Browsing by Author "Manandhar, Dinesh"
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Item Open Access HDAC inhibitors cause site-specific chromatin remodeling at PU.1-bound enhancers in K562 cells.(Epigenetics Chromatin, 2016) Frank, Christopher L; Manandhar, Dinesh; Gordân, Raluca; Crawford, Gregory EBACKGROUND: Small molecule inhibitors of histone deacetylases (HDACi) hold promise as anticancer agents for particular malignancies. However, clinical use is often confounded by toxicity, perhaps due to indiscriminate hyperacetylation of cellular proteins. Therefore, elucidating the mechanisms by which HDACi trigger differentiation, cell cycle arrest, or apoptosis of cancer cells could inform development of more targeted therapies. We used the myelogenous leukemia line K562 as a model of HDACi-induced differentiation to investigate chromatin accessibility (DNase-seq) and expression (RNA-seq) changes associated with this process. RESULTS: We identified several thousand specific regulatory elements [~10 % of total DNase I-hypersensitive (DHS) sites] that become significantly more or less accessible with sodium butyrate or suberanilohydroxamic acid treatment. Most of the differential DHS sites display hallmarks of enhancers, including being enriched for non-promoter regions, associating with nearby gene expression changes, and increasing luciferase reporter expression in K562 cells. Differential DHS sites were enriched for key hematopoietic lineage transcription factor motifs, including SPI1 (PU.1), a known pioneer factor. We found PU.1 increases binding at opened DHS sites with HDACi treatment by ChIP-seq, but PU.1 knockdown by shRNA fails to block the chromatin accessibility and expression changes. A machine-learning approach indicates H3K27me3 initially marks PU.1-bound sites that open with HDACi treatment, suggesting these sites are epigenetically poised. CONCLUSIONS: We find HDACi treatment of K562 cells results in site-specific chromatin remodeling at epigenetically poised regulatory elements. PU.1 shows evidence of a pioneer role in this process by marking poised enhancers but is not required for transcriptional activation.Item Open Access Methods for Comparative Analysis of Chromatin Accessibility and Gene Expression, With Applications to Cellular Reprogramming(2018) Manandhar, DineshCellular reprogramming processes remain poorly characterized at the level of genome- wide chromatin and gene expression changes. Specifically, the extent to which re- programmed cells differ quantitatively from both the starting cells and the target cells is unknown for most reprogramming systems. In addition, direct comparisons between the genome-wide reprogramming efficiencies in systems driven by the over- expression of endogenous versus exogenous master regulator(s) are rarely performed. This thesis presents methods for comparative analyses of genome-wide gene expres- sion and chromatin accessibility data, applied to myogenic reprogramming systems in order to assess reprogramming efficiency and generate testable hypotheses for improving the reprogramming process. First, gene expression and chromatin acces- sibility profiles of MyoD-induced transdifferentiated primary human skin fibroblasts are compared to fibroblasts and myoblasts. Second, similar genome-wide changes are assessed for myogenic conversion of iPS cells driven by overexpression of en- dogenous MyoD versus exogeneous MyoD. Both studies show that (i) while many muscle marker genes are reprogrammed after MyoD overexpression, the genome-wide accessibility and gene expression profiles are still different from those of primary my- oblast or myotube cells; (ii) MyoD induces a continuum of changes in chromatin accessibility, with only a fraction of myogenic chromatin sites gaining a completely reprogrammed accessibility status; and (iii) chromatin-remodeling deficiencies are strongly correlated with incomplete gene expression reprogramming. Classification analyses comparing reprogrammed and non-reprogrammed genes or chromatin sites revealed discriminatory genetic and epigenetic features, suggesting ways to poten- tially improve the reprogramming efficiency. Genomic analysis of transgene MyoD overexpression in iPS cells, compared to endogenous MyoD activation, also showed that MyoD is more “aggressive” in its chromatin opening behavior, showing a large number of off-target chromatin opening events. To further investigate the effects chromatin remodeling events on gene expression in reprogramming studies, a novel cross-cell type gene expression prediction framework (CPGex) is also developed. By integrating and modeling the non-linear combinatorial effects of chromatin accessi- bility as well as the expression levels of regulatory TFs, CPGex is able to weigh the importance of regulatory sites or factors for downstream targeted reprogramming of specific gene(s). The methods described in this thesis can be applied to any cellular reprogramming system in order to quantitatively assess the efficiency of reprogram- ming at the chromatin accessibility and gene expression levels, as well as to generate testable hypothesis for improved genome-wide reprogramming.