Jessica K. Tyler

Epigenetics and aging.

Researchers review how random changes and our environment (for example, diet) determines our life span. Over the past decade, a growing number of studies have revealed that progressive changes to epigenetic information accompany aging in both dividing and nondividing cells. Functional studies in model organisms and humans indicate that epigenetic changes have a huge influence on the aging process. These epigenetic changes occur at various levels, including reduced bulk levels of the core histones, altered patterns of histone posttranslational modifications and DNA methylation, replacement of canonical histones with histone variants, and altered noncoding RNA expression, during both organismal aging and replicative senescence. The end result of epigenetic changes during aging is altered local accessibility to the genetic material, leading to aberrant gene expression, reactivation of transposable elements, and genomic instability. Strikingly, certain types of epigenetic information can function in a transgenerational manner to influence the life span of the offspring. Several important conclusions emerge from these studies: rather than being genetically predetermined, our life span is largely epigenetically determined; diet and other environmental influences can influence our life span by changing the epigenetic information; and inhibitors of epigenetic enzymes can influence life span of model organisms. These new findings provide better understanding of the mechanisms involved in aging. Given the reversible nature of epigenetic information, these studies highlight exciting avenues for therapeutic intervention in aging and age-associated diseases, including cancer.

Nucleosome disassembly during human non-homologous end joining followed by concerted HIRA- and CAF-1-dependent reassembly

The cell achieves DNA double-strand break (DSB) repair in the context of chromatin structure. However, the mechanisms used to expose DSBs to the repair machinery and to restore the chromatin organization after repair remain elusive. Here we show that induction of a DSB in human cells causes local nucleosome disassembly, apparently independently from DNA end resection. This efficient removal of histone H3 from the genome during non-homologous end joining was promoted by both ATM and the ATP-dependent nucleosome remodeler INO80. Chromatin reassembly during DSB repair was dependent on the HIRA histone chaperone that is specific to the replication-independent histone variant H3.3 and on CAF-1 that is specific to the replication-dependent canonical histones H3.1/H3.2. Our data suggest that the epigenetic information is re-established after DSB repair by the concerted and interdependent action of replication-independent and replication-dependent chromatin assembly pathways. DOI: http://dx.doi.org/10.7554/eLife.15129.001

The Overlooked Fact: Fundamental Need for Spike-In Control for Virtually All Genome-Wide Analyses

ABSTRACT Genome-wide analyses of changes in gene expression, transcription factor occupancy on DNA, histone modification patterns on chromatin, genomic copy number variation, and nucleosome positioning have become popular in many modern laboratories, yielding a wealth of information during health and disease states. However, most of these studies have overlooked an inherent normalization problem that must be corrected with spike-in controls. Here we describe the reason why spike-in controls are so important and explain how to appropriately design and use spike-in controls for normalization. We also suggest ways to retrospectively renormalize data sets that were wrongly interpreted due to omission of spike-in controls.

Deficiency of XLF and PAXX prevents DNA double-strand break repair by non-homologous end joining in lymphocytes

ABSTRACT Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway that functions in all phases of the cell cycle. NHEJ repairs genotoxic and physiological DSBs, such as those generated by ionizing radiation and during V(D)J recombination at antigen receptor loci, respectively. DNA end joining by NHEJ relies on the core factors Ku70, Ku80, XRCC4, and DNA Ligase IV. Additional proteins also play important roles in NHEJ. The XRCC4-like factor (XLF) participates in NHEJ through its interaction with XRCC4, and XLF deficiency in humans leads to immunodeficiency and increased sensitivity to ionizing radiation. However, XLF is dispensable for NHEJ-mediated DSB repair during V(D)J recombination in murine lymphocytes, where it may have redundant functions with other DSB repair factors. Paralog of XRCC4 and XLF (PAXX) is a recently identified NHEJ factor that has structural similarity to XRCC4 and XLF. Here we show that PAXX is also dispensable for NHEJ during V(D)J recombination and during the repair of genotoxic DSBs in lymphocytes. However, a combined deficiency of PAXX and XLF blocks NHEJ with a severity comparable to that observed in DNA Ligase IV-deficient cells. Similar to XLF, PAXX interacts with Ku through its C-terminal region, and mutations that disrupt Ku binding prevent PAXX from promoting NHEJ in XLF-deficient lymphocytes. Our findings suggest that the PAXX and XLF proteins may have redundant functions during NHEJ.

The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication. DOI: http://dx.doi.org/10.7554/eLife.18023.001

Impaired cohesion and homologous recombination during replicative aging in budding yeast

How does the genome become unstable during aging? The causal relationship between genomic instability and replicative aging is unclear. We reveal here that genomic instability at the budding yeast ribosomal DNA (rDNA) locus increases during aging, potentially due to the reduced cohesion that we uncovered during aging caused by the reduced abundance of multiple cohesin subunits, promoting increased global chromosomal instability. In agreement, cohesion is lost during aging at other chromosomal locations in addition to the rDNA, including centromeres. The genomic instability in old cells is exacerbated by a defect in DNA double-strand break (DSB) repair that we uncovered in old yeast. This was due to limiting levels of key homologous recombination proteins because overexpression of Rad51 or Mre11 reduced the accumulation of DSBs and largely restored DSB repair in old cells. We propose that increased rDNA instability and the reduced DSB repair capacity of old cells contribute to the progressive accumulation of global chromosomal DNA breaks, where exceeding a threshold of genomic DNA damage ends the replicative life span.

The integrated stress response in budding yeast lifespan extension

Aging is a complex, multi-factorial biological process shared by all living organisms. It is manifested by a gradual accumulation of molecular alterations that lead to the decline of normal physiological functions in a time-dependent fashion. The ultimate goal of aging research is to develop therapeutic means to extend human lifespan, while reducing susceptibility to many age-related diseases including cancer, as well as metabolic, cardiovascular and neurodegenerative disorders. However, this first requires elucidation of the causes of aging, which has been greatly facilitated by the use of model organisms. In particular, the budding yeast Saccharomyces cerevisiae has been invaluable in the identification of conserved molecular and cellular determinants of aging and for the development of approaches to manipulate these aging determinants to extend lifespan. Strikingly, where examined, virtually all means to experimentally extend lifespan result in the induction of cellular stress responses. This review describes growing evidence in yeast that activation of the integrated stress response contributes significantly to lifespan extension. These findings demonstrate that yeast remains a powerful model system for elucidating conserved mechanisms to achieve lifespan extension that are likely to drive therapeutic approaches to extend human lifespan and healthspan.

The role of autophagy in the regulation of yeast life span

The goal of the aging field is to develop novel therapeutic interventions that extend human health span and reduce the burden of age‐related disease. While organismal aging is a complex, multifactorial process, a popular theory is that cellular aging is a significant contributor to the progressive decline inherent to all multicellular organisms. To explore the molecular determinants that drive cellular aging, as well as how to retard them, researchers have utilized the highly genetically tractable budding yeast Saccharomyces cerevisiae. Indeed, every intervention known to extend both cellular and organismal health span was identified in yeast, underlining the power of this approach. Importantly, a growing body of work has implicated the process of autophagy as playing a critical role in the delay of aging. This review summarizes recent reports that have identified a role for autophagy, or autophagy factors in the extension of yeast life span. These studies demonstrate (1) that yeast remains an invaluable tool for the identification and characterization of conserved mechanisms that promote cellular longevity and are likely to be relevant to humans, and (2) that the process of autophagy has been implicated in nearly all known longevity‐promoting manipulations and thus represents an ideal target for interventions aimed at improving human health span.

Mutations that prevent or mimic persistent post-translational modifications of the histone H3 globular domain cause lethality and growth defects in Drosophila

AbstractBackgroundnUnderstanding the function of histone post-translational modifications is the key to deciphering how genomic activities are regulated. Among the least well-understood histone modifications in vivo are those that occur on the surface of the globular domain of histones, despite their causing the most profound structural alterations of the nucleosome in vitro. We utilized a Drosophila system to replace the canonical histone genes with mutated histone transgenes.ResultsMutations predicted to mimic or prevent acetylation on histone H3 lysine (K) 56, K115, K122, and both K115/K122, or to prevent or mimic phosphorylation on H3 threonine (T) 118 and T80, all caused lethality, with the exception of K122R mutants. T118 mutations caused profound growth defects within wing discs, while K115R, K115Q, K56Q, and the K115/K122 mutations caused more subtle growth defects. The H3 K56R and H3 K122R mutations caused no defects in growth, differentiation, or transcription within imaginal discs, indicating that H3 K56 acetylation and K122 acetylation are dispensable for these functions. In agreement, we found the antibody to H3 K122Ac, which was previously used to imply a role for H3 K122Ac in transcription in metazoans, to be non-specific in vivo.ConclusionsOur data suggest that chromatin structural perturbations caused by acetylation of K56, K115, or K122 and phosphorylation of T80 or T118 are important for key developmental processes.

The Histone Chaperones ASF1 and CAF-1 Promote MMS22L-TONSL-Mediated Rad51 Loading onto ssDNA during Homologous Recombination in Human Cells

The access-repair-restore model for the role ofxa0chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, herexa0we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or axa0mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence ofxa0reduced recruitment of the Rad51 loader MMS22L-TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end resection, persistent activation of the ATR-Chk1 pathway, and cell cycle arrest. In agreement, histones occupy ssDNA during DNA repair in yeast.xa0We also uncovered DNA-PKcs-dependent DNA damage-induced ASF1A phosphorylation, which enhances chromatin assembly, promoting MMS22L-TONSL recruitment and, hence, Rad51 loading. We propose that transient assembly of newly synthesized histones onto ssDNA serves to recruit MMS22L-TONSL to efficiently form the Rad51 nucleofilament for strand invasion, suggesting an active role of chromatin assembly in homologous recombination.