Nucleosomal Context of Binding Sites Influences Transcription Factor Binding Affinity and Gene Regulation

Transcription factor (TF) binding to its DNA target site plays an essential role in gene regulation. The location, orientation and spacing of transcription factor binding sites (TFBSs) also affect regulatory function of the TF. However, how nucleosomal context of TFBSs influences TF binding and subsequent gene regulation remains to be elucidated. Using genome-wide nucleosome positioning and TF binding data in budding yeast, we found that binding affinities of TFs to DNA tend to decrease with increasing nucleosome occupancy of the associated binding sites. We further demonstrated that nucleosomal context of binding sites is correlated with gene regulation of the corresponding TF. Nucleosome-depleted TFBSs are linked to high gene activity and low expression noise, whereas nucleosome-covered TFBSs are associated with low gene activity and high expression noise. Moreover, nucleosome-covered TFBSs tend to disrupt coexpression of the corresponding TF target genes. We conclude that nucleosomal context of binding sites influences TF binding affinity, subsequently affecting the regulation of TFs on their target genes. This emphasizes the need to include nucleosomal context of TFBSs in modeling gene regulation

Insights into distinct regulatory modes of nucleosome positioning

<p>Abstract</p> <p>Background</p> <p>The nucleosome is the fundamental unit of eukaryotic genomes. Experimental evidence suggests that the genomic DNA sequence and a variety of protein factors contribute to nucleosome positioning <it>in vivo</it>. However, how nucleosome positioning is determined locally is still largely unknown.</p> <p>Results</p> <p>We found that transcription factor binding sites (TFBSs) with particular nucleosomal contexts show a preference to reside on specific chromosomes. We identified four typical gene classes associated with distinct regulatory modes of nucleosome positioning, and further showed that they are distinguished by transcriptional regulation patterns. The first mode involves the cooperativity between chromatin remodeling and stable transcription factor (TF)-DNA binding that is linked to high intrinsic DNA binding affinities, evicting nucleosomes from favorable DNA sequences. The second is the DNA-encoded low nucleosome occupancy that is associated with high gene activity. The third is through chromatin remodeling and histone acetylation, sliding nucleosomes along DNA. This mode is linked to more cryptic sites for TF binding. The last consists of the nucleosome-enriched organization driven by other factors that overrides nucleosome sequence preferences. In addition, we showed that high polymerase II (Pol II) occupancy is associated with high nucleosome occupancy around the transcription start site (TSS).</p> <p>Conclusions</p> <p>We identified four different regulatory modes of nucleosome positioning and gave insights into mechanisms that specify promoter nucleosome location. We suggest two distinct modes of recruitment of Pol II, which are selectively employed by different genes.</p

Transcriptional Regulation of a Human H4 Histone Gene is Mediated by Multiple Elements Interacting with Similar Transcription Factors: A Dissertation

Synthesis of histone proteins occurs largely during the S phase of the cell cycle and coincides with DNA replication to provide adequate amounts of histones necessary to properly package newly replicated DNA. Controlling transcription from cell cycle dependent and proliferation specific genes, including histone H4, is an important level of regulation in the overall governance of the cell growth process. Coordination of histone gene transcription results from the cumulative effects of cell signaling pathways, dynamic chromatin structure and multiple transcription factor interactions. The research of this dissertation focused on the characterization and identification of transcription factors interacting on the human histone H4 gene FO108. I also focused on the elucidation of regulatory elements within the histone coding region. Our results suggest a possible mechanism by which a transcription factor facilitates reorganization of histone gene chromatin structure. The histone promoter region between -418 nt and -215 nt, Site III, was previously identified as both a positive and negative cis-regulatory element for transcription. Results of in vitroanalyses presented in this dissertation identified multiple transcription factors interacting at Site III. These factors include H4UA-1/YY1, AP-2, AP-2 like factor and distal factor (NF-1 like factor). Transient transfection experiments show that Site III does not confer significant influence on transcription; however, there may exist a physiological role for Site III which would not be detected in these assay systems. We analyzed the histone H4 gene sequences for additional transcription factor binding motifs and identified several putative YY1 binding sites. Using electrophoretic mobility shift assays (EMSA), we found that Site IV, Site I and two elements within the histone H4 coding region are capable of interacting with YY1. In transient transfection experiments using reporter constructs containing either Site III or one of the coding region elements as potential promoter regulatory elements, and an expression vector encoding YY1, we observed levels of expression up to 2.7 fold higher than from the reporters lacking these elements. Therefore, YY1 appears to interact at multiple regulatory sites of the histone gene and can influence transcription through these elements. Prior to this study, the role of the coding region in histone gene expression was not known. To determine if the coding region is involved in regulating transcription, I constructed and tested a series of heterologous reporter constructs containing various sequences of the histone coding region. Results from these experiments demonstrated that the histone coding region contains three repressor elements. Extensive in vitro analysis indicated that the three repressor elements interact with the repressor CDP/cut. Further analysis showed that CDP/cut interactions with the repressor elements are cell cycle regulated and proliferation specific. CDP/cut interactions increase during the cell cycle when histone transcription decreases. These observations are consistent with the hypothesis that CDP/cutis a cell cycle regulated repressor factor which influences transcription of the histone H4 gene as such. The proximal promoter region of the histone H4 gene between -70 nt and +190 nt is devoid of normal nucleosome structure. This same region contains multiple CDP/cut binding sites. We hypothesized that CDP/cut is involved with chromatin remodeling of the histone gene. DNase I footprinting and EMSA results show purified recombinant CDP/cut interacts specifically with the histone promoter reconstituted into nucleosome cores. Thus, CDP/cutmay facilitate the organization of chromatin of the histone gene. In conclusion, the research presented in this dissertation supports the hypothesis that expression from the human histone H4 gene FO108 is regulated by multiple cis-regulatory elements which interact with several proteins. CDP/cut interacts with Site II, the three repressor elements in the histone coding region and at Distal Site I. YY1 interacts at Site IV, Site III, Site I, and twice in the coding region. ATF/CREB interacts with Site IV and Site I. Distal factor interacts with Site III and within the histone coding region. IRF 2 interacts with Site II and Distal Site I. Thus, histone gene expression is probably regulated by transcription factors CDP/cut, YY1, IRF 2 and ATF/CREB interacting with multiple regulatory elements dispersed throughout its promoter and the coding region. Cell cycle regulation of these transcription factors may contribute to cell cycle dependent expression of the histone gene

Molecular mechanisms of nucleosome positioning and DNA methylation in chromatin

In the eukaryotic cell nucleus DNA needs to be highly condensed. The initial level of DNA compaction is mediated by the wrapping of DNA around histone octamers to form nucleosomes. For efficient DNA metabolism, including DNA replication, transcription, repair and recombination, access to the required sequences must be granted. Hence, nucleosomes need to be highly dynamic. This is mediated by ATP-dependent chromatin remodeling complexes. It is still unclear to what extent these enzymes are influenced by local DNA sequences when shifting a nucleosome to different positions. During my PhD thesis I studied ATP-dependent chromatin remodeling factors focusing on the molecular mechanisms of action in dependence on the underlying DNA sequence. I showed that each individual remodeling enzyme possesses distinct nucleosome translocation properties. The direction (outcome) of nucleosome translocation is determined by its underlying DNA sequence and is influenced by other remodeling complex subunits. I demonstrated that nucleosome positioning by two specific motor proteins is determined by the reduced affinity of the remodeling enzyme to the end product of the reaction. In the following, I characterized the kinetic properties of the DNA methyltransferase Dnmt1 in the context of chromatin. DNA methylation is an important epigenetic modification required for a variety of DNA associated processes. Dnmt1 is responsible for the maintenance of methylation patterns. In a second wave of DNA methylation following DNA replication, Dnmt1 needs to access nucleosomal DNA. Using an in vitro approach, I demonstrated that Dnmt1 requires a minimal length of DNA overhangs to bind to mononucleosomes. Furthermore, in vitro mapping of Dnmt1 interactions with its nucleosomal substrate suggests that Dnmt1 needs to contact flanking DNA as well as nucleosomal DNA for efficient binding. Finally, I could show that Dnmt1 methylation activity is inhibited within the nucleosomal core region. Interestingly, addition of recombinant ATP-dependent chromatin remodeling factors abolish the inhibitory effect of the nucleosome, most likely by rendering the nucleosomal DNA accessible to Dnmt1. Taken together, these results suggest a major role for chromatin remodeling enzymes in nucleosome positioning which in turn might be crucial for epigenetic DNA modifications such as DNA methylation

A nucleosome-free dG-dC-rich sequence element promotes constitutive transcription of the essential yeast RIO1 gene

RIO1 is an essential gene that encodes a protein serine kinase and is transcribed constitutively at a very low level. Transcriptional activation of RIO1 dispenses with a canonical TATA box as well as with classical transactivators or specific DNAbinding factors. Instead, a dGdCrich sequence element, that is located 40 to 48 bp upstream the single site of mRNA initiation, is essential and presumably constitutes the basal promoter. In addition, we demonstrate here that this promoter element comprises a nucleosomefree gap which is centered at the dGdC tract and flanked by two positioned nucleosomes. This element is both, necessary and sufficient, for basal transcription initiation at the RIO1 promoter and, thus, constitutes a novel type of core promoter element

Identification and Characterization of Novel Sir3/MeCP2-Chromatin Interactions

The eukaryotic genome is packaged into chromosomes that are made up of a highly organized and heavily regulated structure called chromatin. The proteins involved in the compaction of DNA into this condensed state are mostly understood at the level of the structure of the nucleosome. The higher order arrangement of chromatin and how it effects gene regulation is only partially understood and characterized. The compaction of nucleosomal arrays into 30-nm and higher structures are partially the responsibility of architectural, or structural, chromatin associated proteins. The following dissertation analyzes the individual chromatin contributions of two well studied architectural proteins, the yeast silencing protein Silent Information Regulator 3 (Sir3) and the human transcriptional regulator methyl CpG binding protein 2 (MeCP2). Silencing in yeast is the responsibility of the SIR family of proteins. Classically, the Sir3 protein has been characterized as associating with chromatin through the hypo-acetylated N-termini of the core histones H3 and H4. The Sir3 protein has recently been found to contain a DNA-binding element, my studies characterized Sir3-nucleic acid interactions and showed that Sir3 can bind to chromatin independently of histone N-termini. In contrast, the MeCP2 protein has classically been characterized as a methylated DNA dependent transcriptional repressor, but recent genome-wide analysis reveals MeCP2 distribution can occur on promoters of active genes. Recent in vitro work with MeCP2 and nucleosomal arrays showed a highly ordered, compacted chromatin structure even in the absence of DNA methylation. MeCP2 is of particular biological interest due to the observed link with the neurodevelopmental iii disorder Rett Syndrome (RTT). My studies demonstrated that MeCP2 can bind in vitro to the Ntermini of core histones H2A, H3, and H4. Additionally, the removal of these tails impacted MeCP2-chromatin interactions, and resulted in a reduced level of nucleosomal array condensation. Importantly, the two RTT mutants analyzed here, R133C and R168X, exhibited differential binding to histone N-termini. These results add to the understanding of chromatin organization and arrangement by demonstrating and characterizing additional chromatin contacts for these two chromatin associated proteins

Evidence that the Boundary Element-Associated Factors BEAF-32A and BEAF-32B affect chromatin structure in Drosophila melanogaster

The Boundary Element-Associated Factors, BEAF-32A and BEAF-32B bind to hundreds of loci on Drosophila chromosomes. These proteins function as insulators; they can prevent promoter activation by an enhancer when placed between them and protect transgenes from chromosomal position effects. To gain insight into BEAF function we designed and expressed a transgene encoding a dominant-negative form of BEAF. This peptide, BID, consists of the BEAF self-interaction domain. We demonstrate here that this peptide interferes with BEAF’s ability to bind DNA and prevents it from functioning as an insulator. In addition, expression of BID leads to a global disruption of polytene chromosome structure. Subsequent work using a fly line with a null mutation in the BEAF gene (BEAF AB-KO) also demonstrates a perturbation to polytene chromosome structure, although it is limited to the X-chromosome. Using Micrococcal nuclease and DNase I we analyzed hypersensitive site alterations in the BEAF AB-KO line, and observed alterations that are consistent with the shifting of positioned nucleosomes. This effect appears limited to regions near promoters. Finally, using fluorescently-tagged BEAF-32A and BEAF- 32B we attempt to characterize the localization and behavior of these proteins. We find that they localize very differently on polytene chromosomes, that BEAF-32B disassociates from mitotic chromosomes while BEAF-32A remains associated, and FRAP experiments indicate different recovery dynamics. This data is consistent with a model that BEAF-dependent insulators function by affecting chromatin structure or dynamics

The role of chromatin accessibility in directing the widespread, overlapping patterns of Drosophila transcription factor binding

Abstract Background In Drosophila embryos, many biochemically and functionally unrelated transcription factors bind quantitatively to highly overlapping sets of genomic regions, with much of the lowest levels of binding being incidental, non-functional interactions on DNA. The primary biochemical mechanisms that drive these genome-wide occupancy patterns have yet to be established. Results Here we use data resulting from the DNaseI digestion of isolated embryo nuclei to provide a biophysical measure of the degree to which proteins can access different regions of the genome. We show that the in vivo binding patterns of 21 developmental regulators are quantitatively correlated with DNA accessibility in chromatin. Furthermore, we find that levels of factor occupancy in vivo correlate much more with the degree of chromatin accessibility than with occupancy predicted from in vitro affinity measurements using purified protein and naked DNA. Within accessible regions, however, the intrinsic affinity of the factor for DNA does play a role in determining net occupancy, with even weak affinity recognition sites contributing. Finally, we show that programmed changes in chromatin accessibility between different developmental stages correlate with quantitative alterations in factor binding. Conclusions Based on these and other results, we propose a general mechanism to explain the widespread, overlapping DNA binding by animal transcription factors. In this view, transcription factors are expressed at sufficiently high concentrations in cells such that they can occupy their recognition sequences in highly accessible chromatin without the aid of physical cooperative interactions with other proteins, leading to highly overlapping, graded binding of unrelated factors