國立虎尾科技大學 |

Massively Parallel Characterization of Enhancers in Evolution and Disease.

紀錄類型:	書目-語言資料,手稿 : Monograph/item
正題名/作者:	Massively Parallel Characterization of Enhancers in Evolution and Disease./
作者:	Klein, Jason C.
面頁冊數:	1 online resource (151 pages)
附註:	Source: Dissertation Abstracts International, Volume: 79-12(E), Section: B.
Contained By:	Dissertation Abstracts International79-12B(E).
標題:	Biology. -
電子資源:	click for full text (PQDT)
ISBN:	9780438174511

Massively Parallel Characterization of Enhancers in Evolution and Disease.
Klein, Jason C.

Massively Parallel Characterization of Enhancers in Evolution and Disease. - 1 online resource (151 pages)

Source: Dissertation Abstracts International, Volume: 79-12(E), Section: B.

Thesis (Ph.D.)--University of Washington, 2018.

Includes bibliographical references

On average, protein-coding sequence is over 99.9% identical between humans, yet some individuals develop disease while others do not. Similarly, protein-coding sequences are over 99% identical between human and chimpanzees despite large phenotypic differences. Our genome makes several different phenotypes using the same set of instructions (genes) by regulating the relative timing and expression level of genes throughout development. One way in which our genome does this is through enhancers.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2018

Mode of access: World Wide Web

ISBN: 9780438174511Subjects--Topical Terms:

599573
Biology.
Index Terms--Genre/Form:

554714
Electronic books.

Massively Parallel Characterization of Enhancers in Evolution and Disease.
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500 $a Source: Dissertation Abstracts International, Volume: 79-12(E), Section: B.
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502 $a Thesis (Ph.D.)--University of Washington, 2018.
504 $a Includes bibliographical references
520 $a On average, protein-coding sequence is over 99.9% identical between humans, yet some individuals develop disease while others do not. Similarly, protein-coding sequences are over 99% identical between human and chimpanzees despite large phenotypic differences. Our genome makes several different phenotypes using the same set of instructions (genes) by regulating the relative timing and expression level of genes throughout development. One way in which our genome does this is through enhancers.
520 $a Enhances were first discovered in 1981 as pieces of DNA that are able to increase gene expression independent of their relative position and orientation to the transcriptional start site (TSS). Despite enhancers being implicated in a variety of evolutionary phenotypes and disease, the field still lacks a comprehensive understanding of how they function. Massively parallel reporter assays (MPRAs) have served as an indispensable tool to screen short pieces of DNA for enhancer activity. MPRAs test the ability of thousands to hundreds of thousands of sequences to increase expression of a reporter gene on a plasmid in a single experiment. However, the assay relies on testing short pieces of DNA (<200bp), which may contribute to the assay's relatively low sensitivity. In chapter two of this dissertation, I discuss a protocol I developed to assemble libraries of short (<230bp) DNA fragments into large (up to 700bp) sequences. This protocol has allowed our group and others to screen large synthetic sequences for enhancer activity as well as for protein folding for the first time.
520 $a In chapters three and four, I apply MPRAs to characterize regulatory changes over primate evolution and identify mutations within enhancers that may contribute to a patient's risk for Osteoarthritis. Enhancers have previously been suggested to play a unique role in evolution and speciation due to their decreased pleiotropy and penetrance. In chapter three, I synthesized and screened present day and ancestral orthologs for 348 liver enhancers in order to characterize each enhancer's evolutionary-functional trajectory throughout 40 million years of primate evolution. We identified groups of enhancers with similar trajectories, most of which could be explained by one or two mutational events. We identified and quantified the correlation between sequence and functional divergence of naturally evolving sequence and implicate cytosine deamination within CpGs as a potential driver in enhancer evolution.
520 $a In chapter four, I applied the same assay to identify common polymorphisms that are associated with Osteoarthritis, which alter enhancer activity. From 35 lead GWAS variants, I identified 1605 SNPs associated with Osteoarthritis in European populations. I then synthesized 197bp around the major and minor alleles for each SNP, and screened each sequence for enhancer activity. We confidently measured enhancer activity of both alleles for 753 SNPs, and found six that drove differential enhancer activity at an FDR of 5%. Our lead SNP increases expression of an isoform of HBP1 (a negative regulator of the Wnt-beta-catenin pathway) with an alternative TSS in an osteosarcoma cell line, CRISPR-edited chondrosarcoma cell line, and cartilage from knee Osteoarthritis patients.
520 $a Chapter five discusses ongoing and future projects to systematically compare different enhancer assays to improve sensitivity and specificity of enhancer screens. It also suggests future applications of these assays to better understand the role of gene regulation in evolution and disease.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2018
538 $a Mode of access: World Wide Web
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