Alternative Splicing Products of the Gene for a Human Nuclear Actin-related Protein,hArpNβ/Baf53, that Encode a Protein Isoform,hArpNβS,in the Cytoplasm

A human nuclear actin-related protein, hArpNβ/Baf53, is a component of chromatin remodeling and histone acetyltransferase complexes. We identified two alternative splicing products of the gene for hArpNβ/Baf53. They encoded a protein isoform, hArpNβS; and its fusion product with green fluorescent protein was to be found in the cytoplasm, not the nucleus. The isoforms may contribute to functional regulation of these complexes.     

HIV-1 Tat directly binds to NFkappaB enhancer sequence: role in viral and cellular gene expression

HIV-1 Tat protein reprograms cellular gene expression of infected as well as uninfected cells apart from its primary function of transactivating HIV-1 long terminal repeat (LTR) promoter by binding to a nascent RNA stem–loop structure known as the transactivator response region (TAR). Tat also induces chromatin remodeling of proviral LTR-mediated gene expression by recruiting histone acetyl transferases to the chromatin, which results in histone acetylation. Furthermore several studies have shown convincing evidence that Tat can transactivate HIV-1 gene expression in the absence of TAR, the molecular mechanism of which remains to be elucidated. Here we show a direct interaction of Tat with nuclear factor kappa B (NFκB) enhancer, a global regulatory sequence for many cellular genes both in vitro and in vivo. This interaction not only provides a novel molecular basis to explain TAR-independent transactivation in HIV-1, but also points toward the potential mechanism of Tat- mediated modulation of cellular genes.     

A Novel Approach for Studying Histone H1 Function in Vivo

In this report, we investigate the mechanisms that regulate Drosophila histone H1 expression and its association with chromatin in vivo. We show that histone H1 is subject to negative autoregulation and exploit this result to examine the effects of mutations of the main phosphorylation site of histone H1.     

综述: 表观遗传之染色质重塑

真核细胞中的染色质重塑因子种类繁多,多数以蛋白多聚体的形式存在于细胞中．不同的染色质重塑因子在特定时间定位于特定的核小体上,通过改变染色质结构,影响基因转录活性,进而确保细胞内各种生物学过程的正确运行．另外,染色质重塑因子根据所含功能结构域的不同,大致分为SWI/SNF、ISWI、CHD和INO80四大家族,不同的染色质重塑因子之间既有蛋白质结构和酶活性的相似性,各自又有其特异性．本综述的宗旨在于全面概括和总结染色质重塑因子的分类、结构特点以及其在细胞内的生物学功能,为深入研究染色质重塑因子的生物学功能,尤其是在发育和疾病发生中的作用机制提供理论基础．     

RNA interference as a tool for exploring HIV-1 robustness

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected. After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

Background and Scope

In eukaryotes, chromatin remodelling complexes are shown to be responsible for nucleosome mobility, leading to increased accessibility of DNA for DNA binding proteins. Although the existence of such complexes in plants has been surmised mainly at the genetic level from bioinformatics studies and analysis of mutants, the biochemical existence of such complexes has remained unexplored.

Methods

Histone H1-depleted donor chromatin was prepared by micrococcal nuclease digestion of wheat nuclei and fractionation by exclusion chromatography. Nuclear extract was partially purified by cellulose phosphate ion exchange chromatography. Histone octamer trans-transfer activity was analysed using the synthetic nucleosome positioning sequence in the absence and presence of ATP and its analogues. ATPase activity was measured as ³²Pi released using liquid scintillation counting.

Key Results

ATP-dependent histone octamer trans-transfer activity, partially purified from wheat nuclei using cellulose phosphate, showed ATP-dependent octamer displacement in trans from the H1-depleted native donor chromatin of wheat to the labelled synthetic nucleosome positioning sequence. It also showed nucleosome-dependent ATPase activity. Substitution of ATP by ATP analogues, namely ATPγS, AMP-PNP and ADP abolished the octamer trans-transfer, indicating the requirement of ATP hydrolysis for this activity.

Conclusions

ATP-dependent histone octamer transfer in trans is a recognized activity of chromatin remodelling complexes required for chromatin structure dynamics in non-plant species. Our results suggested that wheat nuclei also possess a typical chromatin remodelling activity, similar to that in other eukaryotes. This is the first report on chromatin remodelling activity in vitro from plants.     

Unravelling the biology of chromatin in health and cancer using proteomic approaches

Introduction: Chromatin remodeling complexes play important roles in the control of genome regulation in both normal and diseased states, and are therefore critical components for the regulation of epigenetic states in cells. Given the role epigenetics plays in cancer, for example, chromatin remodeling complexes are routinely targeted for therapeutic intervention.

Areas covered: Protein mass spectrometry and proteomics are powerful technologies used to study and understand chromatin remodeling. While impressive progress has been made in this area, there remain significant challenges in the application of proteomic technologies to the study of chromatin remodeling. As parts of large multi-subunit complexes that can be heavily modified with dynamic post-translational modifications, challenges in the study of chromatin remodeling complexes include defining the content, determining the regulation, and studying the dynamics of the complexes under different cellular states.

Expert commentary: Impwortant considerations in the study of chromatin remodeling complexes include the complexity of sample preparation, the choice of proteomic methods for the analysis of samples, and data analysis challenges. Continued research in these three areas promise to yield even greater insights into the biology of chromatin remodeling and epigenetics and the dynamics of these systems in human health and cancer.     

Alternative splicing products of the gene for a human nuclear actin-related protein,hArpNbeta/Baf53, that encode a protein isoform,hArpNbetaS, in the cytoplasm

A human nuclear actin-related protein, hArpNbeta/ Baf53, is a component of chromatin remodeling and histone acetyltransferase complexes. We identified two alternative splicing products of the gene for hArpNbeta/ Baf53. They encoded a protein isoform, hArpNbetaS; and its fusion product with green fluorescent protein was to be found in the cytoplasm, not the nucleus. The isoforms may contribute to functional regulation of these complexes.     

Differential requirements for different subfamilies of the mammalian SWI/SNF chromatin remodeling enzymes in myoblast cell cycle progression and expression of the Pax7 regulator

The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) families of ATP-dependent chromatin remodeling enzymes are established co-regulators of gene expression. mSWI/SNF complexes can be assembled into three major subfamilies: BAF (BRG1 or BRM-Associated Factor), PBAF (Polybromo containing BAF), or ncBAF (non-canonical BAF) that are distinguished by the presence of mutually exclusive subunits. The mechanisms by which each subfamily contributes to the establishment or function of specific cell lineages are poorly understood. Here, we determined the contributions of the BAF, ncBAF, and PBAF complexes to myoblast proliferation via knock down (KD) of distinguishing subunits from each complex. KD of subunits unique to the BAF or the ncBAF complexes reduced myoblast proliferation rate, while KD of PBAF-specific subunits did not affect proliferation. RNA-seq from proliferating KD myoblasts targeting Baf250A (BAF complex), Brd9 (ncBAF complex), or Baf180 (PBAF complex) showed mis-regulation of a limited number of genes. KD of Baf250A specifically reduced the expression of Pax7, which is required for myoblast proliferation, concomitant with decreased binding of Baf250A to and impaired chromatin remodeling at the Pax7 gene promoter. Although Brd9 also bound to the Pax7 promoter, suggesting occupancy by the ncBAF complex, no changes were detected in Pax7 gene expression, Pax7 protein expression or chromatin remodeling at the Pax7 promoter upon Brd9 KD. The data indicate that the BAF subfamily of the mSWI/SNF enzymes is specifically required for myoblast proliferation via regulation of Pax7 expression.