Interplay among ATP-Dependent Chromatin Remodelers Determines Chromatin Organisation in Yeast

Cellular DNA is packaged into chromatin, which is composed of regularly-spaced nucleosomes with occasional gaps corresponding to active regulatory elements, such as promoters and enhancers, called nucleosome-depleted regions (NDRs). This chromatin organisation is primarily determined by the activities of a set of ATP-dependent remodeling enzymes that are capable of moving nucleosomes along DNA, or of evicting nucleosomes altogether. In yeast, the nucleosome-spacing enzymes are ISW1 (Imitation SWitch protein 1), Chromodomain-Helicase-DNA-binding (CHD)1, ISW2 (Imitation SWitch protein 2) and INOsitol-requiring 80 (INO80); the nucleosome eviction enzymes are the SWItching/Sucrose Non-Fermenting (SWI/SNF) family, the Remodeling the Structure of Chromatin (RSC) complexes and INO80. We discuss the contributions of each set of enzymes to chromatin organisation. ISW1 and CHD1 are the major spacing enzymes; loss of both enzymes results in major chromatin disruption, partly due to the appearance of close-packed di-nucleosomes. ISW1 and CHD1 compete to set nucleosome spacing on most genes. ISW1 is dominant, setting wild type spacing, whereas CHD1 sets short spacing and may dominate on highly-transcribed genes. We propose that the competing remodelers regulate spacing, which in turn controls the binding of linker histone (H1) and therefore the degree of chromatin folding. Thus, genes with long spacing bind more H1, resulting in increased chromatin compaction. RSC, SWI/SNF and INO80 are involved in NDR formation, either directly by nucleosome eviction or repositioning, or indirectly by affecting the size of the complex that resides in the NDR. The nature of this complex is controversial: some suggest that it is a RSC-bound “fragile nucleosome”, whereas we propose that it is a non-histone transcription complex. In either case, this complex appears to serve as a barrier to nucleosome formation, resulting in the formation of phased nucleosomal arrays on both sides.Fil: Prajapati, Hemant K.. National Instituto of Child Health & Human Development; Estados UnidosFil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Clark, David J.. National Instituto of Child Health & Human Development; Estados Unido

Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell-specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein-protein relationships and protein functions at the chromatin template.Chemical Immunolog

Genetic analysis of cell cycle and chromatin regulation in quiescent fission yeast cells

During proliferation, cells produce their genetic materials to increase the number of cells, while in the absence of nutrients or by the induction of stimulus, the proliferative phase is stopped and entry into quiescence is triggered to increase their chance of survival. Quiescence is a reversible resting phase where cells enter, in case of nutrient deprivation or damage and induced by stimuli. In cancer development, the shift between proliferation and quiescence stage is critical since, for example, tumor cells in dormancy are more resistant to cancer treatments. In the resting phase, energy sources are saved by minimizing or stopping the metabolism and cell division in order to use energy for maintaining cell survival. In this case, cells adapt to the new conditions by gene expression reprogramming, which is mediated by chromatin remodeling mechanisms. Therefore, there is a need to investigate mechanisms to understand genes and pathways affecting quiescence entry and maintenance. To investigate the role of genes in quiescence, using high-throughput flow cytometry analysis, we developed the projects to discover new genes and pathways involved in the vegetative or quiescence stages. To achieve this end, we utilized the fission yeast and Schizosaccharomyces pombe which is a convenient model to study both vegetative and quiescence stages. Then, we performed both DNA content and cell survival analysis on the haploid deletion mutant library. Through these original approaches, gene-deleted mutants were classified according to their phenotypes to disclose mechanisms involved in vegetative and quiescence stages. In the present study, different remodeler complexes such as INO80 C, SWR1 C, and SAGA C were investigated and the effect of these complexes on quiescence entry or maintenance was observed. The results demonstrate the effect of remodeler complexes for reprogramming gene expression patterns, that lead cells to enter quiescence or viability of cells during quiescence. The most interesting complex mainly observed was Ino80. Ino80 ATPase-dependent remodeling complex mediates chromatin remodeling by removing histone variant H2A.Z from chromatin. This remodeler complex is required for the regulation of quiescence-related genes. More remodeler complexes and effective genes, that are related to quiescence entry and maintenance, are explained more in details in the result and discussion section

NMR studies on the nucleosome and chromatin factors

Chromatin is vital to compaction and regulation of the DNA in eukaryotes and is intimately involved in DNA expression, replication, and repair. As one of the cell’s biggest polymers, chromatin forms a multi-scale structure consisting of DNA and protein. At the smallest level, the nucleosome forms the repeating unit of chromatin, consisting of two copies of four different histones and around 150 base pair of DNA. A central question in chromatin biology is how chromatin, DNA binding proteins, chromatin factors and environmental factors work together to regulate all DNA-mediated processes. The underlying molecular mechanisms not only involve interactions between the smallest units, e.g. proteins and the nucleosome, but also the interplay of the complex higher-order organization of chromatin and these molecular interactions. These aspects converge in the case of chromatin remodelers. These enzymes interact with nucleosomes to alter higher-order chromatin structure directly, by reshuffling the position of nucleosomes in the genome. Chapter 1 reviews the basic components of the nucleosome and gives a first glance at factors involved in the higher order organization of chromatin including ionic strength and chromatin remodeling proteins. In chapter 2 we expand on this first glance by reviewing nucleosome-protein interactions in higher order chromatin structures. The increasing availability of high-resolution nucleosome-protein structures allowed us to shed light on how chromatin factors operate in this complex higher order chromatin environment. In chapter 3 we investigate the effect of the currently highest permanent magnetic field of 28.2 Tesla in the context of chromatin. We investigated the performance of methyl-TROSY NMR at 1.2 GHz, using the nucleosome as a test sample. We find that the increased resolution of the 1.2 GHz system allows to resolve small asymmetries in amino acid sidechain conformation between symmetry-related copies of the histone proteins. We further observe significant magnetic field alignment of the nucleosome at 1.2 GHz, giving rise to methyl 1H-13C residual dipolar couplings (RDCs) that can be used for assignment and structural characterization. We show that these histone methyl group RDCs can be used to aid assignment and to determine the overall conformation of the nucleosomal DNA, revealing a significant unwrapping of the DNA from the histone core. In chapter 4 we applied nuclear magnetic resonance to study nucleosomes with mono- and divalent ions using a methyl labeling approach. An increase in ionic strength resulted in a decrease in core residue signals but did not decrease the tail residue signal. Our data point to an increase in effective size of the nucleosome particle due to tail DNA interactions between nucleosome particles. In chapter 5 we studied the conformational dynamics of ISWI and the nucleosome-ISWI complex using methyl-TROSY solution NMR spectroscopy. We find that the free enzyme is highly dynamic throughout the protein. Our data indicate that binding of an active ISWI construct to the nucleosome induces conformational changes through the histone octamer, affecting histone-DNA and histone-histone contacts. These findings provide strong support for histone plasticity during remodeling to facilitate DNA translocation and further highlight the histone octamer as an allosteric unit