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DISSERTATION- SHWETA ARAS.pdf (2.03 MB)

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Role of SWI/SNF Chromatin Remodeling Enzymes in Melanocyte Differentiation and DNA Damage Response to Ultraviolet Radiation

ARAS, SHWETA
http://rave.ohiolink.edu/etdc/view?acc_num=mco1438970443

Abstract Details

2015, Doctor of Philosophy (PhD), University of Toledo, Biomedical Sciences (Cancer Biology).
Melanocytes are pigment producing skin cells which are located in the basal layer of the epidermis. Melanocytes contribute to about 1-2% of the total skin population and are derived from the differentiation of bipotent neural crest cells. In addition to skin, melanocytes are also present in the epithelial surfaces of mucous membranes, the heart, and cochlea of the inner ear where they perform an important function of melanin synthesis. Microphthalmia - Associated Transcription Factor (MITF) acts as the master regulator of melanocyte differentiation by activating expression of melanocyte specific genes thereby regulating melanin synthesis and melanocyte function. The SWI/SNF chromatin remodeling complex has been implicated in the regulation of differentiation of various cell lineages as well as in the maintenance of embryonic pluripotency. We previously determined that MITF interacts with and also requires the activity of the SWI/SNF chromatin remodeling complex to activate melanocyte gene expression. In the first manuscript, we demonstrate that MITF interacts with the SWI/SNF complex through a subunit of the complex known as “BRG1 or BRM Associated Factor 60 or BAF60”. BAF60 forms a key component of the SWI/SNF complex. There exist three types of BAF60 proteins namely BAF60A, BAF60B and BAF60C that are mutually exclusive of each other. In the first study we have shown that MITF interacts physically with all 3 BAF60 subunits namely BAF60A, BAF60B and BAF60C. We further investigated the role of these three BAF60 subunits in the regulation of expression of melanogenic genes and melanin synthesis. We observed that amongst 3 BAF60 proteins, depletion of BAF60A by short interfering RNA (siRNA) abrogated melanin synthesis the most followed by BAF60B and then BAF60C. Tyrosinase (Tyr) expression was affected by all three BAF60s with most effect observed with knockdown of BAF60A followed by BAF60B and then BAF60C. Expression of other two genes namely Tyrosinase related protein 1 (Tyrp1) and Tyrosinase related protein 2 (Tyrp2) was only affected by BAF60A but not others. Thus, BAF60A was found to be the only protein abrogating the expression of all 3 pigmentation genes thereby signifying its role in melanin synthesis and melanocyte differentiation. Chromatin immunoprecipitation (ChIP) experiments show increased enrichment of BAF60A on the promoters of two key melanin synthesizing enzymes namely TYR and (TYRP1) upon differentiation. Moreover, our results indicate that the enrichment of BAF60A on Tyr and Tyrp1 promoters is dependent on MITF as ChIP assay in cells that are depleted of MITF show reduced enrichment of BAF60A on these promoters. Additionally, enrichment of BRG1 as well as chromatin accessibility at promoters and distal regulatory elements of the genes that control expression of these enzymes was abrogated upon siRNA mediated downregulation of BAF60A, indicating the requirement of BAF60A for BRG1 recruitment. Moreover, we also observed that BAF60A-MITF interaction was a direct interaction and this MITF mediated recruitment of BAF60A containing SWI/SNF complex was required to remodel the chromatin structure at the promoters leading to a more accessible chromatin conformation. These observations collectively suggest that MITF physically interacts and recruits BAF60A containing SWI/SNF complexes to the promoters of target genes and thereby activates melanocyte differentiation. In the second manuscript, we have investigated the role of SWI/SNF chromatin remodeling enzymes in base excision repair pathway of UV radiation induced DNA damage. We have shown that ectopic expression of BRG1, the catalytic subunit of the complex reduces accumulation of UV radiation induced oxidative DNA damage by alkaline comet assay. We also observed that ectopic expression of BRG1 induces the formation of γ-H2AX repair foci which is a well established marker of DNA double strand break repair resulting from the excision of oxidative damage. We further investigated the requirement of SWI/SNF subunits such as BRM which is another ATPase of the complex as well as BAF180 and ARID2 which form a part of the PBAF complex and observed that presence of these subunits was essential for BRG1 to reduce accumulation of UV damage. DNA damage response is usually associated with interaction between chromatin modulators and DNA repair machinery. The role SWI/SNF complex plays in BER has not yet been thoroughly studied. We probed for any possible interaction between SWI/SNF ATPase BRG1 and a BER protein APE1. To our knowledge, this is the first report to demonstrate the role of SWI/SNF complex in the base excision repair pathway of UV induced damage in a cellular context by showing an induced interaction between BRG1 and APE1 upon UV irradiation thereby suggesting a crucial role for this chromatin remodeler to facilitate speedy removal of UV induced oxidative lesions. α-MSH has been previously shown to protect melanocytes against UV radiation induced oxidative DNA damage. We further wanted to determine the requirement of SWI/SNF chromatin remodeling complex in this α-MSH mediated protection against DNA damage. Alkaline comet assay and immunofluorescence staining for γ-H2AX repair foci showed that downregulation of both the ATPases of the SWI/SNF complex compromised a-MSH mediated protection against UV induced DNA damage. We observed that in the absence of α-MSH, siBRG1/BRM cells displayed significantly longer comet tails and lesser γ-H2AX foci formation as compared to siControl cells suggesting that SWI/SNF ATPases are required for reducing the accumulation of DNA damage. UV radiation is a major etiological factor involved in the development of melanoma. Eumelanin which is the brown black pigment has been reported in the literature to be photo-protective in nature. On the other hand, its counterpart pheomelanin which is orange to red in color is responsible for red hair color (RHC) phenotype and is often correlated to increased susceptibility to melanoma. Interestingly, the pheomelanin synthesis pathway has been reported to generate reactive oxygen species (ROS) which in turn cause oxidative DNA damage. It is therefore crucial to understand the mechanisms and molecular players involved in base excision repair pathway of oxidative damages and how dysregulation in these mechanisms could lead to increased susceptibility to melanoma.
Ivana de la Serna (Committee Chair)
Kandace Williams (Committee Member)
William Maltese (Committee Member)
James Willey (Committee Member)
Kam Yeung (Committee Member)
134 p.
Biochemistry; Biomedical Research

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Citations

  • ARAS, S. (2015). Role of SWI/SNF Chromatin Remodeling Enzymes in Melanocyte Differentiation and DNA Damage Response to Ultraviolet Radiation [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1438970443

    APA Style (7th edition)

  • ARAS, SHWETA. Role of SWI/SNF Chromatin Remodeling Enzymes in Melanocyte Differentiation and DNA Damage Response to Ultraviolet Radiation. 2015. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1438970443.

    MLA Style (8th edition)

  • ARAS, SHWETA. "Role of SWI/SNF Chromatin Remodeling Enzymes in Melanocyte Differentiation and DNA Damage Response to Ultraviolet Radiation." Doctoral dissertation, University of Toledo, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=mco1438970443

    Chicago Manual of Style (17th edition)

mco1438970443
426
© 2015, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.