Histone Modifications: Insights into Their Influence on Gene Expression.

This year's Albert Lasker Basic Medical Research Award honors David Allis and Michael Grunstein for their pioneering research that highlighted the importance of histones and their post-translational modifications in the direct control of gene expression.

Chasing Histone Biology from Sea Urchins to Yeast.

Together with David Allis, Michael Grunstein just received the Lasker Basic Medical research award. The article that follows is a transcript of a conversation with Jacques Deguine, scientific editor at Cell, that was edited for length. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online.

Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors.

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.

Histone H3 variants and their chaperones during development and disease: contributing to epigenetic control.

Within the nucleus, the interplay between lineage-specific transcription factors and chromatin dynamics defines cellular identity. Control of this interplay is necessary to properly balance stability and plasticity during the development and entire life span of multicellular organisms. Here, we present our current knowledge of the contribution of histone H3 variants to chromatin dynamics during development. We review the network of histone chaperones that governs their deposition timing and sites of incorporation and highlight how their distinct distribution impacts genome organization and function. We integrate the importance of H3 variants in the context of nuclear reprogramming and cell differentiation, and, using the centromere as a paradigm, we describe a case in which the identity of a given genomic locus is propagated across different cell types. Finally, we compare development to changes in stress and disease. Both physiological and pathological settings underline the importance of H3 dynamics for genome and chromatin integrity.

Zygotic genome activation during the maternal-to-zygotic transition.

Embryogenesis depends on a highly coordinated cascade of genetically encoded events. In animals, maternal factors contributed by the egg cytoplasm initially control development, whereas the zygotic nuclear genome is quiescent. Subsequently, the genome is activated, embryonic gene products are mobilized, and maternal factors are cleared. This transfer of developmental control is called the maternal-to-zygotic transition (MZT). In this review, we discuss recent advances toward understanding the scope, timing, and mechanisms that underlie zygotic genome activation at the MZT in animals. We describe high-throughput techniques to measure the embryonic transcriptome and explore how regulation of the cell cycle, chromatin, and transcription factors together elicits specific patterns of embryonic gene expression. Finally, we illustrate the interplay between zygotic transcription and maternal clearance and show how these two activities combine to reprogram two terminally differentiated gametes into a totipotent embryo.

Epigenetic Regulation of DNA Repair Pathway Choice by MacroH2A1 Splice Variants Ensures Genome Stability.

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.

Spt6 Is Required for the Fidelity of Promoter Selection.

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.

Epigenetic Control of Expression Homeostasis during Replication Is Stabilized by the Replication Checkpoint.

DNA replication introduces a dosage imbalance between early and late replicating genes. In budding yeast, buffering gene expression against this imbalance depends on marking replicated DNA by H3K56 acetylation (H3K56ac). Whether additional processes are required for suppressing transcription from H3K56ac-labeled DNA remains unknown. Here, using a database-guided candidate screen, we find that COMPASS, the H3K4 methyltransferase, and its upstream effector, PAF1C, act downstream of H3K56ac to buffer expression. Replicated genes show reduced abundance of the transcription activating mark H3K4me3 and accumulate the transcription inhibitory mark H3K4me2 near transcription start sites. Notably, in hydroxyurea-exposed cells, the S phase checkpoint stabilizes H3K56ac and becomes essential for buffering. We suggest that H3K56ac suppresses transcription of replicated genes by interfering with post-replication recovery of epigenetic marks and assign a new function for the S phase checkpoint in stabilizing this mechanism during persistent dosage imbalance.

Histone Acetylation Inhibits RSC and Stabilizes the +1 Nucleosome.

The +1 nucleosome of yeast genes, within which reside transcription start sites, is characterized by histone acetylation, by the displacement of an H2A-H2B dimer, and by a persistent association with the RSC chromatin-remodeling complex. Here we demonstrate the interrelationship of these characteristics and the conversion of a nucleosome to the +1 state in vitro. Contrary to expectation, acetylation performs an inhibitory role, preventing the removal of a nucleosome by RSC. Inhibition is due to both enhanced RSC-histone interaction and diminished histone-chaperone interaction. Acetylation does not prevent all RSC activity, because stably bound RSC removes an H2A-H2B dimer on a timescale of seconds in an irreversible manner.

Recognition of histone acetylation by the GAS41 YEATS domain promotes H2A.Z deposition in non-small cell lung cancer.

Histone acetylation is associated with active transcription in eukaryotic cells. It helps to open up the chromatin by neutralizing the positive charge of histone lysine residues and providing binding platforms for "reader" proteins. The bromodomain (BRD) has long been thought to be the sole protein module that recognizes acetylated histones. Recently, we identified the YEATS domain of AF9 (ALL1 fused gene from chromosome 9) as a novel acetyl-lysine-binding module and showed that the ENL (eleven-nineteen leukemia) YEATS domain is an essential acetyl-histone reader in acute myeloid leukemias. The human genome encodes four YEATS domain proteins, including GAS41, a component of chromatin remodelers responsible for H2A.Z deposition onto chromatin; however, the importance of the GAS41 YEATS domain in human cancer remains largely unknown. Here we report that GAS41 is frequently amplified in human non-small cell lung cancer (NSCLC) and is required for cancer cell proliferation, survival, and transformation. Biochemical and crystal structural studies demonstrate that GAS41 binds to histone H3 acetylated on H3K27 and H3K14, a specificity that is distinct from that of AF9 or ENL. ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) analyses in lung cancer cells reveal that GAS41 colocalizes with H3K27ac and H3K14ac on the promoters of actively transcribed genes. Depletion of GAS41 or disruption of the interaction between its YEATS domain and acetylated histones impairs the association of histone variant H2A.Z with chromatin and consequently suppresses cancer cell growth and survival both in vitro and in vivo. Overall, our study identifies GAS41 as a histone acetylation reader that promotes histone H2A.Z deposition in NSCLC.

Plant chromatin feels the heat.

Temperature is a key environmental signal regulating plant development, but the mechanisms by which plants sense small changes in ambient temperature have remained elusive. Kumar and Wigge (2010) now reveal that eviction of the histone variant H2A.Z from nucleosomes performs a central role in plant thermosensory perception.

H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis.

Plants are highly sensitive to temperature and can perceive a difference of as little as 1 degrees C. How temperature is sensed and integrated in development is unknown. In a forward genetic screen in Arabidopsis, we have found that nucleosomes containing the alternative histone H2A.Z are essential to perceiving ambient temperature correctly. Genotypes deficient in incorporating H2A.Z into nucleosomes phenocopy warm grown plants, and show a striking constitutive warm temperature transcriptome. We show that nucleosomes containing H2A.Z display distinct responses to temperature in vivo, independently of transcription. Using purified nucleosomes, we are able to show that H2A.Z confers distinct DNA-unwrapping properties on nucleosomes, indicating a direct mechanism for the perception of temperature through DNA-nucleosome fluctuations. Our results show that H2A.Z-containing nucleosomes provide thermosensory information that is used to coordinate the ambient temperature transcriptome. We observe the same effect in budding yeast, indicating that this is an evolutionarily conserved mechanism.

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma.

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

Drosophila H2Av negatively regulates the activity of the IMD pathway via facilitating Relish SUMOylation.

Insects depend on the innate immune response for defense against a wide array of pathogens. Central to Drosophila immunity are antimicrobial peptides (AMPs), released into circulation when pathogens trigger either of the two widely studied signal pathways, Toll or IMD. The Toll pathway responds to infection by Gram-positive bacteria and fungi while the IMD pathway is activated by Gram-negative bacteria. During activation of the IMD pathway, the NF-κB-like transcription factor Relish is phosphorylated and then cleaved, which is crucial for IMD-dependent AMP gene induction. Here we show that loss-of-function mutants of the unconventional histone variant H2Av upregulate IMD-dependent AMP gene induction in germ-free Drosophila larvae and adults. After careful dissection of the IMD pathway, we found that Relish has an epistatic relationship with H2Av. In the H2Av mutant larvae, SUMOylation is down-regulated, suggesting a possible role of SUMOylation in the immune phenotype. Eventually we demonstrated that Relish is mostly SUMOylated on amino acid K823. Loss of the potential SUMOylation site leads to significant auto-activation of Relish in vivo. Further work indicated that H2Av regulates Relish SUMOylation after physically interacting with Su(var)2-10, the E3 component of the SUMOylation pathway. Biochemical analysis suggested that SUMOylation of Relish prevents its cleavage and activation. Our findings suggest a new mechanism by which H2Av can negatively regulate, and thus prevent spontaneous activation of IMD-dependent AMP production, through facilitating SUMOylation of the NF-κB like transcription factor Relish.

Histones participate in base excision repair of 8-oxodGuo by transiently cross-linking with active repair intermediates in nucleosome core particles.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3'-phospho-α,ß-unsaturated aldehyde (PUA) and 5'-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3'-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5'-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5'-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.

An archaeal histone-like protein regulates gene expression in response to salt stress.

Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology. However, the precise function of halophilic histone-like proteins remain unclear. Here we use quantitative phenotyping, genetics, and functional genomics to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging.

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Histone H3K4me1 and H3K27ac play roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

Histone H3K4me1 and H3K27ac are enhancer-specific modifications and are required for enhancers to activate transcription of target genes. However, the reciprocal effects of these histone modifications on each other and their roles in enhancers are not clear. Here to comparatively analyze the role of these modifications, we inhibited H3K4me1 and H3K27ac by deleting the SET domains of histone methyltransferases MLL3 and MLL4 and the HAT domain of histone acetyltransferase p300, respectively, in erythroid K562 cells. The loss of H3K4me1 reduced H3K27ac at the ß-globin enhancer LCR HSs, but H3K27ac reduction did not affect H3K4me1. This unequal relationship between two modifications was revealed in putative enhancers by genome-wide analysis using ChIP-seq. Histone H3 eviction at putative enhancers was weakened by the loss of H3K4me1 but not by the loss of H3K27ac. Chromatin remodeling complexes were recruited into the ß-globin LCR HSs in a H3K4me1-dependent manner. In contrast, H3K27ac was required for enhancer RNA (eRNA) transcription, and H3K4me1 was not enough for it. Forced H3K27ac-induced eRNA transcription without affecting H3K4me1 at the ß-globin LCR HSs. These results indicate that H3K4me1 and H3K27ac affect each other in different ways and play more direct roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

A natural histone H2A variant lacking the Bub1 phosphorylation site and regulated depletion of centromeric histone CENP-A foster evolvability in Candida albicans.

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation.