1,721,030 research outputs found
Characterization and analysis of repetitive centromeres
Get PDFThesis (Ph.D.)--University of Washington, 2017Centromeres are specialized regions of eukaryotic chromosomes that ensure faithful transmission of genetic information at each cell division. The molecular architecture of centromeres is defined by evolutionarily dynamic protein and DNA components, which have been proposed to contribute to the origin of new species, while defects in centromeres have been linked to human disease. Centromeres are embedded in regions composed of large arrays of head-to-tail 'satellite' DNA elements, which are not amenable to many conventional genomic analyses. Here, I describe the development of methods for the analysis of repetitive genomic regions and apply these tools to study primate centromeres, which are composed of ~170-bp alpha-satellite units. Although centromeric DNA is known to be polymorphic in humans, comprehensive cataloguing of variants at centromeres has not been possible. To gain insight into centromeric genetic variation, I developed a method that uses single-molecule sequencing for analyzing characteristic sequence periodicities called higher-order repeats that arise in human centromeres. The application of this approach to catalogue inter-individual, population-scale, and disease-associated structural variation identified extensive polymorphism in centromeres associated with binding sites for CENP-B, a sequence-specific DNA binding protein. This work also defined a set of functionally important alpha-satellite dimeric units that are underrepresented in current centromere models and demonstrated aberrations in centromeric sequence in breast cancer. I suggest a role for CENP-B in the evolution and maintenance of higher-order periodicities in centromeric arrays. Although alpha-satellite is present at the centromeres of most primates, the precise mechanisms of evolution of centromeric DNA and the contribution of genetic sequence to the specification of centromere identity remain unresolved. I examined centromere evolution in primates using a combination of data from different whole-genome sequencing methods. This approach demonstrated the presence of higher-order periodicities in all primates and identified an important role for CENP-B in shaping centromeric repeat organization. Further analysis of alpha-satellite uncovered interspecific variation in the presence of short inverted repeats, which may form hairpin and stem-loop structures. Based on these data, I propose a genetic mechanism for centromere specification that depends on the formation of cruciform or other non-B-form nucleic acid structures. Taken together, this work enables the cataloguing of variation in satellite DNA, defines important evolutionary transitions in primate centromeres, and advances a model for primate centromere evolution and a theory for centromere specification
Epigenomic profiling of human tissues at single-cell resolution
No full textThesis (Ph.D.)--University of Washington, 2022Traditional methods for profiling DNA-protein binding interactions have been limited by low signalto-noise, false positives, and high costs. To overcome these barriers we developed a simple assay,
Cleavage Under Targets & Tagmentation (CUT&Tag), that leverages a transposon based fusion enzyme
to map in situ DNA-protein interactions in small samples of cells at high resolution. We then automated
CUT&Tag to generate hundreds of chromatin profiles for a multitude of histone modifications across
different diseases. Furthermore, we are able to model their cell-type specific gene expression by
integrating the data across multiple histone modifications.
CUT&Tag is characterized by an exceptionally high signal-to-noise ratio and we reasoned that
the method could be used to resolve single-cell chromatin profiles. As a proof-of-concept we
demonstrated that single-cell CUT&Tag resolves both active and repressive chromatin marks in cell lines.
We then leveraged single-cell CUT&Tag to profile thousands of single cells to uncover the heterogeneity
in stem cell development, primary liquid, and solid tumors pre- and post-treatment. Our work is part of a
large-scale effort to build a comprehensive map of all cell types to better understand human health and
improve disease diagnosis and treatment
Twists and Turns of Transcription: Dynamic interplay between RNA Polymerase II and the nucleosome
No full textThesis (Ph.D.)--University of Washington, 2013Transcription regulation underlies basic processes essential to life, including differentiation and development, cell-to-cell communication, and response to environmental stimuli. How the cell achieves a precise gene expression system to maintain cellular identity while allowing for plasticity remains an important biological question. As the interface between DNA and DNA-binding factors, chromatin exerts substantial influence on transcriptional regulation through its fundamental unit, the nucleosome. The following thesis addresses the questions of how nucleosomes influence transcriptional regulation, how RNA Polymerase II (Pol II) affects nucleosome stability and dynamics, and how Pol II overcomes the nucleosomal barrier. Using the heat shock response as a model for transcriptional regulation, we found that nucleosomes of activated genes increased in turnover, while those of repressed genes exhibited decreased turnover, suggesting that the act of transcription causes nucleosome turnover. This causality challenges the role of histone modifications in regulating gene expression, as modifications must be re-established after each turnover event. Furthermore, we discovered that the transcription-driven nucleosome turnover is partly mediated by the torsional stress on the DNA generated during Pol II translocation. Nucleosomes were destabilized as Pol II generates positive torsion ahead, and stabilized by the negative torsion behind, providing a mechanism for efficient Pol II progression while maintaining chromatin structure and organization
High-resolution studies of the chromatin and transcription landscape
Get PDFThesis (Ph.D.)--University of Washington, 2014Transcription from genomic DNA is regulated in many ways and governs cellular identity, growth, and homeostasis. If transcription becomes deregulated, it can serve a critical role in the development of cancer and other human pathologies, however complete understanding of this process is lacking. In eukaryotes, transcriptional regulation involves a balance between repressive packaging of the genome into nucleosomes and enabling access to regulatory proteins as well as RNA polymerase II (RNAPII). Nucleosomes are strong physical barriers to transcription in vitro that cause RNAPII to backtrack and arrest. Yet, in vivo RNAPII must transcribe across many nucleosomes for every gene at a very high rate. How this happens and what mechanisms enable RNAPII transit through nucleosomes has long remained unclear. Here we show that the nucleosomes of active genes have a distinct histone composition which involves the replacement of both canonical H2A histones with histone variant H2A.Z. These homotypic H2A.Z nucleosomes show evidence of disruption during transcriptional elongation, suggesting that they are formed through transcription mediated turnover and then replacement with H2A.Z. Homotypic H2A.Z nucleosomes have unique physical properties and interact with distinct chromatin remodelers from canonical nucleosomes. Hence, we developed a single-nucleotide resolution approach to map RNAPII genome-wide and determine the nature of transcription through these nucleosomes. We show that the entry site to the nucleosome is most refractory, contrary to existing models based on transcription in vitro, and that the first nucleosome from the transcription start site (+1) is a much larger barrier than downstream nucleosomes that causes RNAPII to backtrack. Nucleosome occupancy positively correlates with the magnitude of the barrier, however our results suggest that an evolved function of H2A.Z is to ease the inhibitory nature of nucleosomes on transcription. This helps to explain why H2A.Z is most enriched where the barrier is the largest and also why it is essential in development where transcriptional fidelity is critical
Expansion of human centromeric arrays in cells undergoing break-induced replication
No full textThesis (Ph.D.)--University of Washington, 2024Human centromeres are located within highly homogeneous mega-sized α-satellite arrays and evolve rapidly, which can lead to variation in array lengths and sequences. Proposed mechanisms for such alterations are homology directed repair mechanisms including unequal cross-over between sister chromatids, gene conversion, and break-induced replication. However, the underlying molecular mechanisms responsible for the massive, complex, and rapid sequence turn over, length variation, and homogeneous organization of centromeric arrays have not been experimentally validated. This dissertation project investigates whether centromeric array expansion and contraction can occur within limited somatic cell divisions and the molecular mechanisms responsible for this change. This thesis work has demonstrated that centromeric array length can change in somatic cells (in ~20 cell divisions) of different cell lines, with various magnitudes, in a chromosome specific manner. In addition, centromeric arrays expand more frequently than contract, which can counteract the loss of SSA of DSBs at centromeres leading to an overall increase in array length. Large contractions can occur, but usually only when the array length is significantly longer than the population average. Finally, this array length change does not occur without the BIR essential proteins RAD52 and PIF1, indicating that BIR can drive centromere sequence evolution in cells undergoing BIR. This project provided key insights into a longstanding fundamental question: how centromere sequences evolve
Centromeric localization and adaptive evolution of an arabidopsis histone H3 variant
Get PDFCentromeric H3-like histones, which replace histone H3 in the centromeric chromatin of animals and fungi, have not been reported in plants. We identified a histone H3 variant from Arabidopsis thaliana that encodes a centromere-identifying protein designated HTR12. By immunological detection, HTR12 localized at centromeres in both mitotic and meiotic cells. HTR12 signal revealed tissue- and stage-specific differences in centromere morphology, including a distended bead-like structure in interphase root tip cells. The anti-HTR12 antibody also detected spherical organelles in meiotic cells. Although the antibody does not label centromeres in the closely related species Arabidopsis arenosa, HTR12 signal was found on all centromeres in allopolyploids of these two species. Comparison of the HTR12 genes of A. thaliana and A. arenosa revealed striking adaptive evolution in the N-terminal tail of the protein, similar to the pattern seen in its counterpart in Drosophila. This finding suggests that the same evolutionary forces shape centromeric chromatin in both animals and plants.Fil: Talbert, Paul B.. Howard Hughes Medical Institute; Estados UnidosFil: Masuelli, Ricardo Williams. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Howard Hughes Medical Institute; Estados UnidosFil: Tyagi, Anand P.. Howard Hughes Medical Institute; Estados Unidos. University of Washington; Estados UnidosFil: Comai, Luca. University of Washington; Estados UnidosFil: Henikoff, Steven. Howard Hughes Medical Institute; Estados Unido
Evidence of influence of genomic DNA sequence on human X chromosome inactivation.
No full textA significant number of human X-linked genes escape X chromosome inactivation and are thus expressed from both the active and inactive X chromosomes. The basis for escape from inactivation and the potential role of the X chromosome primary DNA sequence in determining a gene's X inactivation status is unclear. Using a combination of the X chromosome sequence and a comprehensive X inactivation profile of more than 600 genes, two independent yet complementary approaches were used to systematically investigate the relationship between X inactivation and DNA sequence features. First, statistical analyses revealed that a number of repeat features, including long interspersed nuclear element (LINE) and mammalian-wide interspersed repeat repetitive elements, are significantly enriched in regions surrounding transcription start sites of genes that are subject to inactivation, while Alu repetitive elements and short motifs containing ACG/CGT are significantly enriched in those that escape inactivation. Second, linear support vector machine classifiers constructed using primary DNA sequence features were used to correctly predict the X inactivation status for >80% of all X-linked genes. We further identified a small set of features that are important for accurate classification, among which LINE-1 and LINE-2 content show the greatest individual discriminatory power. Finally, as few as 12 features can be used for accurate support vector machine classification. Taken together, these results suggest that features of the underlying primary DNA sequence of the human X chromosome may influence the spreading and/or maintenance of X inactivation
Chromatin landscape of the "dark matter of the genome": centromeres of S. cerevisiae and repeat sequences of D. melanogaster.
Get PDFThesis (Ph.D.)--University of Washington, 2014The chromatin landscape plays a major role in defining cell phenotypes through transcriptional regulation and specification of the main features of chromosome, such as centromeres and pericentric heterochromatin. Studies of the chromatin landscape so far have been mostly confined to the protein-coding part of the genome. In this work I present a study of the chromatin landscape of two non-coding regions: centromeres in budding yeast Saccharomyces cerevisiae and pericentric repeat sequences of the fruit fly Drosophila melanogaster. I have shown that the centromere of budding yeast contains a nucleosome with a special structure, called a hemisome. This finding eliminated other previously proposed models of the centromeric nucleosome and reconciled previous conflicting observations. I also developed a method to quantify enrichment of repeat sequences in Chip-Seq experiments and used it to construct an epigenetic map of heterochromatin in D. melanogaster using public datasets Drosophila Genetic Reference Panel (DGRP) and modENCODE. This analysis yielded several unexpected biologically interesting findings such as preferential association of HP1a protein with transposable elements and depletion of nucleosomes from AT-rich short repeats sequences
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
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