1,721,037 research outputs found
An Open and Shut Genome: The Dynamic Relationship between DNA Sequence, Transcription Factors, and Chromatin in S. cerevisiae
Get PDFTranscription factors bind to regulatory regions to help activate or repress genes. Compaction of the genome into nucleosomes helps reduce the physical space that must be sampled to find in vivo targets, yet forces transcription factors and histones to compete for access to DNA. The equilibrium of this competition can be altered via post-translational modifications of the histone core and regulation of nucleosome position. I examined the differential contributions of DNA sequence, transcription factors, and chromatin to the regulation of gene expression in the model organism, Saccharomyces cerevisiae. While transcription factors bind DNA in a sequence specific fashion, the contribution of DNA in determining in vivo usage of consensus sequences is specific to the biological role of the transcription factor itself. Chromatin shows limited changes in different growth conditions, but localized changes surrounding transcription factor binding sites are evident. Chromatin stability appears to be regulated by the underlying DNA sequence of the genome and modified by active mechanisms such as post-translational modifications. Together, these processes establish stable chromatin in the body of genes and unstable chromatin at promoters. Depletion of nucleosomes is sufficient to alter transcription of 50% of the yeast genome, suggesting that chromatin plays a major role in regulating gene expression via regulating DNA accessibility to transcription factors as well as less direct effects, such as blocking assembly of the transcriptional machinery
The role of Set2, transcription factor residence, and nucleosome spacing in the dynamic access of genomic information
Get PDFDNA is a heteropolymer that serves as a mutable form of storage for genomic information. Nucleosomes condense genomes by wrapping 147 bp of negatively charged DNA around a positively charged histone core. Histone modifications and selective placement of nucleosomes expand and allow for regulated access of the information content in DNA. Understanding and predicting the placement and organization of nucleosomes, as well as the dynamics of genome utilization, is therefore critical for expanding our knowledge of life. A complex set of machinery regulates RNA polymerase II passage through a nucleosomal template. Loss of the histone H3K36 methyltransferase, SET2, leads to aberrant (cryptic) transcription initiation from within the coding region of genes due to an inability to regulate chromatin reassembly following transcription. We used whole genome microarrays to map and identify sites of aberrant transcription initiation in set2Δ. We developed a statistically principled algorithm to show there is no evidence that cryptic initiation occurs more frequently in long or infrequently transcribed genes. I adapted an assay to study the residence dynamics of the S. cerevisiae transcription factor, Rap1, genome-wide. Rap1 binds with a long residence at highly transcribed genes promoters. These sites typically have a high Rap1 affinity motif and low in vitro affinity for the formation of nucleosomes. In contrast, we find that sites with short Rap1 binding typically have high nucleosome occupancy and fast histone turnover. We propose that an active regulated competition between transcription factors and nucleosomes can regulate transcription factor residence and function. The HMGB class of proteins is known to influence the dynamics of nucleosomes and transcription factors. We mapped the distribution of the major nuclear HMGB containing proteins by ChIP-seq, genome accessibility using FAIRE-seq, and mapped nucleosomes using MNase-seq in an HMGB mutant. We identified linker length differences between several strains. This linker length change allowed us to identify invariant nucleosome boundaries and test the underlying principles of nucleosome positioning in S. cerevisiae. Collectively, these studies provide a richer picture of how DNA access is regulated by complex nucleosome-mediated mechanisms.Doctor of Philosoph
The genomic distribution and function of NFI and histone variant H2A.Z during C. elegans development
Get PDFDevelopment relies on precise spatial and temporal access of the transcription regulatory machinery to genomic information. Throughout development, transcription factors bind a discrete set of specific targets that cannot be predicted through sequence alone. Identification and access to regulatory elements is heavily influenced by the heterogeneous chromatin landscape that packages eukaryotic genomes. Local chromatin environments may specify a genomic index of sequence availability and genome function. To begin to examine this genomic index in development, I mapped the occupancy of a developmentally essential variant histone, H2A.Z, and a model transcription factor, NFI-1. In one of the first utilizations of Chromatin ImmunoPrecipitation on genomic DNA microarrays (ChIP-chip) in C. elegans, I show that the histone variant H2A.Z occupies a subset of promoters that tend to be required for development and occupied by RNA Polymerase II. As in other metazoans, we show that C. elegans H2A.Z is required for proper development. H2A.Z identifies transcription start sites in both canonical genes and genes likely to be independently regulated within operons. Fewer sites of H2A.Z occupancy exist on the X chromosome, though the data suggests no direct role for H2A.Z in dosage compensation. Our data suggests H2A.Z plays a vital role in establishing or maintaining a specialized chromatin environment at developmental promoters. We then mapped the in vivo binding of the animal-specific transcription factor, NFI. Despite a overabundance of the discovered binding motif in the genome, NFI binds few sites in vivo. There is little difference in the in vitro and in vivo sequence affinity of NFI. In vivo NFI sites have low nucleosome occupancy, suggesting that nucleosomes positioned at NFI binding sites may be susceptible to loss. C. elegans NFI targets are conserved, and 84% of the C. briggsae homologs have NFI promoter motifs. This study provides a basis for understanding NFI function and recruitment to a paucity of in vivo sites, despite an abundance of sequence motifs. These studies present the first genome-wide maps of a chromatin component and transcription factor in C. elegans and provide a foundation for future studies of chromatin and transcription factor-genome interactions in a native developmental context
Chromatin Profiles of Human Cells in Health and Disease Using FAIRE
Get PDFBreast cancer is a heterogenous disease comprised of molecularly distinct subtypes with diverse clinical outcomes. Understanding the molecular composition of each subtype will aid in the effective diagnosis and treatment of breast cancer. The composition and activity of subtype-selective regulatory pathways operate, in part, through binding of proteins at distinct sites throughout the genome, often referred to as regulatory elements, to govern levels of gene expression. One of the characteristics of these binding events is the displacement of nucleosomes. Here we have developed a technique called FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements), which is capable of the genome-wide identification of active regulatory elements in human cells based on the nucleosome-depleted nature of these sites. Using FAIRE we have identified the genome-wide set of active regulatory elements in the luminal and basal-like tumor subtypes. Here most of the active regulatory elements were distinct to each subtype and tended to occur not at transcriptional start sites, but at distal regulatory elements. Many of these unique sites also reflected the activity of the regulatory mechanisms present in a given subtype. For example, in the hormone-responsive luminal cells we detected strong FAIRE signals at estrogen-receptor alpha binding sites, whereas the signals are diminished or absent in the hormone nonresponsive basal-like cells. These distal regulatory elements tended to be clustered to form distinct genomic domains containing the set of all expressed genes in the respective subtype, regardless of whether the gene was differentially expressed between the subtypes. This suggests that the combination of gene expression and the subtype-selective active regulatory elements provides an expanded understanding of the molecular complexity between subtypes. The subtype-selective regulatory elements were also enriched with sequence motifs for DNA-binding proteins, which included factors known to be active in the respective subtypes. The remaining sequence motifs should serve as a useful starting point for the identification of additional candidates that distinguish the subtypes, especially for the relatively uncharacterized basal-like subtype. We also used FAIRE to investigate the set of active regulatory elements associated with the transformation of a mammary epithelial cell line to a cancerous phenotype, which included a subset of the population becoming cancer stem cells. The transformed state was achieved with only modest changes in the set of active regulatory elements (5%). The transformation can instead be attributed to relatively subtle changes in the expression of transcription factors that share a common DNA-binding site, which through both competitive and cooperative interactions at existing regulatory elements alter regulatory interactions and global expression levels. Examination of the cancer stem cells, isolated by flow-cytometry, using FAIRE revealed quite unexpectedly that these cells were in fact derived from a separate starting population. Although these findings ultimately left us with many unanswered questions, it provided us an opportunity to explore the properties regulatory components underlying the cancer stem cell phenotype. Together, these findings indicate that FAIRE will be a powerful tool for discovery of the molecular characteristics underlying cancer and that FAIRE holds promise as a clinical diagnostic tool
Histone H3 Lysine 36 modification distinguishes transcribed and non-transcribed regions of the S. cerevisiae genome
Get PDFChromatin plays a dynamic role in regulating gene transcription. Regulation of accessibility of DNA template is mediated in part by nucleosome occupancy such that nucleosomes are relatively depleted upstream of genes and relatively enriched in the coding regions. One of the factors that influence this differential nucleosome occupancy is histone post translational modifications. One such modification is dimethylation of histone H3 at Lysine 36 (H3K36me2). It is mediated by Set2, a histone methyl transferase (HMT) in yeast which had been shown to associate with RNA polymerase II (RNA pol-II) during transcription elongation at individual loci. To study the role of Set2 in gene regulation, I sought to determine the genome wide localization of H3K36me2. Using chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip), we show that H3K36me2 is predominantly localized to RNA pol-II transcribed regions and is depleted in the regulatory (promoter) regions genome-wide. Mating loci, telomeres, RNA pol-III transcribed regions have scarce or low levels of H3K36me2. H3K36me2 modification begins within RNA pol-II transcribed ORFs at approximately same location, independent of the length of the ORF. This further confirms that Set2 associates with RNA pol-II after the initiation phase of transcription. Levels of H3K36me2 do not correlate with the transcriptional frequencies of genes. However, genes that are transcribed at some detectably level tend to have higher levels of H3K36me2 than genes that are completely repressed. H3K36me2 therefore acts as a mark that demarcates coding and regulatory regions. The function of such a mark became clear with the finding by other groups that localization of Set2 and H3K36me2 at coding regions was essential for maintaining the fidelity of transcriptional initiation. Absence of Set2 leads to hyperacetylation in the coding regions and, as a consequence, aberrant initiation events. My studies show that H3K36me2 is a chromatin mark that demarcates functionally distinct regions of the genome by marking the coding regions specifically. Studies by others show that this localization of H3K36me2 is important for maintaining proper chromatin structure. H3 Lysine 36 is also acetylated and ChIP-chip analysis showed that H3K36ac is enriched in the promoter regions in the entire yeast genome. The function of H3K36ac is not yet known but it is possible that one way H3K36me2 is restricted to the coding regions by acetylating this residue in the regulatory regions. Another way organisms demarcate specific functional boundaries is by restricting tri methyl Lysine 4 at histone H3 (H3K4me3) to the 5' end of coding regions. Ctk1, a kinase that has been shown to phosphorylate Serine 2 of C-terminal domain (CTD) of RNA pol-II was shown to regulate the levels of H3K4me3. Ctk1 is required for the recruitment of Set2 to RNA pol-II. My genome wide studies show that absence of Ctk1 causes spreading of H3K4me3 into the 3' region of ORFs globally resulting in disruption of chromatin structure within the ORFs and occurrence of aberrant transcription initiation. These studies show that specific histone modification patterns are important for maintaining chromatin structure. Organisms have developed multiple mechanisms to ensure proper localization of these modifications disruption of which could cause disturbances in transcriptional programs
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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