GSBS Dissertations and Theses

Approval Date

12-18-2014

Document Type

Doctoral Dissertation

Academic Program

Cell Biology

Department

NeuroNexus Neuroscience Institute

First Thesis Advisor

Jeanne B. Lawrence, PhD

Keywords

Cell Aging, Chromatin, Heterochromatin, Satellite DNA, Down Syndrome, Genetic Epigenesis, Protein Unfolding

Subjects

Dissertations, UMMS; Cell Aging; Chromatin; Heterochromatin; DNA, Satellite; Down Syndrome; Epigenesis, Genetic; Protein Unfolding

Abstract

Cellular senescence is thought to play an essential role in many biological functions including tumor suppression and organismal aging. Senescent cells, which are permanently removed from the cell cycle, can be found both in vivo in many different tissue types and in vitro within cultures of non-immortalized cells. Despite their inability to proliferate, these cells persist and remain metabolically active for indefinite periods of time. This physiologic process occurs in response to a variety of cellular insults including oxidative stress, shortened telomeres, constitutive oncogene expression, and DNA damage, and can be initiated by upregulation of one of the two known senescent pathways, involving p16/Rb or p53/p21. The senescent cell phenotype is also characterized by changes to cell and nuclear morphology and to the secretory profile of the cell.

Related to changes in nuclear morphology, epigenetic modifications to the packaging of DNA are thought to be key to the initiation and maintenance of the senescence program. While a large number of earlier studies focused on the findings that senescent cells gain regions of condensed heterochromatin, often in the form of Senescent Associated Heterochromatin Foci (SAHF), this thesis work shows that there is a marked loss of heterochromatin in the peri/centromeric regions of the genome. In fact, both α-satellite and satellite II sequences across the genome distend in a striking and unanticipated fashion; this can be readily visualized by fluorescence in situ hybridization (FISH) as their structure changes from a condensed spot to highly elongated and fine thread-like signals. We have termed this exceptional decondensation of constitutive heterochromatin Senescence Associated Distension of Satellites (SADS). Importantly, a series of experiments shows that SADS is both a consistent and an early event in the cell senescence process, which occurs as a result of every senescence induction method examined. We also observed that this distension was characteristic of both human and murine cells and in vivo in human benign Prostatic Intraepithelial Neoplasia (PIN) tissue. Furthermore, unlike SAHF formation, SADS can occur due to the activation of either of the two senescence pathways, p16/Rb or p53/p21.

Additionally, the cytological dimensions of the thread-like satellite signals indicates that SADS represents “unraveling” of DNA on an unprecedented scale. Thus, it was surprising that this event was not facilitated by changes to several canonical histone modifications associated with condensed heterochromatin, namely H3K9Me3, H3K27Me3, or H3K4Me3, nor is it caused by loss of DNA methylation. Consequently, we believe that this marked distension of satellite DNA is due to changes in higher-order folding of the chromatin fiber. This is important for understanding fundamental events in the cell senescence process, but also provides a unique system for study of chromatin packaging that may provide new insights into the organization of DNA well beyond nucleosome packaging and the ten nanometer fiber. In fact, initial super resolution images of SADS suggest that the satellite sequences may be organized into domains or “globules”. Hence, we suggest that the changes to satellite sequence packaging may be facilitated by changes to higher-order nuclear structural proteins, such as LaminB1, which is reduced in senescent cells.

Finally, this work provides analysis of the literature and preliminary experiments to consider the possibility that there are increased levels of cell senescence in Down syndrome (trisomy 21) cells. As individuals with Down syndrome (DS) experience many manifestations of premature aging (including early-onset Alzheimer’s Disease), have a resistance to solid tumor formation, are more susceptible to oxidative stress, and are trisomic for several genes implicated in causing senescence, our analysis provides plausibility for the hypothesis that accelerated rates of senescence may play a significant role in DS physiology. We also provide results of preliminary studies and outline the next steps for experimentation, using DS fibroblasts and a unique genetically engineered DS iPS cell system. As a final note, the quantification of cell senescence in trisomic versus disomic cells for these experiments relies substantially on the new single-cell marker of senescence discovered and established by this theses work, the Senescence-Associated Distension of Satellites.

DOI

10.13028/M2K30F

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Copyright is held by the author, with all rights reserved.

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