Targeting H4K20 methylation to rejuvenate aged stem cell epigenome and regenerative function.

ABSTRACT
As we age, the intrinsic ability of stem cells to self-renew and differentiate to maintain tissue integrity dramatically declines.
Therefore, understanding the processes leading to stem cell dysfunction with age is essential for the future development of
novel, effective stem cell-based therapies to treat disorders associated with aging. Therefore, my long-term goal is to
elucidate the epigenetic mechanisms of stem cell aging, manipulate them to rejuvenate aged tissue, and promote healthy
aging. More specifically, the insight provided by this proposal would be used to devise strategies to rejuvenate muscle and
hematopoietic stem cell function, and therefore promote skeletal muscle recovery and reduce age-associated systemic low-
grade chronic inflammation. To accomplish this objective, we will utilize mouse genetic models, models of skeletal muscle
degenerative injury and moderate exercise (voluntary wheel running; VWR), cell culture systems, imaging analysis, small
molecule inhibitors, flow cytometry analysis, physiological measures of recovery, genomics, and epigenomics (Cleavage
Under Targets and Tagmentation; CUT&Tag). In aged mice, both muscle stem cell (MuSC) and hematopoietic stem and
progenitor cell (HSPC) quiescence is disrupted, leading to reduced regenerative capacity. Recent studies used VWR to
restore quiescence and rejuvenate both MuSC and HSPC function in aged mice. The epigenetic landscape in both stem cell
populations changes dramatically, yet the mechanisms underlying these events as well as their contribution to age-associated
dysfunction remain understudied. The lysine methyltransferase 5a (Kmt5a) is the sole enzyme catalyzing monomethylation
of lysine 20 on histone H4 (H4K20me1), which is required for subsequent di- and tri-methylation by Kmt5b and Kmt5c,
respectively. Methylation of H4K20 is critical for chromatin organization and regulation of transcription, yet its role in adult
stem cells is entirely unknown, especially in the context of aging. Our preliminary data show that Kmt5a and H4K20me1
decrease in aged MuSCs. Specific deletion of Kmt5a in MuSCs recapitulates aging phenotype by decreasing the pool of
stem cells, suggesting disruption of quiescence and impaired self-preservation of the pool. Using the recently developed
epigenomic technique CUT&Tag, we assessed H4K20me1 in adult and aged quiescent MuSCs and found that H4K20me1
is mostly located at the genes’ transcriptional start site and significantly decreases with age. Further analysis revealed that
age-associated loss of H4K20me1 silenced numerous Notch genes including Rbpj, critical to maintaining MuSC quiescence.
Significantly, Kmt5a inhibition and subsequent loss of H4K20me1 in MuSCs led to decreased RNA Polymerase II serine 2
phosphorylation, suggesting the impaired release of promoter-proximal pausing and therefore potent gene silencing. Thus,
we propose to examine if the loss of Kmt5a, and consequently H4K20me1, in aging MuSCs contributes to the disruption of
their quiescence state. Also, we will determine the role of Kmt5a in regulating RNA Polymerase II promoter-proximal
pausing, and how this proposed mechanism contributes to controlling MuSC fate and function. Last, we will determine if
moderate exercise using a VWR model can rejuvenate MuSC and HSPC epigenome through the restoration of H4K20
methylation. The specific aims of this proposal are: 1) Determine the role of Kmt5a in MuSC quiescence regulation during
aging and 2) Determine the impact of VWR on Kmt5a-mediated epigenetic remodeling in aged MuSC and aged HSPC.

Grant Number: 5R00AG071736-04
NIH Institute/Center: NIH
Principal Investigator: Romeo Blanc