FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

Structural insights into the human NuA4/TIP60 acetyltransferase and chromatin remodeling complex.

The human nucleosome acetyltransferase of histone H4 (NuA4)/Tat-interactive protein, 60 kilodalton (TIP60) coactivator complex, a fusion of the yeast switch/sucrose nonfermentable related 1 (SWR1) and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4, H2A, and H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the E1A binding protein P400 (EP400) subunit serves as a scaffold holding the different functional modules in specific positions, creating a distinct arrangement of the actin-related protein (ARP) module. EP400 interacts with the transformation/transcription domain-associated protein (TRRAP) subunit by using a footprint that overlaps with that of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.

Assessing contributions of DNA sequences at the 3' end of a yeast gene on yFACT, RNA polymerase II, and nucleosome occupancy.

OBJECTIVE: In past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3' end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3' end of genes in promoting yFACT dissociation upon transcription termination. RESULTS: We generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3' end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene's 3' end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3' ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture.

DNA polymerase δ subunit Pol32 binds histone H3-H4 and couples nucleosome assembly with Okazaki fragment processing.

During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.

Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.

Single-nucleosome imaging unveils that condensins and nucleosome-nucleosome interactions differentially constrain chromatin to organize mitotic chromosomes.

For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast.

BACKGROUND: Human hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability. RESULTS: By comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability. CONCLUSION: Our findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.

Cell-free DNA from germline TP53 mutation carriers reflect cancer-like fragmentation patterns.

Germline pathogenic TP53 variants predispose individuals to a high lifetime risk of developing multiple cancers and are the hallmark feature of Li-Fraumeni syndrome (LFS). Our group has previously shown that LFS patients harbor shorter plasma cell-free DNA fragmentation; independent of cancer status. To understand the functional underpinning of cfDNA fragmentation in LFS, we conducted a fragmentomic analysis of 199 cfDNA samples from 82 TP53 mutation carriers and 30 healthy TP53-wildtype controls. We find that LFS individuals exhibit an increased prevalence of A/T nucleotides at fragment ends, dysregulated nucleosome positioning at p53 binding sites, and loci-specific changes in chromatin accessibility at development-associated transcription factor binding sites and at cancer-associated open chromatin regions. Machine learning classification resulted in robust differentiation between TP53 mutant versus wildtype cfDNA samples (AUC-ROC = 0.710-1.000) and intra-patient longitudinal analysis of ctDNA fragmentation signal enabled early cancer detection. These results suggest that cfDNA fragmentation may be a useful diagnostic tool in LFS patients and provides an important baseline for cancer early detection.

Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4.

Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. In vivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.

CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.

The budding yeast Fkh1 Forkhead associated (FHA) domain promotes a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

Structure of the human TIP60 complex.

Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.

An integrated structural model of the DNA damage-responsive H3K4me3 binding WDR76:SPIN1 complex with the nucleosome.

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.

A DNA condensation code for linker histones.

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

A repressive H3K36me2 reader mediates Polycomb silencing.

In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.

Near millimolar concentration of nucleosomes in mitotic chromosomes from late prometaphase into anaphase.

Chromosome compaction is a key feature of mitosis and critical for accurate chromosome segregation. However, a precise quantitative analysis of chromosome geometry during mitotic progression is lacking. Here, we use volume electron microscopy to map, with nanometer precision, chromosomes from prometaphase through telophase in human RPE1 cells. During prometaphase, chromosomes acquire a smoother surface, their arms shorten, and the primary centromeric constriction is formed. The chromatin is progressively compacted, ultimately reaching a remarkable nucleosome concentration of over 750 µM in late prometaphase that remains relatively constant during metaphase and early anaphase. Surprisingly, chromosomes then increase their volume in late anaphase prior to deposition of the nuclear envelope. The plateau of total chromosome volume from late prometaphase through early anaphase described here is consistent with proposals that the final stages of chromatin condensation in mitosis involve a limit density, such as might be expected for a process involving phase separation.

Microscale Thermophoresis Analysis of Chromatin Interactions.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya.Microscale thermophoresis (MST) is a biophysical technique for the study of biomolecular interactions. It has seen increasing application in recent years thanks to its solution-based nature, rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events.Here, we describe the use of MST in the study of chromatin interactions. The emphasis lies on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB, the sequential binding of core histone complexes to histone chaperone APLF, the impact of the nucleosomal context on the recognition of histone modifications, and the binding of a viral peptide to the nucleosome. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples.

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes.

Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction.

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1's ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDR's interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1-nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.

Interpretable deep residual network uncovers nucleosome positioning and associated features.

Nucleosomes represent elementary building units of eukaryotic chromosomes and consist of DNA wrapped around a histone octamer flanked by linker DNA segments. Nucleosomes are central in epigenetic pathways and their genomic positioning is associated with regulation of gene expression, DNA replication, DNA methylation and DNA repair, among other functions. Building on prior discoveries that DNA sequences noticeably affect nucleosome positioning, our objective is to identify nucleosome positions and related features across entire genome. Here, we introduce an interpretable framework based on the concepts of deep residual networks (NuPoSe). Trained on high-coverage human experimental MNase-seq data, NuPoSe is able to learn sequence and structural patterns associated with nucleosome organization in human genome. NuPoSe can be also applied to unseen data from different organisms and cell types. Our findings point to 43 informative features, most of them constitute tri-nucleotides, di-nucleotides and one tetra-nucleotide. Most features are significantly associated with the nucleosomal structural characteristics, namely, periodicity of nucleosomal DNA and its location with respect to a histone octamer. Importantly, we show that features derived from the 27 bp linker DNA flanking nucleosomes contribute up to 10% to the quality of the prediction model. This, along with the comprehensive training sets, deep-learning architecture, and feature selection method, may contribute to the NuPoSe's 80-89% classification accuracy on different independent datasets.