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 of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly, via knockdown of the histone chaperones ASF1 or CAF-1 or a mutation that prevents ASF1A binding to histones, hinders Rad51 loading onto ssDNA during homologous recombination. This is a consequence of reduced 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. We 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.

MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

Chaperoning histones during DNA replication and repair.

Nuclear DNA is tightly packaged into chromatin, which profoundly influences DNA replication, transcription, repair, and recombination. The extensive interactions between the basic histone proteins and acidic DNA make the nucleosomal unit of chromatin a highly stable entity. For the cellular machinery to access the DNA, the chromatin must be unwound and the DNA cleared of histone proteins. Conversely, the DNA has to be repackaged into chromatin afterward. This review focuses on the roles of the histone chaperones in assembling and disassembling chromatin during the processes of DNA replication and repair.

Thinking Outside the Cell: Replicating Replication In Vitro.

Benchmark studies by Yeeles et al. (2017), Kurat et al. (2017), and Devbhandari et al. (2017) have reconstituted rapid and regulated budding yeast DNA replication on naked and chromatinized templates in vitro, providing key insights into this fundamentally important process.

Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair.

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. We find that Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.

Reprogramming Carbohydrate Metabolism in Cancer and Its Role in Regulating the Tumor Microenvironment.

Altered metabolism has become an emerging feature of cancer cells impacting their proliferation and metastatic potential in myriad ways. Proliferating heterogeneous tumor cells are surrounded by other resident or infiltrating cells, along with extracellular matrix proteins, and other secretory factors constituting the tumor microenvironment. The diverse cell types of the tumor microenvironment exhibit different molecular signatures that are regulated at their genetic and epigenetic levels. The cancer cells elicit intricate crosstalks with these supporting cells, exchanging essential metabolites which support their anabolic processes and can promote their survival, proliferation, EMT, angiogenesis, metastasis and even therapeutic resistance. In this context, carbohydrate metabolism ensures constant energy supply being a central axis from which other metabolic and biosynthetic pathways including amino acid and lipid metabolism and pentose phosphate pathway are diverged. In contrast to normal cells, increased glycolytic flux is a distinguishing feature of the highly proliferative cancer cells, which supports them to adapt to a hypoxic environment and also protects them from oxidative stress. Such rewired metabolic properties are often a result of epigenetic alterations in the cancer cells, which are mediated by several factors including, DNA, histone and non-histone protein modifications and non-coding RNAs. Conversely, epigenetic landscapes of the cancer cells are also dictated by their diverse metabolomes. Altogether, this metabolic and epigenetic interplay has immense potential for the development of efficient anti-cancer therapeutic strategies. In this book chapter we emphasize upon the significance of reprogrammed carbohydrate metabolism in regulating the tumor microenvironment and cancer progression, with an aim to explore the different metabolic and epigenetic targets for better cancer treatment.

Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging.

All eukaryotic cells divide a finite number of times, although the mechanistic basis of this replicative aging remains unclear. Replicative aging is accompanied by a reduction in histone protein levels, and this is a cause of aging in budding yeast. Here we show that nucleosome occupancy decreased by 50% across the whole genome during replicative aging using spike-in controlled micrococcal nuclease digestion followed by sequencing. Furthermore, nucleosomes became less well positioned or moved to sequences predicted to better accommodate histone octamers. The loss of histones during aging led to transcriptional induction of all yeast genes. Genes that are normally repressed by promoter nucleosomes were most induced, accompanied by preferential nucleosome loss from their promoters. We also found elevated levels of DNA strand breaks, mitochondrial DNA transfer to the nuclear genome, large-scale chromosomal alterations, translocations, and retrotransposition during aging.

Nucleosomes Find Their Place in Life.

To carry epigenetic information, the chromatin structure must be accurately rebuilt after its deconstruction during genomic replication. Using an elegant, novel approach, Vasseur et al.[1] reveal that transcription plays a key role in sculpting the chromatin after DNA replication.

Is Gcn4-induced autophagy the ultimate downstream mechanism by which hormesis extends yeast replicative lifespan?

The number of times a cell divides before irreversibly arresting is termed replicative lifespan. Despite discovery of many chemical, dietary and genetic interventions that extend replicative lifespan, usually first discovered in budding yeast and subsequently shown to apply to metazoans, there is still little understanding of the underlying molecular mechanisms involved. One unifying theme is that most, if not all, interventions that extend replicative lifespan induce "hormesis", where a little inflicted damage makes cells more able to resist similar challenges in the future. One of the many cellular changes that occur during hormesis is a global reduction in protein synthesis, which has been linked to enhanced longevity in many organisms. Our recent study in budding yeast found that it was not the reduction in protein synthesis per se, but rather the subsequent induction of the conserved Gcn4 transcriptional regulator and its ability to induce autophagy that was responsible for extending replicative lifespan. We propose that Gcn4-dependent induction of autophagy occurring downstream of reduced global protein synthesis may be a unifying molecular mechanism for many interventions that extend replicative lifespan.

Proteomic identification of histone post-translational modifications and proteins enriched at a DNA double-strand break.

Here, we use ChAP-MS (chromatin affinity purification with mass spectrometry), for the affinity purification of a sequence-specific single-copy endogenous chromosomal locus containing a DNA double-strand break (DSB). We found multiple new histone post-translational modifications enriched on chromatin bearing a DSB from budding yeast. One of these, methylation of histone H3 on lysine 125, has not previously been reported. Among over 100 novel proteins enriched at a DSB were the phosphatase Sit4, the RNA pol II degradation factor Def1, the mRNA export protein Yra1 and the HECT E3 ligase Tom1. Each of these proteins was required for resistance to radiomimetics, and many were required for resistance to heat, which we show here to cause a defect in DSB repair in yeast. Yra1 and Def1 were required for DSB repair per se, while Sit4 was required for rapid inactivation of the DNA damage checkpoint after DSB repair. Thus, our unbiased proteomics approach has led to the unexpected discovery of novel roles for these and other proteins in the DNA damage response.

Elevated histone expression promotes life span extension.

Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here, we report a mechanism whereby altering chromatin structure regulates life span. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived, and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the histone information regulator (Hir) complex or overexpression of histones dramatically extends life span via a pathway that is distinct from previously known pathways of life span extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends life span.

Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation.

The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. Here we demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. We show that autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and we propose that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP.

CBP/p300-mediated acetylation of histone H3 on lysine 56.

Acetylation within the globular core domain of histone H3 on lysine 56 (H3K56) has recently been shown to have a critical role in packaging DNA into chromatin following DNA replication and repair in budding yeast. However, the function or occurrence of this specific histone mark has not been studied in multicellular eukaryotes, mainly because the Rtt109 enzyme that is known to mediate acetylation of H3K56 (H3K56ac) is fungal-specific. Here we demonstrate that the histone acetyl transferase CBP (also known as Nejire) in flies and CBP and p300 (Ep300) in humans acetylate H3K56, whereas Drosophila Sir2 and human SIRT1 and SIRT2 deacetylate H3K56ac. The histone chaperones ASF1A in humans and Asf1 in Drosophila are required for acetylation of H3K56 in vivo, whereas the histone chaperone CAF-1 (chromatin assembly factor 1) in humans and Caf1 in Drosophila are required for the incorporation of histones bearing this mark into chromatin. We show that, in response to DNA damage, histones bearing acetylated K56 are assembled into chromatin in Drosophila and human cells, forming foci that colocalize with sites of DNA repair. Furthermore, acetylation of H3K56 is increased in multiple types of cancer, correlating with increased levels of ASF1A in these tumours. Our identification of multiple proteins regulating the levels of H3K56 acetylation in metazoans will allow future studies of this critical and unique histone modification that couples chromatin assembly to DNA synthesis, cell proliferation and cancer.

The histone shuffle: histone chaperones in an energetic dance.

Our genetic information is tightly packaged into a rather ingenious nucleoprotein complex called chromatin in a manner that enables it to be rapidly accessed during genomic processes. Formation of the nucleosome, which is the fundamental unit of chromatin, occurs via a stepwise process that is reversed to enable the disassembly of nucleosomes. Histone chaperone proteins have prominent roles in facilitating these processes as well as in replacing old histones with new canonical histones or histone variants during the process of histone exchange. Recent structural, biophysical and biochemical studies have begun to shed light on the molecular mechanisms whereby histone chaperones promote chromatin assembly, disassembly and histone exchange to facilitate DNA replication, repair and transcription.

Histone exchange and histone modifications during transcription and aging.

The organization of the eukaryotic genome into chromatin enables DNA to fit inside the nucleus while also regulating the access of proteins to the DNA to facilitate genomic functions such as transcription, replication and repair. The basic repeating unit of chromatin is the nucleosome, which includes 147 bp of DNA wrapped 1.65 times around an octamer of core histone proteins comprising two molecules each of H2A, H2B, H3 and H4. Each nucleosome is a highly stable unit, being maintained by over 120 direct protein-DNA interactions and several hundred water mediated ones. Accordingly, there is considerable interest in understanding how processive enzymes such as RNA polymerases manage to pass along the coding regions of our genes that are tightly packaged into arrays of nucleosomes. Here we present the current mechanistic understanding of this process and the evidence for profound changes in chromatin dynamics during aging. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Epigenetic regulation of genomic integrity.

Inefficient and inaccurate repair of DNA damage is the principal cause of DNA mutations, chromosomal aberrations, and carcinogenesis. Numerous multiple-step DNA repair pathways exist whose deployment depends on the nature of the DNA lesion. Common to all eukaryotic DNA repair pathways is the need to unravel the compacted chromatin structure to facilitate access of the repair machinery to the DNA and restoration of the original chromatin state afterward. Accordingly, our cells utilize a plethora of coordinated mechanisms to locally open up the chromatin structure to reveal the underlying DNA sequence and to orchestrate the efficient and accurate repair of DNA lesions. Here we review changes to the chromatin structure that are intrinsic to the DNA damage response and the available mechanistic insight into how these chromatin changes facilitate distinct stages of the DNA damage repair pathways to maintain genomic stability.

A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

Multivalent binding of the tardigrade Dsup protein to chromatin promotes yeast survival and longevity upon exposure to oxidative damage.

Tardigrades are remarkable in their ability to survive extreme environments. The damage suppressor (Dsup) protein is thought responsible for their extreme resistance to reactive oxygen species (ROS) generated by irradiation. Here we show that expression of Ramazzottius varieornatus Dsup in Saccharomyces cerevisiae reduces oxidative DNA damage and extends the lifespan of budding yeast exposed to chronic oxidative genotoxicity. This protection from ROS requires either the Dsup HMGN-like domain or sequences C-terminal to same. Dsup associates with no apparent bias across the yeast genome, using multiple modes of nucleosome binding; the HMGN-like region interacts with both the H2A/H2B acidic patch and H3/H4 histone tails, while the C-terminal region binds DNA. These findings give precedent for engineering an organism by physically shielding its genome to promote survival and longevity in the face of oxidative damage.