Project Summary
Complex organisms face daunting “epigenetic challenges”. How is a single genome interpreted to instruct over
one thousand distinct cell fates? How do extracellular signals rapidly and robustly turn on select genes in the
three billion base-pair genome? Epigenetic mechanisms underlie balanced blood cell differentiation and the
speed and scope of cellular responses to pathogens or tissue damage—features that define immunity, tolerance,
and survival during infection. Critical to understanding the mechanisms that “solve” these epigenetic challenges
is the study of histones, proteins that package and regulate the genome. The focus of this project is to reveal the
function of histones and histone post-translational modifications (PTMs) in mammalian organisms. Of particular
interest is Histone variant H3.3, which represents 2 of 15 copies of H3 in the genome but is enriched in
dynamically regulated chromatin such as enhancers, promotors and gene bodies. Additionally, H3.3 is the only
H3 that is expressed in a DNA synthesis independent fashion. For these reasons we have focused on studying
the function of H3.3 residues and modifications in hematopoietic development and immune cell function as these
systems reflect complex mammalian development and rapid cellular responses, and are highly relevant to health
and disease.
Preliminary experiments focused on the function of co-transcriptional modification H3.3S31ph, and loss
of this mark abrogates the ability of a macrophage cell line (RAW264.7s) to respond to LPS. To examine which
other H3.3 residues and modifications are required for this rapid transcriptional response, I have developed a
novel knockout and replacement system in BMDMs (Aim 1). Early results have shown that mutation of certain
lysine residues to arginine (H3.3K4R, H3.3K36R) leads to decreased stimulation-induced transcription, whereas
others (H3.3K9R, H3.3K27R) have no effect. To validate the functional relevance of these results, we have
shown the requirement of H3.3 for in vivo immune response to listeria. Our results will inform ongoing studies to
define dedicated mechanisms for rapid transcription.
Additionally, we will use this model of knockout and replacement to determine the function of H3.3 and
key residues in hematopoietic development (Aim 2). Initial experiments shown the requirement for H3.3 in
hematopoietic stem cell survival, and macrophage differentiation. Targeted and unbiased screening of histone
“readers, writers, and erasers” will enable us to link H3.3 mutant phenotypes to chromatin regulatory pathways
and factors. Together these studies will elucidate how epigenetic mechanisms can regulate cellular differentiation
and the speed and scope of cellular responses. By advancing basic knowledge of the epigenetic mechanisms
regulating these cellular processes, the proposed research will have broad implications for basic biology and
disease, as well as direct implications in bacterial infection and patients with H3.3 pathway mutations.