Nucleosome Positioning and Its Role in Gene Regulation in Yeast

Nucleosome, composed of a 147-bp segment of DNA helix wrapped around a histone protein octamer, serves as the basic unit of chromatin. Nucleosome positioning refers to the relative position of DNA double helix with respect to the histone octamer. The positioning has an important role in transcription, DNA replication and other DNA transactions since packing DNA into nucleosomes occludes the binding site of proteins. Moreover, the nucleosomes bear histone modifications thus having a profound effect in regulation. Nucleosome positioning and its roles are extensively studied in model organism yeast. In this chapter, nucleosome organization and its roles in gene regulation are reviewed. Typically, nucleosomes are depleted around transcription start sites (TSSs), resulting in a nucleosome-free region (NFR) that is flanked by two well-positioned H2A.Z-containing nucleosomes. The nucleosomes downstream of the TSS are equally spaced in a nucleosome array. DNA sequences, especially 10–11 bp periodicities of some specific dinucleotides, partly determine the nucleosome positioning. Nucleosome occupancy can be determined with high throughput sequencing techniques. Importantly, nucleosome positions are dynamic in different cell types and different environments. Histones depletions, histones mutations, heat shock and changes in carbon source will profoundly change nucleosome organization. In the yeast cells, upon mutating the histones, the nucleosomes change drastically at promoters and the highly expressed genes, such as ribosome genes, undergo more change. The changes of nucleosomes tightly associate the transcription initiation, elongation and termination. H2A.Z is contained in the +1 and −1 nucleosomes and thus in transcription. Chaperon Chz1 and elongation factor Spt16 function in H2A.Z deposition on chromatin. The chapter covers the basic concept of nucleosomes, nucleosome determinant, the techniques of mapping nucleosomes, nucleosome alteration upon stress and mutation, and Htz1 dynamics on chromatin

Characterization of Histone Mutants Associated with Mitotic Defects in Saccharomyces cerevisiae

Nucleosomes, the basic unit and the building blocks of chromatin have an essential role in the tight packaging of DNA into higher order chromatin. Work from our lab and others have provided information on the contributions of different histone proteins and specific domains within the nucleosome made to create the centromeric chromatin structure required for normal chromosome segregation during mitosis. The DNA entry/exit site is a particular region of the nucleosome where histone H2A, H3 and H4 form critical interactions that appear to be essential for the association of Sgo1, a tension sensing protein that monitors kinetochore-microtubule attachment. In our study, we first characterized histone H2A mutants with respect to their chromosome segregation phenotypes. Three mutations that show such phenotypes were in the C-terminal region of H2A, which is located in the DNA entry/exit region of the nucleosome, in close proximity to H3 and H4 residues that show severe chromosome segregation defects when mutated. We then created a double mutant strain that incorporated two single mutations, one in H2A and one in H4, to study their combined effect in chromosome segregation and normal cell cycle progression. The H2A N115S residue, positioned in the C-terminal tail of histone H2A, and H4 K44Q, positioned in H4 L1 histone fold domain, both falling in the region of DNA entry/exit point of the nucleosome, were incorporated in our double mutant. We found that the incorporation of the H2A N115S mutation alleviated the growth defect of the H4 K44Q single mutant and fully suppressed its increase-in-ploidy phenotype. We also found that overexpression of Sgo1 suppressed the sensitivity of both single mutants and the double mutant to the microtubule depolymerizing drug benomyl. We conclude that histone-histone interactions within the DNA entry/exit point of the nucleosome are particularly important in chromosome segregation, most likely in establishing centromere-kinetochore attachments during mitosis. In addition, and consistent with a previously noted role of the DNA entry/exit point, this nucleosomal region creates a unique surface required for the recruitment of Sgo1, and perhaps other proteins, such as components of the CPC, required for normal microtubule attachment at the centromere

Evolution of nucleosome positioning and gene regulation in yeasts : a genomic and computational approach

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 107-111).Chromatin organization plays a major role in gene regulation and can affect the function and evolution of new transcriptional programs. Here, we present the first multi-species comparative genomic analysis of the relationship between chromatin organization and gene expression by measuring mRNA abundance and nucleosome positions genome-wide in 13 Ascomycota yeast species. Our work introduces a host of new computational tools for studying chromatin structure, function, and evolution. We improved on existing methods for detecting nucleosome positions and developed a new approach for identifying nucleosome-free regions (NFRs) and characterizing chromatin organization at gene promoters. We used a general statistical approach for studying the evolution of chromatin and gene regulation at a functional level. We also introduced a new technique for discovering the DNA binding motifs of transacting General Regulatory Factors (GRFs) and developed a new technique for quantifying the relative contribution of intrinsic sequence, GRFs, and transcription to establishing NFRs. And finally, we built a computational framework to quantify the evolutionary interplay between nucleosome positions, transcription factor binding sites, and gene expression. Through our analysis, we found large conservation of global and functional chromatin organization. Chromatin organization has also substantially diverged in both global quantitative features and in functional groups of genes. We find that global usage of intrinsic anti-nucleosomal sequences such as PolyA varies over this phylogeny, and uncover that PolyG tracts also intrinsically repel nucleosomes. The specific sequences bound by GRFs are also highly plastic; we experimentally validate an evolutionary handover from Cbfl in pre-WGD yeasts to Rebi in post-WGD yeast. We also identify five mechanisms that couple chromatin organization to evolution of gene regulation, including (i) compensatory evolution of alternative modifiers associated with conserved chromatin organization; (ii) a gradual transition from constitutive to transregulated NFRs; (iii) a loss of intrinsic anti-nucleosomal sequences accompanying changes in chromatin organization and gene expression, (iv) repositioning of motifs from NFRs to nucleosome-occluded regions; and (v) the expanded use of NFRs by paralogous activator-repressor pairs. Our multi-species dataset and general computational framework provide a foundation for future studies on how chromatin structure changes over time and in evolution.by Alexander Minchev Tsankov.Ph.D

Dissecting cis and trans Determinants of Nucleosome Positioning: A Dissertation

Eukaryotic DNA is packaged in chromatin, whose repeating subunit, the nucleosome, consists of an octamer of histone proteins wrapped by about 147bp of DNA. This packaging affects the accessibility of DNA and hence any process that occurs on DNA, such as replication, repair, and transcription. An early observation from genome-wide nucleosome mapping in yeast was that genes had a surprisingly characteristic structure, which has motivated studies to understand what determines this architecture. Both sequence and trans acting factors are known to influence chromatin packaging, but the relative contributions of cis and trans determinants of nucleosome positioning is debated. Here we present data using genetic approaches to examine the contributions of cis and trans acting factors on nucleosome positioning in budding yeast. We developed the use of yeast artificial chromosomes to exploit quantitative differences in the chromatin structures of different yeast species. This allows us to place approximately 150kb of sequence from any species into the S.cerevisiae cellular environment and compare the nucleosome positions on this same sequence in different environments to discover what features are variant and hence regulated by trans acting factors. This method allowed us to conclusively show that the great preponderance of nucleosomes are positioned by trans acting factors. We observe the maintenance of nucleosome depletion over some promoter sequences, but partial fill-in of NDRs in some of the YAC v promoters indicates that even this feature is regulated to varying extents by trans acting factors. We are able to extend our use of evolutionary divergence in order to search for specific trans regulators whose effects vary between the species. We find that a subset of transcription factors can compete with histones to help generate some NDRs, with clear effects documented in a cbf1 deletion mutant. In addition, we find that Chd1p acts as a potential “molecular ruler” involved in defining the nucleosome repeat length differences between S.cerevisiae and K.lactis. The mechanism of this measurement is unclear as the alteration in activity is partially attributable to the N-terminal portion of the protein, for which there is no structural data. Our observations of a specialized chromatin structure at de novo transcriptional units along with results from nucleosome mapping in the absence of active transcription indicate that transcription plays a role in engineering genic nucleosome architecture. This work strongly supports the role of trans acting factors in setting up a dynamic, regulated chromatin structure that allows for robustness and fine-tuning of gene expression

Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin

abstract: Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin. I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions. SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug. Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.Dissertation/ThesisDoctoral Dissertation Biological Design 201

The Identification of Genetic and Epigenetic Changes that Contribute to Type 1 Diabetes

Type 1 diabetes (T1D) results from an immune cell mediated destruction of insulin-producing pancreatic β cells. Currently there is no cure for T1D. The exact cause for T1D is unknown but growing evidence points to the contribution of both genetic and environmental factors, leading to a breakdown in immunological tolerance normally maintained by Regulatory T (Treg) cells. The exact environmental contributions to T1D progression are not well characterised but emerging studies suggest that they may alter the immune system via epigenetic modification. Recent data strongly link the breakdown in tolerance in T1D and other autoimmune diseases to alterations in the transcriptional program in CD4+ T cells, however, the molecular mechanisms are not well understood. This work proposes that in T1D causal genetic risk SNPs alter the gene expression patterns in CD4+ Treg and or T helper cells by either disrupting or creating new TF (transcription factor) binding sites in regulatory elements (enhancers) located in genetic susceptibility regions and this may combine with environmentally induced epigenetic change and alter chromatin accessibility. Current methods to identify the functional consequences and mechanisms of these changes are complex, time consuming and expensive as generally they can only examine one TF/binding site at a time, involve TF binding site prediction, which has a high degree of false positives/negatives and require large quantities of starting material making them challenging for application on limited clinical samples. To overcome these limitations, and to functionally annotate genetic risk of T1D, this study employs genome wide approaches including ATAC-seq and RNA-seq to compare the DNA accessibility and transcriptomes in CD4+ Treg and Th (Helper T)/Tconv (Conventional T) cells isolated from individuals with established T1D and sibling-matched healthy controls. By incorporating case-control ATAC-seq and TF footprints this study prioritises 111 and 96 T1D-associated SNPs in Treg and Tconv cells, respectively, that may play a role in mediating the disease susceptibility and subsequently contributing to the loss of tolerance in T1D. Using a bioinformatic pipeline to integrate case-control ATAC-seq differentially accessible peaks and RNA-seq differentially expressed genes with Hi-C 3D connectivity maps this study identifies 42 and 21 dysregulated gene targets in Treg and Tconv cells, respectively. Those targets include TIGIT, MAF and IL2 and the enhancers regulating those loci showed differential accessibility and are enriched for T1D SNPs and differential TF footprint signals. One theory to explain such observation is T1D SNPs and epigenetic alterations may alter or disrupt TF occupancy at these loci contributing to dysregulated target gene regulation. This study identifies changes in chromatin structure in T1D samples relative to healthy controls, enabling the identification of changes driven by both genetic and epigenetic variation that correlates with an altered transcriptional program in T1D. T1D associated SNPs at these regions can then be correlated with alterations in TF binding and putative epigenetically modified T1D regions can be validated in follow-up functional assays to demonstrate causality. This study captures chromatin and transcriptional changes between T1D and healthy individuals but it does not have the capability to distinguish if the changes are the driver or the consequence of the disease because the case cohort contains only established T1D from a single time point. In order to infer causality those changes would need to be tracked and validated over a timeline of disease progression in a longitudinal cohort. Nonetheless, this work provides a novel 3D genomic approach to functionally annotating the genetic risk and epigenetic changes that directly or indirectly result in altered gene expression, and promising preliminary data warranting further investigation on the causal functional role of the dysregulated gene targets in T1D.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 202

Kinetics and mechanisms of Ikaros-mediated transcriptional regulation

The Ikaros family of zinc finger transcription factors is essential for B cell development, and frequently mutated in B cell malignancies. Our lab has previously identified Ikaros target genes in pre-B cells by combining Ikaros ChIP-seq binding data and gene expression profiling. To address the kinetics and mechanisms of Ikaros-mediated transcriptional regulation, I have used an inducible Ikaros system, which allows for the monitoring of cellular and molecular changes during Ikaros-mediated gene silencing at high temporal resolution. Within minutes of Ikaros induction, the Ikaros-regulated model loci Igll1 and Myc showed decreased promoter accessibility and RNA polymerase II (RNAPII) occupancy. These early events were followed by changes in nucleosome composition, including an increased histone H2B/H3 ratio, the deposition of the histone variant H2A.Z, and decreased active histone acetylation marks. Histone deacetylation was not required to initiate down-regulation of Igll1 and Myc transcription, since treatment with the histone deacetylase inhibitor Trichostatin A did not interfere with Ikaros-mediated gene silencing. I next elucidated the mechanistic relationship between the early events of decreased promoter accessibility and decreased RNAPII occupancy. Addition of Triptolide resulted in the removal of RNAPII from the Igll1 and Myc promoters, but did not affect nucleosome occupancy and its regulation mediated by Ikaros. This suggested that Ikaros regulates nucleosome positioning and occupancy directly, and not through effects on RNAPII. Consistent with this hypothesis, Ikaros-mediated gene silencing was delayed by RNAi-mediated knockdown of chromatin remodeler Mi-2β (Chd4), the ATPase subunit of the Mi-2/NuRD complex. Hence, Ikaros-initiated chromatin remodelling was identified as one of the earliest events during Ikaros-mediated gene silencing, and was required for rapid transcriptional down-regulation of Ikaros target genes.Open Acces

Clustered ChIP-Seq-defined transcription factor binding sites and histone modifications map distinct classes of regulatory elements

<p>Abstract</p> <p>Background</p> <p>Transcription factor binding to DNA requires both an appropriate binding element and suitably open chromatin, which together help to define regulatory elements within the genome. Current methods of identifying regulatory elements, such as promoters or enhancers, typically rely on sequence conservation, existing gene annotations or specific marks, such as histone modifications and p300 binding methods, each of which has its own biases.</p> <p>Results</p> <p>Herein we show that an approach based on clustering of transcription factor peaks from high-throughput sequencing coupled with chromatin immunoprecipitation (Chip-Seq) can be used to evaluate markers for regulatory elements. We used 67 data sets for 54 unique transcription factors distributed over two cell lines to create regulatory element clusters. By integrating the clusters from our approach with histone modifications and data for open chromatin, we identified general methylation of lysine 4 on histone H3 (H3K4me) as the most specific marker for transcription factor clusters. Clusters mapping to annotated genes showed distinct patterns in cluster composition related to gene expression and histone modifications. Clusters mapping to intergenic regions fall into two groups either directly involved in transcription, including miRNAs and long noncoding RNAs, or facilitating transcription by long-range interactions. The latter clusters were specifically enriched with H3K4me1, but less with acetylation of lysine 27 on histone 3 or p300 binding.</p> <p>Conclusion</p> <p>By integrating genomewide data of transcription factor binding and chromatin structure and using our data-driven approach, we pinpointed the chromatin marks that best explain transcription factor association with different regulatory elements. Our results also indicate that a modest selection of transcription factors may be sufficient to map most regulatory elements in the human genome.</p

How a cell decides its own fate: a single-cell view of molecular mechanisms and dynamics of cell-type specification

Biological and Soft Matter Physic