Histones, the main protein component of chromatin, are essential for storing and organizing the genome in a compact yet accessible manner. DNA wraps tightly around histone oligomers to form nucleosomes which can store unused portions of DNA and regulate accessibility to the replication or transcription machinery. By affecting chromatin structure histones and their modifying enzymes make up a complex network with important regulatory roles in DNA replication, mitosis, and gene expression. In addition to the canonical histones 1-4, there are specialized histone variants with important regulatory functions in transcription or DNA repair for example. The histone variant H3.3 is deposited during replication independent assembly of nucleosomes and is typically enriched in active genes. Although only differing from canonical H3 by four amino acid residues, H3.3 can play important functions in gene expression and differentiation (1). These differing residues affect H3.3 function by allowing interaction with specific nucleosome assembly machinery or through post translational modification (1). While the localization of H3.3 at active genes and promoters is strong evidence for their role in transcription regulation, their exact function is unknown. H3.3 is thought to be important for the inheritance of epigenetic chromatin marks (1).
While much of the work examining H3.3 localization and dynamics has been done using epitope tagged constructs, H3.3 antibodies have been effectively used in various applications. A study by Lin et. al. from the University of San Francisco used an H3.3 antibody for immunofluorescence and was able to monitor endogenous H3.3 levels separately from canonical H3 (2). A genome-wide analysis of H3.3 incorporation during myogenic differentiation used monoclonal H3.3 antibody for ChIP-Seq (3). In addition to cell differentiation, H3.3 is important for chromatin dynamics during spermatogenesis. Using an H3.3 antibody, the Knoepfler group characterized sperm development in their knockout mouse model with a disrupted H3.3 gene locus (4). The knockout mouse displayed abnormal germ cell development and an infertility phenotype (4). These studies demonstrate the utility of H3.3 antibodies to monitor endogenously expressed protein using various techniques. This detection strategy allows the monitoring of chromatin dynamics without altering normal cellular conditions with overexpressed fusion proteins.
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