Tyrosine phosphorylation of histone H2A by CK2 regulates transcriptional elongation.

Post-translational histone modifications have a critical role in regulating transcription, the cell cycle, DNA replication and DNA damage repair. The identification of new histone modifications critical for transcriptional regulation at initiation, elongation or termination is of particular interest. Here we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals. This regulation is based on the phosphorylation of a highly conserved tyrosine residue, Tyr 57, in histone H2A and is mediated by the unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of Tyr 57 in H2A in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA-polymerase-II-transcribed coding genes and active enhancers. Mutation of Tyr 57 causes a loss of H2B mono-ubiquitination as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and the H2A(Y57F) mutation enhance H2B deubiquitination activity of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA complex during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A.

Multigenerational silencing dynamics control cell aging.

Cellular aging plays an important role in many diseases, such as cancers, metabolic syndromes, and neurodegenerative disorders. There has been steady progress in identifying aging-related factors such as reactive oxygen species and genomic instability, yet an emerging challenge is to reconcile the contributions of these factors with the fact that genetically identical cells can age at significantly different rates. Such complexity requires single-cell analyses designed to unravel the interplay of aging dynamics and cell-to-cell variability. Here we use microfluidic technologies to track the replicative aging of single yeast cells and reveal that the temporal patterns of heterochromatin silencing loss regulate cellular life span. We found that cells show sporadic waves of silencing loss in the heterochromatic ribosomal DNA during the early phases of aging, followed by sustained loss of silencing preceding cell death. Isogenic cells have different lengths of the early intermittent silencing phase that largely determine their final life spans. Combining computational modeling and experimental approaches, we found that the intermittent silencing dynamics is important for longevity and is dependent on the conserved Sir2 deacetylase, whereas either sustained silencing or sustained loss of silencing shortens life span. These findings reveal that the temporal patterns of a key molecular process can directly influence cellular aging, and thus could provide guidance for the design of temporally controlled strategies to extend life span.

Functions for diverse metabolic activities in heterochromatin.

Growing evidence demonstrates that metabolism and chromatin dynamics are not separate processes but that they functionally intersect in many ways. For example, the lysine biosynthetic enzyme homocitrate synthase was recently shown to have unexpected functions in DNA damage repair, raising the question of whether other amino acid metabolic enzymes participate in chromatin regulation. Using an in silico screen combined with reporter assays, we discovered that a diverse range of metabolic enzymes function in heterochromatin regulation. Extended analysis of the glutamate dehydrogenase 1 (Gdh1) revealed that it regulates silent information regulator complex recruitment to telomeres and ribosomal DNA. Enhanced N-terminal histone H3 proteolysis is observed in GDH1 mutants, consistent with telomeric silencing defects. A conserved catalytic Asp residue is required for Gdh1's functions in telomeric silencing and H3 clipping. Genetic modulation of α-ketoglutarate levels demonstrates a key regulatory role for this metabolite in telomeric silencing. The metabolic activity of glutamate dehydrogenase thus has important and previously unsuspected roles in regulating chromatin-related processes.

Critical genomic regulation mediated by Enhancer of Polycomb.

Enhancer of Polycomb (EPC) was first identified for its contributions to development in Drosophila and was soon-thereafter purified as a subunit of the NuA4/TIP60 acetyltransferase complex. Since then, EPC has often been left in the shadows as an essential, yet non-catalytic subunit of NuA4/TIP60; however, its deep conservation and disease association make clear that it warrants additional attention. In fact, recent studies in yeast demonstrated that its Enhancer of Polycomb, Epl1, was just as important for gene expression and acetylation as is the catalytic subunit of NuA4. Despite its conservation, studies of EPC have often remained siloed between organisms. Here, our goal is to provide a cohesive view of the current state of the EPC literature as it stands among the major model organisms in which it has been studied. EPC is involved in multiple processes, beginning with its cardinal role in regulating global and targeted histone acetylation. EPC also frequently serves as an important interaction partner in these basic cellular functions, as well as in multicellular development, such as in hematopoiesis and skeletal muscle differentiation, and in human disease. Taken together, a unifying theme from these studies highlights EPC as a critical genomic regulator.

Homocitrate synthase connects amino acid metabolism to chromatin functions through Esa1 and DNA damage.

The enzyme homocitrate synthase (HCS) catalyzes the first step in lysine biosynthesis, and early biochemical data placed it in the cytoplasm or mitochondria, where most amino acid synthesis occurs. It was therefore surprising when refined fractionation techniques and specific immunoreagents clearly demonstrated its localization to the nucleus. These observations raised the question of whether HCS had a function within the nucleus independent of lysine synthesis. We demonstrate that HCS encoded by LYS20 in yeast is linked to the key process of DNA damage repair through the essential MYST family histone acetyltransferase Esa1 and the H2A.Z histone variant. This discovery indicates that HCS has a role in addition to amino acid synthesis, and that it functions in nuclear activities involving chromatin regulation that are distinct from its previously established role in lysine biosynthesis. The chromatin-linked roles are dependent on nuclear localization of Lys20, but are independent of HCS catalytic activity. Thus, Lys20 appears to have evolved as a bifunctional protein that connects cellular metabolism with chromatin functions.

A moonlighting metabolic protein influences repair at DNA double-stranded breaks.

Catalytically active proteins with divergent dual functions are often described as 'moonlighting'. In this work we characterize a new, chromatin-based function of Lys20, a moonlighting protein that is well known for its role in metabolism. Lys20 was initially described as homocitrate synthase (HCS), the first enzyme in the lysine biosynthetic pathway in yeast. Its nuclear localization led to the discovery of a key role for Lys20 in DNA damage repair through its interaction with the MYST family histone acetyltransferase Esa1. Overexpression of Lys20 promotes suppression of DNA damage sensitivity of esa1 mutants. In this work, by taking advantage of LYS20 mutants that are active in repair but not in lysine biosynthesis, the mechanism of suppression of esa1 was characterized. First we analyzed the chromatin landscape of esa1 cells, finding impaired histone acetylation and eviction. Lys20 was recruited to sites of DNA damage, and its overexpression promoted enhanced recruitment of the INO80 remodeling complex to restore normal histone eviction at the damage sites. This study improves understanding of the evolutionary, structural and biological relevance of independent activities in a moonlighting protein and links metabolism to DNA damage repair.

Balancing chromatin remodeling and histone modifications in transcription.

Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive crosstalk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relations between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the imitation-switch (ISWI) and chromodomain helicase DNA-binding protein 1 (CHD1) chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast.

MYSTs mark chromatin for chromosomal functions.

The MYST family of lysine acetyltransferases has been intensely studied because of its broad conservation and biological significance. In humans, there are multiple correlations between the enzymes and development and disease. In model organisms, genetic and biochemical studies have been particularly productive because of mechanistic insights they provide in defining substrate specificity, the complexes through which the enzymes function, and the sites of their activity within the genome. Established and emerging data from yeast reveal roles for the three MYST enzymes in diverse chromosomal functions. In particular, recent studies help explain how MYST complexes coordinate with other modifiers, the histone variant H2A.Z, and remodeling complexes to demarcate silent and active chromosomal domains, facilitate transcription, and enable repair of DNA damage.

Functional antagonism between Sas3 and Gcn5 acetyltransferases and ISWI chromatin remodelers.

Chromatin-modifying enzymes and ATP-dependent remodeling complexes have been intensely studied individually, yet how these activities are coordinated to ensure essential cell functions such as transcription, replication, and repair of damage is not well understood. In this study, we show that the critical loss of Sas3 and Gcn5 acetyltransferases in yeast can be functionally rescued by inactivation of ISWI remodelers. This genetic interaction depends on the ATPase activities of Isw1 and Isw2, suggesting that it involves chromatin remodeling activities driven by the enzymes. Genetic dissection of the Isw1 complexes reveals that the antagonistic effects are mediated specifically by the Isw1a complex. Loss of Sas3 and Gcn5 correlates with defective RNA polymerase II (RNAPII) occupancy at actively transcribed genes, as well as a significant loss of H3K14 acetylation. Inactivation of the Isw1a complex in the acetyltransferase mutants restores RNAPII recruitment at active genes, indicating that transcriptional regulation may be the mechanism underlying suppression. Dosage studies and further genetic dissection reveal that the Isw1b complex may act in suppression through down-regulation of Isw1a. These studies highlight the importance of balanced chromatin modifying and remodeling activities for optimal transcription and cell growth.

Non-canonical chromatin-based functions for the threonine metabolic pathway.

The emerging class of multi-functional proteins known as moonlighters challenges the "one protein, one function" mentality by demonstrating crosstalk between biological pathways that were previously thought to be functionally discrete. Here, we present new links between amino acid metabolism and chromatin regulation, two biological pathways that are critical for cellular and organismal homeostasis. We discovered that the threonine biosynthetic pathway is required for the transcriptional silencing of ribosomal DNA (rDNA) in Saccharomyces cerevisiae. The enzymes in the pathway promote rDNA silencing through distinct mechanisms as a subset of silencing phenotypes was rescued with exogenous threonine. In addition, we found that a key pathway enzyme, homoserine dehydrogenase, promotes DNA repair through a mechanism involving the MRX complex, a major player in DNA double strand break repair. These data further the understanding of enzymes with non-canonical roles, here demonstrated within the threonine biosynthetic pathway, and provide insight into their roles as potential anti-fungal pharmaceutical targets.

STUbLs in chromatin and genome stability.

Chromatin structure and function is based on the dynamic interactions between nucleosomes and chromatin-associated proteins. In addition to the other post-translational modifications considered in this review issue of Biopolymers, ubiquitin and SUMO proteins also have prominent roles in chromatin function. A specialized form of modification that involves both, referred to as SUMO-targeted ubiquitin ligation, or STUbL [Perry, Tainer, and Boddy, Trends Biochem Sci, 2008, 33, 201-208], has significant effects on nuclear functions, ranging from gene regulation to genomic stability. Intersections between SUMO and ubiquitin in protein modification have been the subject of a recent comprehensive review [Praefcke, Hofmann, and Dohmen, Trends Biochem Sci, 2012, 37, 23-31]. Our goal here is to focus on features of enzymes with STUbL activity that have been best studied, particularly in relation to their nuclear functions in humans, flies, and yeasts. Because there are clear associations of disease and development upon loss of STUbL activities in metazoans, learning more about their function, regulation, and substrates will remain an important goal for the future.

Functional tug of war between kinases, phosphatases, and the Gcn5 acetyltransferase in chromatin and cell cycle checkpoint controls.

Covalent modifications of chromatin regulate genomic structure and accessibility in diverse biological processes such as transcriptional regulation, cell cycle progression, and DNA damage repair. Many histone modifications have been characterized, yet understanding the interactions between these and their combinatorial effects remains an active area of investigation, including dissecting functional interactions between enzymes mediating these modifications. In budding yeast, the histone acetyltransferase Gcn5 interacts with Rts1, a regulatory subunit of protein phosphatase 2A (PP2A). Implicated in the interaction is the potential for the dynamic phosphorylation of conserved residues on histone H2B and the Cse4 centromere-specific histone H3 variant. To probe these dynamics, we sought to identify kinases which contribute to the phosphorylated state. In a directed screen beginning with in silico analysis of the 127 members of yeast kinome, we have now identified 16 kinases with genetic interactions with GCN5 and specifically found distinct roles for the Hog1 stress-activated protein kinase. Deletion of HOG1 (hog1Δ) rescues gcn5Δ sensitivity to the microtubule poison nocodazole and the lethality of the gcn5Δ rts1Δ double mutant. The Hog1-Gcn5 interaction requires the conserved H2B-T91 residue, which is phosphorylated in vertebrate species. Furthermore, deletion of HOG1 decreases aneuploidy and apoptotic populations in gcn5Δ cells. Together, these results introduce Hog1 as a kinase that functionally opposes Gcn5 and Rts1 in the context of the spindle assembly checkpoint and suggest further kinases may also influence GCN5's functions.

Engineering longevity-design of a synthetic gene oscillator to slow cellular aging.

Synthetic biology enables the design of gene networks to confer specific biological functions, yet it remains a challenge to rationally engineer a biological trait as complex as longevity. A naturally occurring toggle switch underlies fate decisions toward either nucleolar or mitochondrial decline during the aging of yeast cells. We rewired this endogenous toggle to engineer an autonomous genetic clock that generates sustained oscillations between the nucleolar and mitochondrial aging processes in individual cells. These oscillations increased cellular life span through the delay of the commitment to aging that resulted from either the loss of chromatin silencing or the depletion of heme. Our results establish a connection between gene network architecture and cellular longevity that could lead to rationally designed gene circuits that slow aging.

The glucanosyltransferase Gas1 functions in transcriptional silencing.

Transcriptional silencing is a crucial process that is mediated through chromatin structure. The histone deacetylase Sir2 silences genomic regions that include telomeres, ribosomal DNA (rDNA) and the cryptic mating-type loci. Here, we report an unsuspected role for the enzyme Gas1 in locus-specific transcriptional silencing. GAS1 encodes a beta-1,3-glucanosyltransferase previously characterized for its role in cell wall biogenesis. In gas1 mutants, telomeric silencing is defective and rDNA silencing is enhanced. We show that the catalytic activity of Gas1 is required for normal silencing, and that Gas1's role in silencing is distinct from its role in cell wall biogenesis. Established hallmarks of silent chromatin, such as Sir2 and Sir3 binding, H4K16 deacetylation, and H3K56 deacetylation, appear unaffected in gas1 mutants. Thus, another event required for telomeric silencing must be influenced by GAS1. Because the catalytic activity of Gas1 is required for telomeric silencing, Gas1 localizes to the nuclear periphery, and Gas1 and Sir2 physically interact, we propose a model in which carbohydrate modification of chromatin components provides a new regulatory element that may be critical for chromatin function but which is virtually unexplored in the current landscape of chromatin analysis.

Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with challenges to proteostasis.

Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

Structural basis for L-lysine feedback inhibition of homocitrate synthase.

The alpha-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the alpha-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by l-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with l-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the (alpha/beta)(8) TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the epsilon-ammonium group of l-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the l-lysine epsilon-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of l-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.

Cell cycle roles for GCN5 revealed through genetic suppression.

The conserved acetyltransferase Gcn5 is a member of several complexes in eukaryotic cells, playing roles in regulating chromatin organization, gene expression, metabolism, and cell growth and differentiation via acetylation of both nuclear and cytoplasmic proteins. Distinct functions of Gcn5 have been revealed through a combination of biochemical and genetic approaches in many in vitro studies and model organisms. In this review, we focus on the unique insights that have been gleaned from suppressor studies of gcn5 phenotypes in the budding yeast Saccharomyces cerevisiae. Such studies were fundamental in the early understanding of the balance of counteracting chromatin activities in regulating transcription. Most recently, suppressor screens have revealed roles for Gcn5 in early cell cycle (G1 to S) gene expression and regulation of chromosome segregation during mitosis. Much has been learned, but many questions remain which will be informed by focused analysis of additional genetic and physical interactions.

Crystal structure and functional analysis of homocitrate synthase, an essential enzyme in lysine biosynthesis.

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed alpha/beta topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.

Advances in quantitative biology methods for studying replicative aging in Saccharomyces cerevisiae.

Aging is a complex, yet pervasive phenomenon in biology. As human cells steadily succumb to the deteriorating effects of aging, so too comes a host of age-related ailments such as neurodegenerative disorders, cardiovascular disease and cancer. Therefore, elucidation of the molecular networks that drive aging is of paramount importance to human health. Progress toward this goal has been aided by studies from simple model organisms such as Saccharomyces cerevisiae. While work in budding yeast has already revealed much about the basic biology of aging as well as a number of evolutionarily conserved pathways involved in this process, recent technological advances are poised to greatly expand our knowledge of aging in this simple eukaryote. Here, we review the latest developments in microfluidics, single-cell analysis and high-throughput technologies for studying single-cell replicative aging in S. cerevisiae. We detail the challenges each of these methods addresses as well as the unique insights into aging that each has provided. We conclude with a discussion of potential future applications of these techniques as well as the importance of single-cell dynamics and quantitative biology approaches for understanding cell aging.

A programmable fate decision landscape underlies single-cell aging in yeast.

Chromatin instability and mitochondrial decline are conserved processes that contribute to cellular aging. Although both processes have been explored individually in the context of their distinct signaling pathways, the mechanism that determines which process dominates during aging of individual cells is unknown. We show that interactions between the chromatin silencing and mitochondrial pathways lead to an epigenetic landscape of yeast replicative aging with multiple equilibrium states that represent different types of terminal states of aging. The structure of the landscape drives single-cell differentiation toward one of these states during aging, whereby the fate is determined quite early and is insensitive to intracellular noise. Guided by a quantitative model of the aging landscape, we genetically engineered a long-lived equilibrium state characterized by an extended life span.