Role of the SWI/SNF Chromatin Remodeling Complex in Regulation of Inflammation Gene Expression

Molecular Biology - The process of inflammation is the body’s natural defense response to the penetration of foreign substances and molecules from the outside. Many proteins, signaling...     

绝缘子调控基因的表达

绝缘子在调控真核基因时空特异表达的过程中起着至关重要的作用.它的主要功能是增强子阻断和异染色质屏障.已经有竞争、阻断和成环等模型描述其增强子阻断功能;而它的异染色质屏障功能主要是通过影响染色质组蛋白的翻译后修饰来实现.已经确定的绝缘子包括果蝇基因组中的染色质特化结构(specialized chromatin structures, scs)和scs、gypsy、鸡珠蛋白β基因座上游的DNaseⅠ高敏感位点cHS4以及小鼠或人Igf2/H19基因座上的印记控制区(imprinting control region, ICR)和DNA甲基化区域(DNA methylated regions, DMR)元件等.许多转录因子参与绝缘子的基因调控作用,例如脊椎动物中的CCCTC结合因子(CCCTC binding factor,CTCF).利用基因组学和生物信息学等方法,还可以在基因组中发现新的绝缘子元件.     

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host''s chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.     

Dual Roles of the Mammalian GARP Complex in Tethering and SNARE Complex Assembly at the trans-Golgi Network

Tethering factors and SNAREs control the last two steps of vesicular trafficking: the initial interaction and the fusion, respectively, of transport vesicles with target membranes. The Golgi-associated retrograde protein (GARP) complex regulates retrograde transport from endosomes to the trans-Golgi network (TGN). Although GARP has been proposed to function as a tethering factor at the TGN, direct evidence for such a role is still lacking. Herein we report novel and specific interactions of the mammalian GARP complex with SNAREs that participate in endosome-to-TGN transport, namely, syntaxin 6, syntaxin 16, and Vamp4. These interactions depend on the N-terminal regions of Vps53 and Vps54 and the SNARE motif of the SNAREs. We show that GARP functions upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. However, interactions of GARP with SNAREs are insufficient to promote retrograde transport, because deletion of the C-terminal region of Vps53 precludes GARP function without affecting GARP-SNARE interactions. Finally, we present in vitro data consistent with a tethering role for GARP, which is disrupted by deletion of the Vps53 C-terminal region. These findings indicate that GARP orchestrates retrograde transport from endosomes to the TGN by promoting vesicle tethering and assembly of SNARE complexes in consecutive, independent steps.Conveyance of cargo among organelles of the secretory and endosomal-lysosomal pathways is mediated by transport vesicles that bud from a donor compartment and fuse with an acceptor compartment in a specific and regulated manner (2, 25, 42). The accuracy and efficiency of vesicle fusion with the target compartment are provided by the concomitant actions of at least three protein families: tethers, small GTPases, and SNAREs. The general view is that a transport vesicle first finds its target organelle through interaction with tethering factors and then fuses with it through assembly of SNARE proteins while small GTPases of the Rab and Arl subfamilies orchestrate multiple steps of the overall process (1, 38, 44). The mechanistic details, however, are far from being completely understood and might vary depending on the transport pathway considered.Tethering represents the first step in the interaction between a transport vesicle and its target membrane and results in the formation of physical links between two membranes that are bound to fuse. Two types of tethering factor, long coiled-coil proteins (e.g., p115, GCC185, and GM-130) and multisubunit complexes (e.g., HOPS/Vps-C, exocyst, COG, and GARP/VFT) have been implicated in nearly all vesicular transport routes (19, 38), although their direct role in connecting two opposing membranes has been documented for only a few (7, 40). Fusion is triggered by the assembly of SNAREs on the transport vesicle (v-SNAREs) with their cognate SNAREs on the target membranes (t-SNAREs) to form a SNARE pin or SNARE complex (12, 35). SNARE complex assembly involves the formation of a four-helix bundle that drives fusion of the two lipid bilayers (10, 14). Small GTPases participate in the initial recruitment of tethering factors and other peripherally associated effectors to specific locations on membranes, as well as in the subsequent fusion events (21). For example, the long coiled-coil protein GCC185 binds different GTPases, Rab9 on transport vesicles through the middle part and Rab6 and Arl1 at the trans-Golgi network (TGN) through the C-terminal part, thereby facilitating the recognition and connection of both membrane-bound compartments (11, 33). Other coiled-coil tethers have the ability to bind several different Rabs through domains that are not required for Golgi apparatus targeting. This supports a general model for a tentacular Golgi complex in which coiled-coil proteins capture and retain Rab-containing vesicles (33).In addition to bringing together transport vesicles with target organelles, tethers may also regulate SNARE complex assembly, thus coordinating these two steps of vesicular transport. Several examples of tether-SNARE interactions have been reported, but no consensus for a mechanism of interaction or functional significance has yet emerged. For example, the HOPS complex associates with v- and t-SNARE complexes on Saccharomyces cerevisiae vacuoles both before and after fusion (37). Sec6p, a member of the exocyst complex, binds to the plasma membrane t-SNARE Sec9p, preventing its interaction with the cognate t-SNARE Sso1p (34). The COG complex binds the Golgi t-SNARE syntaxin 5 and enhances intra-Golgi SNARE complex stability (29). The long coiled-coil protein p115 also stimulates SNARE complex assembly (30).The Golgi-associated retrograde protein (GARP) complex, also named the Vps fifty-three (VFT) complex, together with COG and the exocyst, belongs to the quatrefoil family of multisubunit tethering complexes (43), a structurally diverse group of peripheral membrane protein assemblies. Defects in the GARP, COG, or exocyst complexes cause accumulation of untethered vesicles that are scattered throughout the cytoplasm and contain different cargo proteins (18, 20, 45, 47). Direct proof of a tethering function for the GARP complex is still lacking, although its inactivation leads to defects consistent with a prominent role in the fusion of endosome-derived transport intermediates with the TGN (4-6, 20, 31). The yeast GARP complex is composed of four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Mutations in any of these subunits impair the retrieval of the secretory vesicle v-SNARE Snc1p and the carboxypeptidase Y receptor, Vps10p, from endosomes (5, 23, 32). The mammalian GARP complex also comprises Vps52, Vps53, and Vps54 subunits, but no Vps51 subunit has been identified to date (13). Depletion of the mammalian GARP complex prevents the delivery of Shiga toxin B subunit and the retrieval of TGN-localized proteins, such as TGN46, from endosomes to the TGN (20). Moreover, GARP depletion blocks the recycling of the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the TGN, leading to missorting of the CI-MPR cargo, lysosomal hydrolases, into the extracellular space (20). The essential nature of mammalian GARP function in endosome-to-TGN transport is highlighted by the embryonic lethality of mice with ablation of the Vps54 subunit gene (27) and the motor neuron degeneration of Wobbler mice bearing a Vps54 hypomorphic mutation (27).In yeast, the GARP subunit Vps51p specifically binds to the conserved N-terminal regulatory domain of the t-SNARE Tlg1p (5, 32). This finding led to the proposal that GARP tethers endosome-derived vesicles through its interaction with Tlg1p. However, deletions or point mutations that eliminate the binding of Vps51p to Tlg1p do not show any functional phenotype in vivo (8). Binding of Tlg1p to Vps51p is thus not essential for GARP-mediated vesicle tethering. In this work, we set out to study the possible link between the mammalian GARP complex and SNAREs. We found that GARP specifically and directly interacts with SNAREs that participate in the endosome-to-TGN retrograde route (i.e., syntaxin 6 [Stx6], Stx16, and Vamp4). These interactions depend on the fusion-inducing SNARE “motif” of the SNAREs and the N-terminal regions of Vps53 and Vps54. Functional analyses place the GARP complex upstream of the SNAREs, regulating their localization and assembly into SNARE complexes. In addition, we demonstrate that the GARP complex has a vesicle tethering function independent of its interaction with the SNAREs.     

一种新的慢病毒--JDV的基因表达与调控

JDV是一种新近分离的慢病毒,与HIV,BIV有很高的同源性,引起牛急性传染病,有很高致病性和死亡率,本文介绍了JDV分子生物学研究方面的最新进展。     

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2-8-17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells.     

A Role of Polycomb Group Genes in the Regulation of Gap Gene Expression in Drosophila

Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.     

Preliminary Assessment of the Impact of MicroRNA-Mediated Regulation on Coding Sequence Evolution in Mammals

Despite prior claims to the contrary, several lines of evidence suggest that selection acts on synonymous mutations in mammals. What might be the mechanisms for such selection? Here I attempt to quantify the constraints on the evolution of the coding sequence resulting from regulation of mRNA by microRNAs (miRNAs) that antisense-bind to the coding region of mRNAs. I employ a set of genes recently experimentally verified to be the target of a miRNA, all with putative antisense pairing domains within the coding sequence. Although very small (～22 nucleotides), 2 of 13 pairing domains show evidence of significantly slow sequence evolution. This, along with evidence that these genes are regulated by the miRNA under consideration, provides the first good candidate domains for intra-CDS pairing of a miRNA in mammals. When analyzed en masse, the putative pairing domains have a significantly reduced rate of synonymous evolution (～35% lower than null). However, given the size and rarity of pairing domains within the coding sequence, the effects that such constraint has on estimates of the mutation rate are small enough to be ignored (probably less than 1% reduction). The pairing sites also have low K_a values and the selection on the synonymous sites is unlikely to lead to misleading reports of localized high K_a/K_s ratios.     

A Role for the Geomagnetic Field in Cell Regulation

We advance the hypothesis that biological systems utilize the geomagnetic field (GMF) for functional purposes by means of ion cyclotron resonance-like (ICR) mechanisms. Numerous ICR-designed experiments have demonstrated that living things are sensitive, in varying degrees, to magnetic fields that are equivalent to both changes in the general magnetostatic intensity of the GMF, as well as its temporal perturbations. We propose the existence of ICR-like cell regulation processes, homologous to the way that biochemical messengers alter the net biological state through competing processes of enhancement and inhibition. In like manner, combinations of different resonance frequencies all coupled to the same local magnetic field provide a unique means for cell regulation. Recent work on ultraweak ICR magnetic fields by Zhadin and others fits into our proposed framework if one assumes that cellular systems generate time-varying electric fields of the order 100 mV/cm with bandwidths that include relevant ICR frequencies.     

A Role for the TOC Complex in Arabidopsis Root Gravitropism

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.     

长非编码RNA在基因调控和肿瘤形成中的作用

哺乳动物中,只有小部分基因转录成为编码蛋白质的RNA,大量的基因则转录为不能编码蛋白质的RNA,即ncRNA。长非 编码RNA(lncRNAs)是分子长度在200-100000 nt 之间的一类ncRNA。lncRNAs 的数量超过蛋白质编码基因的数量。目前,对长非 编码RNA(lncRNAs)的生物学特性,转录调控以及其在肿瘤发生发展中的作用机制的研究任然是RNA研究的热点。lncRNAs 通 过控制染色质重塑,转录调控和录转录后调控而在基因的转录调节中发挥了重要作用。lncRNAs 与多种肿瘤相关,并且在抑制因 素和促进因素中都具有重要的作用。众多文献报道的结果表明lncRNAs 参与调控基因表达,在正常细胞与肿瘤细胞的转换中起 到至关重要的作用。