications across mammalian genomes in various cell types. Although distinct genomic regions and functional elements have been strongly associated with specific histone modifications, an overwhelming number of combinatorial patterns have also been observed across the genome. While peaks of enrichment in ChIP-chip and ChIP-seq data may suggest stable and predictive 'landmarks' across the genomic landscape, studies from transcribed genes indicate a more dynamic model of how these data may be interpreted. In light of such studies, which show highly dynamic methylation, acetylation and phosphorylation of histone H3 during gene transcription, we consider the extent to which genome-wide maps of chromatin state could be interpreted as 'snapshots' of heterogeneous profiles deriving from dynamic modification processes. Rather than acting as static 'epigenetic' landmarks, histone modifications may function as dynamic and transient operational marks supporting specific steps in diverse processes throughout the mammalian genome

Stress-activated MAP kinases in chromatin and transcriptional complexes

Stress-activated MAP kinases (SAPKs) are activated by stressors or by certain physiological stimuli and mediate an intracellular response appropriate to the change in environment. Long-term adaptation requires reprogramming of transcription and one of the most significant actions of SAPK cascades is therefore induction of gene expression. SAPKs and their downstream kinases phosphorylate many chromatin-associated and transcription factors. Further, they can induce localised histone modification by regulating histone acetyltransferases and deacetylases. p38/SAPK2 also elicits phosphorylation of the nucleosomal proteins histone H3 and HMGN1 (previously HMG-14) via the downstream mitogen- and stress-stimulated kinases MSK1/2. Finally, recent evidence indicates a novel non-enzymatic SAPK function in transcriptional complexes, suggesting a more structural role. The yeast SAPK Hog1p is recruited to a proportion of its target genes on activation and localises beyond the promoter into coding regions. The observation that Hog1p interacts with elongating RNA polymerase II in addition to several transcriptional elongation factors has led to the suggestion that this SAPK may behave like an elongation factor at some target genes. The generality of this new function is discussed. © 2007 Springer-Verlag Berlin Heidelberg

Signalling to chromatin through post-translational modifications of HMGN.

The DNA of eukaryotic genomes is highly packaged by its organisation into chromatin, the fundamental repeating unit of which is the nucleosome core particle, consisting of 147 base pairs of DNA wrapped around an octamer of two copies each of the four core histone proteins H2A, H2B, H3 and H4 (K. Luger, A.W. Mader, R.K. Richmond, D.F. Sargent, T.J. Richmond, Crystal structure of the nucleosome core particle at 2.8 A resolution, Nature 389 (1997) 251-260 [1] and references therein). Accessibility of DNA within chromatin is a central factor that affects DNA-dependent nuclear function such as transcription, replication, recombination and repair. To integrate complex signalling networks associated with these events, many protein and multi-protein complexes associate transiently with nucleosomes. One class of such are the High-Mobility Group (HMG) proteins which are architectural DNA and nucleosome-binding proteins that may be subdivided into three families; HMGA (HMGI/Y/C), HMGB (HMG1/2) and HMGN (HMG14/17). The structure of chromatin and nucleosomes can be altered, both locally and globally, by interaction with such architectural proteins thereby influencing accessibility of DNA. This chapter deals with the HMGN protein family, specifically their post-translational modification as part of regulatory networks. We focus particularly on HMGN1, the most extensively studied family member to date, and to a lesser extent on HMGN2. We critically evaluate evidence for the role of post-translational modification of these proteins in response to different signals, exploring the sites and potential significance of such modification