Roles Of Chromatin Remodeling Factors And Protosilencer In Gene Siliencing And Heterochromatin Structure

The basic unit of chromatin is the nucleosome, which is composed of two copies each of the four core histones H2A.H2B, H32. H42 and approximately 146 bp of DNA wrapped in about two turns around a histone octamer complex. Chromatin is divided into different regions, which are loosely structured euchromatin and highly condensed chromatin heterochromatin. Gene expression is normal in euchromatin,but gene expression is silent in heterochromatin which is also known as silent chromatin. Heterochromatin in Saccharomyces cerevisiae exists at HML and HMR loci as well as subtelomeric region. Like its metazoan counterpart, Saccharomyces cerevisiae heterochromatin consists of highly like its metazoan counterpart, its heterochromatin consists of highly ordered and stably positioned nucleosomes. It also bears characteristic histone modifications such as hypoacetylation and hypomethylation, DNA replication occurs in later S phase.Establishment of HM loci silencing is initiated at silencer, which are composed of two or three combinations of binding sites for proteins Rap1 protein, Abf1 protein, and the ORC. These binding sites are called protosilencer. It was proposed that silencer-binding proteins recruit a complex of SIR complex (Sir2p/Sir3p/Sir4p), which is an integral part of the silent chromatin. Sir2p was found to be a histone deacetylase, Sir3p/Sir4p interact with the N-terminal tails of histones H3 and H4, and there is evidence that Sir3p (hence the SIR complex) has a much higher affinity for hypoaceylated histones. So Sir2p recruited to the silencer deacetylates histones in an adjacent nucleosome enabling it to bind another SIR complex with high affinity, the nucleosome-bound SIR complex in turn deacetylates the neighboring nucleosome allowing it to recruit a new SIR complex,In this manner, the SIR complex promotes its own propagation along the chromatin to form heterochromatin.Although S. cerevisiae silencers function through a common mechanism, they exhibit different efficiencies in gene silencing. At the HMR locus, HMR-E can silence the HMRa genes on its own, whereas HMR-I can't, or play an auxiliary role. At the HML locus, on the other hand, either HML-E or HML-I alonecan silence the HMLa genes. When ectopically inserted near the MAT locus, HMR-E was shown to be stronger than HML-E. A possible explanation for the difference of the silencers concerning their potencies of silencing is that each silencer consists of a unique sequence, and maybe we will benefit a lot from studying protosilencer.In addition, there are huge difference between enchromatin and heterchromatin, and converting chromatin form one form to another need its remodeling, but none of the Sir proteins has chromatin remodeling activity. Fortunately, some evidences show that chromatin remodeling proteins Fun30, Snf2, Isw1 are involved in transcriptional silencing, how any of these factors contributes to gene silencing and heterochromatin structure is not known.Our finding that protsilencer alone can not produce gene silencing; with the help of HML-E silencer, protosilencer Abf1p and ORC binding sites can produce gene silencing on on its both sides while Rap1 binding site only have gene silencing on its right side, these results show that protosilencer play auxiliary role on gene silencing, and Rap1 binding site not only play auxiliary role on gene silencing, but also has boundary acitivity. We also found that increasing the copy number Abf1 binding sites can enhance the role of gene silencing with helping of HML-E silencer, and it is amazing that two copies of the Abf1 binding site alone can have gene silencing without HML-E silencer, but the reasons for the change need further study; we also found that Raplp binding sites varys degrees of gene silencing in different locations, and only has gene silencing effect in its original position, and the possible explanation for the difference maybe because of the sequence around it can increased its role in gene silecing.In order to study the intrinsic reason of gene silencing of protosilencer, frist we adopted the topology assay to analyze the supericoil chromatin DNA. We found that the ORC binding site and Abf1p binding site together with HML-E silencer formed higher proportion of negative supercoils, but the Rap1 binding site together with HML-E silencer form lower percentage DNA negative supercoils; we also found if we increases the copy number of Abfl binding site which is together with HML-E silencer, negative supercoiled DNA would increase, especially at three copies almost all of DNA circle will be negative supercoils; We found that the Abf1p binding site t with help of HML-E silencer, they would form more negative DNA supercoils at original location of Rap1 binding site than other locations. The DNA supercoils data is consistent with previous analysis of gene silencing.Moreover, we mapped nucleosomes within HM1-E and protosilencer by micrococcal nuclease digestion and indirect end labeling. The results show that the chromatin nucleosome arrangement between HML-E silencer and ORC binding site or Abfl binding site are similar with HML-E with the HMR-E silencer, but the chromatin nucleosome arrangement between HML-E silencer and Rapl binding site are similar with HML-E alone. In addition, nucleosomes arrangement near the protosilencer in sir- strains is similar with Sir+ strains, so it is indicated that the protosilencer changes nucleosome around it does not depend on the Sir proteins.In addition, there is evidence implicating chromatin remodeling proteins play an important role on heterochromatin formation, but their exact roles are not clear. We demonstrate that the Fun30p and IswIp chromatin remodeling factors are similarly required for gene silencing at the HML locus, but they differentially contribute to the structure and stability of HML heterochromatin in Saccharomyces cerevisiae. In the absence of Fun30p, only a partially silenced structure is established at HML. This structure likely represents an intermediate state of heterochromatin that is between fully silenced and derepressed chromatin structures. Moreover, Fun30p removal reduces the rate of de novo establishment of heterochromatin, suggesting that Fun30p assists the silencing machinery in forming mature heterochromatin. On the other hand, IswIp is dispensable for the formation of heterochromatin structure, but is instead critically required for maintaining its stability. Therefore, chromatin remodeling proteins may rearrange nucleosomes during the formation of heterochromatin, or serve to stabilize/maintain heterochromatin structure.