Development of a Targeted Chromatin Associated Protein Purification Method Identifies Novel Histone Gene Regulators
- Author(s): Tsui, Chiahao Kevin
- Advisor(s): Tjian, Robert
- et al.
Regulation of gene expression is fundamental to many biological processes such as lineage specification during embryonic development or metabolic responses to environmental cues. Classic in vitro biochemical experiments have been successful at identifying various components that are necessary for controlling gene expression, such as transcription factors, and co-activators as well as components of the machinery that regulate RNA processing, stability and degradation. However, detailed mechanistic understanding of gene regulatory mechanisms for many specific genes and gene networks remains unknown. Recently, many labs have attempted to couple mass spectrometry with purification methods that enrich for a specific genomic locus to identify potential novel regulators associated with specific chromosomal targets of interest. Here, I describe the development of a novel dCAS9 mediated purification method for proteins associated with specific cis-regulatory chromatin elements. First, I established the proof of principle by identifying specific human telomere associated proteins. I then further advanced this “reverse-ChIP” strategy by applying it to the de novo identification of novel regulators linked to the Drosophila histone gene locus.
Chapter 1 provides an overview of what is currently known and unknown about gene regulation at the Drosophila histone cluster as well as a summary of recent advances in the field of “reverse-ChIP” techniques. Chapter 2 describes my development of the dCAS9 mediated purification method for proteins specifically associated with targeted chromatin elements, including the purification and identification of proteins bound to human telomeres. Chapter 3 details how I’ve adapted the dCAS9 mediated purification method to identify novel regulators of the Drosophila histone cluster.