MAY 03, 2018 11:00 AM PDT

Performing ChIP Using Low Amounts of Frozen Mouse Brain Tissue - An Investigation of How Stress Alters 5hmC by Mediating Transcription Factor (TF) Binding

Sponsored by: Covaris
Speaker
  • Neuroscience Ph.D. Candidate at the University of Wisconsin-Madison
    Biography
      Sisi Li is currently a Ph.D. candidate in the lab of Dr. Reid Alisch at the University of Wisconsin-Madison in the Neuroscience Training Program. Her research focuses on the role of the environmentally sensitive novel epigenetic mark, 5-hydroxymethylcytosine (5hmC) in response to stress leading to neuropsychiatric disorders. Since joining the lab in 2013, she has produced three co-first author publications and seven publications over her research career. She has given 15 oral and poster presentations within the university and at conferences across the country and is currently working on her fourth and fifth first author publications.

      Notably, she has collaborated with 13 different laboratories and worked in depth with technical experts in three core facilities across the UW-Madison campus specializing in confocal microscopy imaging, genetic biotechnology and flow cytometry/cell sorting. Her final project and the last portion of her dissertation work is focused on developing a deeper understanding of the molecular mechanisms linking stress, 5hmC, and transcription factor binding using chromatin immunoprecipitation (ChIP)-qPCR with the help of Covaris products to establish that 5hmC can induce alterations in gene regulation by mediating the chemistry of transcription factor binding. Her ultimate goal is to inform the development of new specific and more personalized therapeutic drug treatment options for patients affected by stress-induced neuropsychiatric disorders.

    Abstract

    DATE: May 3, 2018

    TIME: 11:00AM PDT, 2:00PM EDT

     

    While stress is one of the leading causes of neuropsychiatric disorders, the molecular underpinnings of how stress induces alterations in brain chemistry leading to mental illness remains poorly understood. Thus, my research has focused on the novel environmentally sensitive DNA methylation mark 5-hydryoxymethycytosine (5hmC) which is highly enriched in the brain, sensitive to stress, and associated with active neuronal transcription.

     

    During my dissertation work, we have:

    • Observed that stress causes a significant increase in 5hmC levels on the important stress related glucocorticoid receptor gene (Nr3c1).
    • Demonstrated that genome profiling of 5hmC revealed stress-related disruptions across the whole genome, and genes containing differentially hydroxylmethylated regions (DhMR) exhibited significant enrichment in neuronal pathways, suggesting biological relevance.
    • Identified genes with changes in both 5hmC levels and gene expression through subsequent overlapping with RNA-Seq data from the same mice/brain tissue.
    • Discovered an enrichment of known binding sites for transcription factors, suggesting that 5hmC may regulate gene expression by mediating transcription factor binding in both males and females.

     

    With this information at hand, using chromatin immunoprecipitation (ChIP) from a limited amount of brain tissue, we showed that stress resulted in a significant decrease in the binding of a transcription factor to one of the candidate genes with the highest altered 5hmC level due to stress. We further supported our ChIP-qPCR findings using electrophoretic shift assay (EMSA) and western blot. Together, the data from these molecular analyses suggest that 5hmC alters TF binding in response to stress.

     

    In this webinar, the speaker will:

    • Discuss research background and present ChIP-qPCR, RNA-Seq, and EMSA data.
    • Provide tips on performing ChIP with low amounts of frozen brain tissue, a very difficult tissue type to process.
    • Describe how to minimize tissue loss and the importance of formaldehyde fixation in ChIP.
    • How using Covaris AFA and tissue ChIP protocol helped me in my research.

     


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