Distinct effects of nuclear membrane localization on gene transcription silencing in Drosophila S2 cells and germ cells

Nuclear envelope proteins have important roles in chromatin organization and signal-dependent transcriptional regulation. A previous study reported that the inner nuclear membrane protein, Otefin （Ote）, was essential for germline stem cell （GSC） maintenance via interaction with Smad complex. The interaction of Ore with the Smad complex recruits the barn locus to the nuclear periphery and subsequently results in bam transcriptional silencing, revealing that nuclear peripheral localization is essential for barn gene regulation. However, it remains unknown whether the nuclear peripheral localization is sufficient for barn silencing. To address this issue, we have established a tethering system, in which the Gal4 DNA binding domain （DBD） of the Flag：Gal4 DBD：Ote△LEM fusion protein physically interacts with the Gal4 binding sites upstream of bamP-gfp to artificially recruit the reporter gene gfp to the nuclear membrane. Our data demonstrated that the nuclear peripheral localization seemed to affect the expression of the target naked gene in S2 ceils. By contrast, in Drosophila germ cells, the nuclear membrane localization was not sufficient for gene silencing.

Features,Mechanisms and Applies of Post-transcriptional Gene Silencing in Transgenic Plants

Since transgene silencing was found in transgenic plants,many scholars have studied it extensively and considered that it has three functional mechanisms:post dependent gene silencing,transcriptional gene silencing,post transcriptional gene silencing.At the moment,people have mainly focused on the study of post transcriptional gene silencing and found its features:extensivity,conduction and peculiarity,also put forward some hypothesis for its mechanisms,for example,RNA threshold model,aberrant RNA model,inter or intra molecular base pairing model and so on.Furthermore,post transcriptional gene silencing is being applied in gene engineering of plants.Recently the people have found that post transcriptional gene silencing has bearing on capacity plants resisting virus.Many researchers have studied post transcriptional gene silencing,but there are some questions which need be solved in the future.This article summarizes progresses in features,mechanisms,applies of post transcriptional gene silencing about transgenic plants.

Dual activities of a silencing information regulator complex in yeast transcriptional regulation and DNA-damage response

The Saccharomyces cerevisiae silencing information regulator(SIR)complex contains up to four proteins,namely Sir1,Sir2,Sir3,and Sir4.While Sir2 encodes a NAD-dependent histone deacetylase,other SIR proteins mainly function as structural and scaffold components through physical interaction with various proteins.The SIR complex displays different conformation and composition,including Sir2 homotrimer,Sir1-4 heterotetramer,Sir2-4 heterotrimer,and their derivatives,which recycle and relocate to different chromosomal regions.Major activities of the SIR complex are transcriptional silencing through chromosomal remodeling and modulation of DNA double-strand-break repair pathways.These activities allow the SIR complex to be involved in mating-type maintenance and switching,telomere and subtelomere gene silencing,promotion of nonhomologous end joining,and inhibition of homologous recombination,as well as control of cell aging.This review explores the potential link between epigenetic regulation and DNA damage response conferred by the SIR complex under various conditions aiming at understanding its roles in balancing cell survival and genomic stability in response to internal and environmental stresses.As core activities of the SIR complex are highly conserved in eukaryotes from yeast to humans,knowledge obtained in the yeast may apply to mammalian Sirtuin homologs and related diseases.

Nuclear reprogramming: the zygotic transcription program is established through an“erase-and-rebuild” strategy

Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors （CFs） disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naive chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the ＂erase-and-rebuild＂ process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper （Gao T, et al., Cell Res 2007; 17：135-150.） that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition.

The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis

Although DNA methylation is known to play an important role in the silencing of transposable elements （TEs） and introduced transgenes, the mechanisms that generate DNA methylation-independent transcrip- tional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation （RdDM） is required for the silencing of the RD29A-LUC transgene in the Arabidopsis rosl mutant back- ground with defective DNA demethylase. Loss of function of ARGONAUTE 4 （AGO4） gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the rosl/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC trans- gene expression in the rosl/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/ U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcrip- tional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.

Sumoylation of SUVR2 contributes to its role in transcriptional gene silencing

The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remodeling proteins CHR19,CHR27, and CHR28(CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing

Tomato yellow leaf curl virus(TYLCV)is known to encode 6 canonical viral proteins.Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions.The fifth ORF of TYLCV in the complementary sense,which we name C5,is evolutionarily conserved,but little is known about its expression and function during viral infection.Here,we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody.Ectopic expression of C5 using a potato virus X(PVX)vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species(ROS)in Nicotiana benthamiana plants.C5 was able to effectively suppress local and systemic post-transcriptional gene silencing(PTGS)induced by single-stranded GFP but not double-stranded GFP,and reversed the transcriptional gene silencing(TGS)of GFP.Furthermore,the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants.Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5.Collectively,this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor,hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

RNA silencing movement in plants

Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.

Nitrogen starvation induces genome-wide activation of transposable elements in Arabidopsis

Nitrogen(N)availability is a major limiting factor for plant growth and agricultural productivity.Although the gene regulation network in response to N starvation has been extensively studied,it remains unknown whether N starvation has an impact on the activity of transposable elements(TEs).Here,we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions.Through genetic screening of idm1-14 suppressors,we cloned GLU1,which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway.We found that glutamate synthase 1(GLU1)and its functional homologs GLU2 and glutamate transport 1(GLT1)are redundantly required for TE silencing,suggesting that N metabolism can regulate TE activity.Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation.Genetic analysis indicated that N starvationinduced TE activation is also independent of other well-established epigenetic mechanisms,including histone methylation and heterochromatin decondensation.Our results provide new insights into the regulation of TE activity under stressful environments in planta.