An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation.

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.

Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L.

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.

Single-Cell Chromatin Modification Profiling Reveals Increased Epigenetic Variations with Aging.

Post-translational modifications of histone proteins and exchanges of histone variants of chromatin are central to the regulation of nearly all DNA-templated biological processes. However, the degree and variability of chromatin modifications in specific human immune cells remain largely unknown. Here, we employ a highly multiplexed mass cytometry analysis to profile the global levels of a broad array of chromatin modifications in primary human immune cells at the single-cell level. Our data reveal markedly different cell-type- and hematopoietic-lineage-specific chromatin modification patterns. Differential analysis between younger and older adults shows that aging is associated with increased heterogeneity between individuals and elevated cell-to-cell variability in chromatin modifications. Analysis of a twin cohort unveils heritability of chromatin modifications and demonstrates that aging-related chromatin alterations are predominantly driven by non-heritable influences. Together, we present a powerful platform for chromatin and immunology research. Our discoveries highlight the profound impacts of aging on chromatin modifications.

Excess Translation of Epigenetic Regulators Contributes to Fragile X Syndrome and Is Alleviated by Brd4 Inhibition.

Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.

Resetting the Yeast Epigenome with Human Nucleosomes.

Humans and yeast are separated by a billion years of evolution, yet their conserved histones retain central roles in gene regulation. Here, we "reset" yeast to use core human nucleosomes in lieu of their own (a rare event taking 20 days), which initially only worked with variant H3.1. The cells adapt by acquiring suppressor mutations in cell-division genes or by acquiring certain aneuploid states. Converting five histone residues to their yeast counterparts restored robust growth. We reveal that humanized nucleosomes are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy, globally reduce RNA content, and slow adaptation to new conditions by delaying chromatin remodeling. These humanized yeasts (including H3.3) pose fundamental new questions about how chromatin is linked to many cell processes and provide a platform to study histone variants via yeast epigenome reprogramming.

The Inherent Asymmetry of DNA Replication.

Semiconservative DNA replication has provided an elegant solution to the fundamental problem of how life is able to proliferate in a way that allows cells, organisms, and populations to survive and replicate many times over. Somewhat lost, however, in our admiration for this mechanism is an appreciation for the asymmetries that occur in the process of DNA replication. As we discuss in this review, these asymmetries arise as a consequence of the structure of the DNA molecule and the enzymatic mechanism of DNA synthesis. Increasing evidence suggests that asymmetries in DNA replication are able to play a central role in the processes of adaptation and evolution by shaping the mutagenic landscape of cells. Additionally, in eukaryotes, recent work has demonstrated that the inherent asymmetries in DNA replication may play an important role in the process of chromatin replication. As chromatin plays an essential role in defining cell identity, asymmetries generated during the process of DNA replication may play critical roles in cell fate decisions related to patterning and development.

Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation.

Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.

Distinct silencer states generate epigenetic states of heterochromatin.

Heterochromatic loci can exhibit different transcriptional states in genetically identical cells. A popular model posits that the inheritance of modified histones is sufficient for inheritance of the silenced state. However, silencing inheritance requires silencers and therefore cannot be driven by the inheritance of modified histones alone. To address these observations, we determined the chromatin architectures produced by strong and weak silencers in Saccharomyces. Strong silencers recruited Sir proteins and silenced the locus in all cells. Strikingly, weakening these silencers reduced Sir protein recruitment and stably silenced the locus in some cells; however, this silenced state could probabilistically convert to an expressed state that lacked Sir protein recruitment. Additionally, changes in the constellation of silencer-bound proteins or the concentration of a structural Sir protein modulated the probability that a locus exhibited the silenced or expressed state. These findings argued that distinct silencer states generate epigenetic states and regulate their dynamics.

A giant virus genome is densely packaged by stable nucleosomes within virions.

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

Topography of histone H3-H4 interaction with the Hat1-Hat2 acetyltransferase complex.

Chaperones influence histone conformation and intermolecular interaction in multiprotein complexes, and the structures obtained with full-length histones often provide more accurate and comprehensive views. Here, our structure of the Hat1-Hat2 acetyltransferase complex bound to Asf1-H3-H4 shows that the core domains of H3 and H4 are involved in binding Hat1 and Hat2, and the N-terminal tail of H3 makes extensive interaction with Hat2. These findings expand the knowledge about histone-protein interaction and implicate a function of Hat2/RbAp46/48, which is a versatile histone chaperone found in many chromatin-associated complexes, in the passing of histones between chaperones.

Immune Sensing of Cell Death through Recognition of Histone Sequences by C-Type Lectin-Receptor-2d Causes Inflammation and Tissue Injury.

The immune system monitors the health of cells and is stimulated by necrosis. Here we examined the receptors and ligands driving this response. In a targeted screen of C-type lectin receptors, a Clec2d reporter responded to lysates from necrotic cells. Biochemical purification identified histones, both free and bound to nucleosomes or neutrophil extracellular traps, as Clec2d ligands. Clec2d recognized poly-basic sequences in histone tails and this recognition was sensitive to post-translational modifications of these sequences. As compared with WT mice, Clec2d-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model. In macrophages, Clec2d localized to the plasma membrane and endosomes. Histone binding to Clec2d did not stimulate kinase activation or cytokine production. Rather, histone-bound DNA stimulated endosomal Tlr9-dependent responses in a Clec2d-dependent manner. Thus, Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage.

Simultaneous profiling of multiple chromatin proteins in the same cells.

Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here, we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell-type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different chromatin proteins.

Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients.

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of 'cell cycle genes', which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis.

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.

Control of noncoding RNA production and histone levels by a 5' tRNA fragment.

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs," are an emerging class of regulatory RNAs with poorly understood functions. We recently identified a role for one specific tRF-5' tRF-Gly-GCC, or tRF-GG-as a repressor of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs-snoRNAs, scaRNAs, and snRNAs-that are dependent on Cajal bodies for stability and activity. Among these noncoding RNAs, regulation of the U7 snRNA by tRF-GG modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, on activity of a histone 3' UTR reporter, and ultimately on MERVL regulation could all be suppressed by manipulating U7 RNA levels. We additionally show that the related RNA-binding proteins hnRNPF and hnRNPH bind directly to tRF-GG, and are required for Cajal body biogenesis, positioning these proteins as strong candidates for effectors of tRF-GG function in vivo. Together, our data reveal a conserved mechanism for 5' tRNA fragment control of noncoding RNA biogenesis and, consequently, global chromatin organization.

Histone oxidation as a new mechanism of metabolic control over gene expression.

The emergence of aerobic respiration created unprecedented bioenergetic advantages, while imposing the need to protect critical genetic information from reactive byproducts of oxidative metabolism (i.e., reactive oxygen species, ROS). The evolution of histone proteins fulfilled the need to shield DNA from these potentially damaging toxins, while providing the means to compact and structure massive eukaryotic genomes. To date, several metabolism-linked histone post-translational modifications (PTMs) have been shown to regulate chromatin structure and gene expression. However, whether and how PTMs enacted by metabolically produced ROS regulate adaptive chromatin remodeling remain relatively unexplored. Here, we review novel mechanistic insights into the interactions of ROS with histones and their consequences for the control of gene expression regulation, cellular plasticity, and behavior.

DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments.

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore-microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone-humanized yeasts to counterbalance human histone-induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.

Sirtuins in Epigenetic Silencing and Control of Gene Expression in Model and Pathogenic Fungi.

Fungi, including yeasts, molds, and mushrooms, proliferate on decaying matter and then adopt quiescent forms once nutrients are depleted. This review explores how fungi use sirtuin deacetylases to sense and respond appropriately to changing nutrients. Because sirtuins are NAD+-dependent deacetylases, their activity is sensitive to intracellular NAD+ availability. This allows them to transmit information about a cell's metabolic state on to the biological processes they influence. Fungal sirtuins are primarily known to deacetylate histones, repressing transcription and modulating genome stability. Their target genes include those involved in NAD+ homeostasis, metabolism, sporulation, secondary metabolite production, and virulence traits of pathogenic fungi. By targeting different genes over evolutionary time, sirtuins serve as rewiring points that allow organisms to evolve novel responses to low NAD+ stress by bringing relevant biological processes under the control of sirtuins.

Demethylation of the Protein Phosphatase PP2A Promotes Demethylation of Histones to Enable Their Function as a Methyl Group Sink.

Dysregulation of chromatin methylation is associated with defects in cellular differentiation as well as a variety of cancers. How cells regulate the opposing activities of histone methyltransferase and demethylase enzymes to set the methylation status of the epigenome for proper control of gene expression and metabolism remains poorly understood. Here, we show that loss of methylation of the major phosphatase PP2A in response to methionine starvation activates the demethylation of histones through hyperphosphorylation of specific demethylase enzymes. In parallel, this regulatory mechanism enables cells to preserve SAM by increasing SAH to limit SAM consumption by methyltransferase enzymes. Mutants lacking the PP2A methyltransferase or the effector H3K36 demethylase Rph1 exhibit elevated SAM levels and are dependent on cysteine due to reduced capacity to sink the methyl groups of SAM. Therefore, PP2A directs the methylation status of histones by regulating the phosphorylation status of histone demethylase enzymes in response to SAM levels.