In Vitro Assay to Study Histone Ubiquitination During Transcriptional Regulation.
In mammals, gene expression is largely controlled at the transcriptional level. In response to environmental or intrinsic signaling, gene expression is often fine-tuned by epigenetic modifications, including DNA methylation and histone modifications. One such histone modification is ubiquitination that predominately occurs in mono-ubiquitinated forms on histone H2A and H2B. We recently identified and characterized a novel E3 ligase called TRIM37 that ubiquitinates H2A. This study highlights the consequence of aberrant histone ubiquitination at the promoters of tumor suppressor genes in breast cancer. Regulatory mechanism by which TRIM37 and other auxiliary proteins are involved in the initiation and progression of breast cancer is of utmost importance toward generating effective therapeutics. Here, we describe a detailed step-by-step process of carrying out in vitro ubiquitination assay using purified histone proteins or reconstituted nucleosomes and affinity-purified recombinant E3 ligase like TRIM37. These experimental procedures are largely based on our studies in mammalian cells and will be a useful tool to identify substrate for E3 ubiquitin ligase as well as characterizing new E3 ligases.
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. js2yr@virginia.edu.
Jogender
J
Tushir-Singh
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA. sb5fk@virginia.edu.
Sanchita
S
Bhatnagar
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D057075:Enzyme Assays; D044127:Epigenesis, Genetic; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009707:Nucleosomes / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054875:Ubiquitination
D006657:Histones; D009707:Nucleosomes; D011994:Recombinant Proteins; D044767:Ubiquitin-Protein Ligases
E3 ubiquitin ligase; H2A; Histones; In vitro assay; Nucleosomes; Recombinant protein; Ubiquitination