NoRC, a novel chromatin remodeling complex involved in ribosomal RNA gene silencing

Regulation of gene expression takes place in the nucleus in a highly structured and condensed nucleoprotein environment, called chromatin (Felsenfeld and Groudine, 2003; Khorasanizadeh, 2004; Vaquero et al., 2003). A broad group of factors regulates the properties of chromatin; e.g. by covalently modifying histones and / or by ATP-dependent chromatin remodeling, thereby allowing or preventing gene expression. The mammalian genome contains hundreds of gene copies encoding precursor ribosomal RNA and the transcription of these genes is highly regulated with respect to cellular metabolism (Grummt, 2003). However, even in actively growing cells, only a subset of the rRNA genes are actively transcribed, exhibiting an accessible chromatin conformation (Conconi et al., 1989). In a chromatin context, the activation of rDNA genes involves the transcription termination factor TTF-I (Längst et al., 1998; Längst et al., 1997a). However, the silenced rDNA gene fraction remains in an inaccessible heterochromatic state throughout the cell cycle (Conconi et al., 1989). Until recently, the onset of silencing and the mechanisms that maintain the inactive state of rRNA genes were less understood. Recent studies, including the work presented in this thesis, provide insights into the molecular mechanism of ribosomal RNA gene silencing (Lawrence et al., 2004; Németh et al., 2004; Santoro and Grummt, 2001; Santoro et al., 2002; Strohner et al., 2004; Zhou et al., 2002). Accumulating evidence indicates that the combined action of chromatin modifying mechanisms such as chromatin remodeling, histone modification and DNA methylation contribute to the process of rRNA gene silencing. Here I present data demonstrating an active role of the chromatin remodeling complex NoRC in rDNA gene silencing and propose dual functions of TTF-I in rDNA regulation in chromatin, namely involvement in both activation and silencing of rDNA transcription. 4.1 NoRC, a novel chromatin remodeling complex In this doctoral study, a novel protein complex, composed of the nucleolar protein Tip5 and the ATPase Snf2h, was purified using convential chromatography and affinity purification methods. A detailed chromatin remodeling analysis revealed that this complex is able to induce mononucleosome movement in an ATP and histone H4 tail dependent fashion. Finally, this Tip5-Snf2h complex was termed NoRC (nucleolar remodeling complex), a novel member of the ISWI family of ATP-dependent chromatin remodeling complexes (Strohner et al., 2001). To dissect its functions, the NoRC complex was reconstituted from its recombinant subunits Tip5 and Snf2h, using the baculo virus driven expression system. Reconstitution confirmed the direct interaction between Tip5 and Snf2h. Furthermore, recombinant and cellular NoRC display similar sizes in gel filtration columns. Recombinant NoRC exhibits chromatin stimulated ATPase activity and mobilizes nucleosomes in an energy-dependent manner. Both activities are histone H4 tail dependent. NoRC and its subunits Tip5 and Snf2h were compared in different DNA / Nucleosome binding assays. NoRC shows preferred binding to structured (bent) DNA, e.g. a region within the mouse rDNA promoter, and interacts with mononucleosomes in electrophoretic mobility shift assays (EMSA). While no stable interaction with core nucleosomes could be detected in EMSA, ATPase assays and DNase I protection assays noticeably pinpointed to NoRC / nucleosome interactions with both nucleosomal and protruding linker DNA. 4.2 NoRC specifically represses rDNA transcription in chromatin The functional consequences of the Tip5 / TTF-I interaction were assessed and the influence on chromatin structure of the rDNA promoter in an in vitro system was determined. Tip5 in NoRC interacts with the N-terminal part of full length TTF-I and unmasks its DNA binding site. This interaction is required both for binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter in chromatin. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome. To elucidate a potential role of NoRC in rDNA transcriptional regulation, we used an in vitro transcription system with an rDNA minigene reconstituted into chromatin. These studies revealed a specific function for NoRC in rDNA transcriptional repression on chromatin templates. In contrast, NoRC had no effect on DNA transcription. Transcription experiments were then performed with chromatin templates reconstituted from recombinant histones lacking individual histone tails. The results indicate that NoRC-mediated rDNA gene repression is dependent on the histone H4 tail, suggesting an involvement of chromatin remodeling. Further transcription experiments revealed that NoRC-mediated repression occurs prior to preinitiation complex formation and does not affect activated rDNA genes. NoRC stably associates with the silenced gene, and these early steps of rDNA repression do not depend on DNA and histone modifications (Strohner et al., 2004). NoRC showed preferred binding to a structured (bent) region within the mouse rDNA promoter. Methylation of a single CpG dinucleotide within this region abrogated rDNA transcription in chromatin (Santoro and Grummt, 2001), but did not influence DNA binding of NoRC. Furthermore, nucleosomal DNA is less methylated than free DNA, but chromatin remodeling enhances methylation. The results suggest an important role for the chromatin remodeling complex NoRC in the establishment of rDNA silencing. NoRC then contributes to maintenance of the silenced state throughout the cell cycle by interacting with DNA and histone modifying enzymes. Transcriptional repression by chromatin remodeling factors seems to be a common mechanism to stably inhibit gene expression

RNA Is an Integral Component of Chromatin that Contributes to Its Structural Organization

Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%–5% of total chromatin-associated nucleic acids, are polyA− and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s) are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with α-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity

Highly efficient gene expression based on chromatin engineering

制度:新 ; 報告番号:甲3108号 ; 学位の種類:博士(理学) ; 授与年月日:2010/4/22 ; 早大学位記番号:新537

RNA synthesis and processing in isolated HeLa cell nuclei

Isolated HeLa cell nuclei have been characterised in terms of their ability to transcribe, process and transport RNA. In terms of transcription, it was found that all three RNA polymerases were active in the isolated nuclei. The size and nuclear location of the products of RNA polymerase I and III suggested that transcription by these polymerases was occurring normally in vitro. However, the RNA synthesised by RNA polymerase II was found to be much smaller than expected from the reported size of HeLa cell transcription units, when analysed in denaturing gradients. This was in contrast to the results of Sarma et al (1976) which showed that the size of RNA synthesised by RNA polymerase II in isolated HeLa cell nuclei is large when analysed under non-denaturing conditions. A number of possible reasons for this small size of RNA were examined. The results obtained indicate that this small size of RNA polymerase II product was probably not due to;- i. A slow elongation rate by RNA polymerase II resulting in a "nascent transcript profile." ii. Degradation of RNA in the isolated nuclei. iii. The absence of nuclear and cytoplasmic factors during incubation of the nuclei. iv. Degradation of the DM template during isolation and incubation of nuclei. It was found, however, that the state of the chromatin template was important in determining the size of RM transcribed. Thus, addition of acetyl CoA to isolated nuclei, which acetylated the histones, caused an increase in the size of RNA polymerase II product. On the other hand methylation of histones with Ado-Met vitro was correlated with a decrease in the size of RM. It was also found that the ionic content of the incubation medium affected the size of RNA transcript synthesised by RNA polymerase II. In particular, substituting 90 mM (NH4)2SO4 for 75 mM KCl in the incubation medium increased the size of RNA. This effect is discussed in terms of recent results which suggest that the small size of RNA polymerase II transcript commonly observed in vitro might be due to premature termination of transcription. The small RNA transcribed by RNA polymerase II in vitro appears to be stable. However, hnRNA prelabelled in vivo reduced in size during incubation of isolated nuclei. Some of this RNA is released from the nuclei during incubation. This release of RNA was examined to determine whether it represented the specific transport of mRNA. Although the size of released RNA particles, their ability, and the size of released RNA were consistent with mRNA transport, other features were more consistent with the leakage of hnRNP particles. The released RNA resembled hnRNA in terms of binding to poly(U) Sepharose, and the protein associated with the released RNA were similar to hnRHP particle proteins. Although the released RNA had messenger activitiy in a wheat germ cell free translation system, it is not possible to rule out mRNA contamination as the cause of this stimulation. It therefore appears that the isolated nuclei system of Sarma et al (1976) may not be ideal either for examining the transcription or the processing and transport of mRNA. On the other hand, the results obtained in the present study suggest ways in which this system might be modified in order to achieve full length transcription by RNA polymerase II in vitro, and study the processing and transport of these transcripts

Distinct domains of erythroid Kruppel-like factor modulate chromatin remodeling and transactivation at the endogenous beta-globin gene promoter

Characterization of the mechanism(s) of action of trans-acting factors in higher eukaryotes requires the establishment of cellular models that test their function at endogenous target gene regulatory elements. Erythroid Kruppel-like factor (EKLF) is essential for beta -globin gene transcription. To elucidate the in vivo determinants leading to transcription of the adult beta -globin gene, functional domains of EKLF were examined in the context of chromatin remodeling and transcriptional activation at the endogenous locus. Human EKLF (hEKLF) sequences, linked to an estrogen-responsive domain, were studied with an erythroblast cell line lacking endogenous EKLF expression (J2e Delta eklf). J2e Delta eklf cells transduced with hEKLF demonstrated a dose-dependent rescue of beta -globin transcription in the presence of inducing ligand. Further analysis using a series of amino-terminal truncation mutants of hEKLF identified a distinct internal domain, which is sufficient for transactivation. Interestingly, studies of the chromatin structure of the beta -promoter revealed that a smaller carboxy-terminal domain generated an open promoter configuration. In vitro and in vivo binding studies demonstrated that this region interacted with BRG1, a component of the SWI/SNF chromatin remodeling complex. However, further study revealed that BRG1 interacted with an even smaller domain of EKLF, suggesting that additional protein interactions are required for chromatin remodeling at the endogenous beta -promoter. Taken together, our findings support a stepwise process of chromatin remodeling and coactivator recruitment to the beta -globin promoter in vivo. The J2e Delta eklf inducible hEKLF system will be a valuable tool for further characterizing the temporal series of events required for endogenous beta -globin gene transcription

Structure of lampbrush chromosome loops during different states of transcriptional activity as visualized in the presence of physiological salt concentrations

Lampbrush chromosomes of amphibian oocytes were isolated in the presence of near-physiological salt concentrations, to preserve their native state, and studied by electron microscopy of ultrathin s~dions. The transcriptional state of the lampbrush chromosomes was experimentally modulated by incubating the oocytes for various time periods in medium containing actinomycin D. The observations show that the structure of the lateral loops changes rapidly in response to alterations in transcriptional activity. During decreasing transcriptional activity and reduced packing density of transcripts, the chromatin axis first condensed into nucleosomes and then into an approximately 30 nm thick higher order chromatin fiber. Packaging of the loop axis into supranucleosomal structures may contribute to the foreshortening and retraction of the loops observed during inhibition of transcription and in later stages of meiotic prophase. The increasing packing density of the DNA during the retraction process of the loops could also be visualized by immunofluorescence microscopy using antibodies to DNA. The dependence of the loop chromatin structure on transcriptional activity is discussed in relation to current views of mechanisms involved in gene activation