Epigenetic mechanisms of mammalian tissue aging

National Institute On Aging

Investigators

Linked publications, trials & patents

Abstract

Investigating the role of chromatin effector proteins in muscle stem cell aging The skeletal muscle constitutes 35% of the body mass and is responsible for motor activity, thermoregulation and energy storage. Muscle stem cells (MuSCs) were first identified in electron micrographs of frog muscle as satellite cells that remain in juxtaposition to myofibers under the basal lamina. Under normal circumstances, the majority of MuSCs are in a mitotically quiescent state (QSCs) with little turnover. Over time, QSCs undergo primarily chronological aging. In response to muscle injury, however, a proportion of the QSCs become activated to enter the cell cycle (ASCs). Hence, population studies of aged MuSCs may reflect both chronological and replicative aging. The percentage of MuSCs drops dramatically in aged mice and human muscle and the cells take longer time to activate and reenter the cell cycle. MuSC ablation studies have established their role as chief myogenic progenitors in the adult. A partial picture of the epigenomic landscape in aged QSCs and ASCs is available through the work of Rando and colleagues. They explored DNA and histone methylation status (H3K4me3, H3K27me3, bivalency and H3K36me3) and concomitant gene expression changes during aging. This work identified JMJD3 as an important demethylase that increases in old QSCs and contributes to an enrichment of H3K27me3. Our ongoing efforts propose to complete the chromatin landscape of aging MuSCs by incorporating other dynamic histone modifications (histone acetylation) and key epigenetic factors such as BRD4, EP300 and CBP that mark enhancers. We have successfully isolated MuSCs in the lab and generated CUT&RUN libraries. We are currently analyzing the data. Using the above landscape as a foundation, we will compile a list of epigenetic effectors that can be targeted in MuSCs to reverse age-related changes in chromatin and improve regenerative potential. We will use constructs that harbor guide RNAs targeting protein domains in epigenetic enzymes to screen for key players in stem cell aging. Alternatively, we will screen a panel of epigenetic inhibitors that improve differentiation potential in old muscle stem cells Finally, positive candidates from the screen(s) will be followed up by manipulating aged MuSCs, transplanting into old mice and testing improvement in stem cell function with a panel of in vitro and in vivo assays. Taken together, this work will identify key epigenetic players in MuSC function and point to therapeutic avenues to improve age-related muscle degeneration. Single-cell analysis of the aging and regenerating liver The liver is the primary metabolic organ responsible for nutrient metabolism, detoxification, production of important hormones and immune function. The liver has the remarkable ability to regenerate after resection, although this ability declines strongly with age. It is not fully clear if there is a stem cell population in the adult liver, particularly given that all hepatocytes show hypertrophy and that rodent hepatocytes can re-enter cell cycle at least once after 70% hepatectomy (PH), negating the need for stem cells. However, other studies that employ lineage tracing and transplantation have revealed clonogenic origins from peri-portal regions. With the advent of single-cell genomics technology, we think we will now be able to resolve this question. Our primary objective in this Aim is to characterize cell state changes and identify populations that are disrupted during normal aging and contribute to impaired regeneration in old mice. Our long-term goal is to isolate these deficient cells from old mice, reset their chromatin and transplant them back for improved liver function. We have successfully performed several PH surgeries in young and old mice and characterized chromatin changes using bulk assays. We have also generated single-cell chromatin accessibility profiles of these livers. Currently, we are correlating our bulk and single-cell findings. Taken together, this work will both identify key mechanisms that induce cell composition changes and functional decline in aging liver. Investigating the role of histone acetyltransferases in brain aging Previous work in senescent cells has shown the critical role of p300 (but not paralogous CBP) in seeding the formation of new enhancers that drive senescence genes. Importantly, depletion of p300 delays the onset of senescence and could be a potential anti-aging target. Interestingly, both CBP and p300 are highly expressed in different brain regions. Conventional, conditional and brain-specific knockouts of CBP function in adult mice show a range of motor, memory and learning defects. In contrast, p300 knockouts are less deleterious. Additionally, the role of these critical histone acetyltransferases in the brain during aging remains unexplored. Thus, the major goal of this Aim is to investigate the roles of paralogous histone acetyltransferase enzymes, p300 and CBP, in brain aging. We have used AAV-Cre mediated knockout of p300 in young and old floxed mice and perform several neurological and behavioral tests for assessing learning and memory in mice. We will ultimately perform further downstream chromatin analysis to ascertain the function of p300 particularly at enhancer regions in the aging brain at both single-cell and bulk levels. Taken together, this work will establish whether p300 is a viable anti-aging target in the brain and provide molecular insights into how p300 exerts action at the non-coding genome to influence age-related decline in brain function. In fiscal year 2021, we have continued our investigations as outlined in the directions above. We have collaborated with Dr. Vittorio Sartorelli to isolate muscle stem cells with FACS and have generated trial ChIP-seq libraries that will be sequenced soon. We have also performed single-cell chromatin analysis of the aging and regenerating liver and are currently analyzing this data using standard computational pipelines. Additionally, we have generated mass spec data identifying key modifications that are increased with age, bulk RNA-seq data and ChIP-seq data from liver samples. To probe the role of histone acetylation in age-related loss of learning and memory, we have performed stereotaxic injections knocking out p300 in young and old mice and identified a loss of cognitive flexibility in p300 knockout mice. Integration of these multi-omic and behavioral datasets will provide key insights into the molecular mechanisms of tissue aging.