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In single cells, variably sized nanoscale chromatin structures are observed, but it is unknown whether these form a cohesive framework that regulates RNA transcription. Here, we demonstrate that the human genome is an emergent, self-assembling, reinforcement learning system. Conformationally defined heterogeneous, nanoscopic packing domains form by the interplay of transcription, nucleosome remodeling, and loop extrusion. We show that packing domains are not topologically associated domains. Instead, packing domains exist across a structure-function life cycle that couples heterochromatin and transcription in situ, explaining how heterochromatin enzyme inhibition can produce a paradoxical decrease in transcription by destabilizing domain cores. Applied to development and aging, we show the pairing of heterochromatin and transcription at myogenic genes that could be disrupted by nuclear swelling. In sum, packing domains represent a foundation to explore the interactions of chromatin and transcription at the single-cell level in human health.

Facultative heterochromatin is marked by the repressive histone modification H3K27me3 in eukaryotes. Deposited by the PRC2 complex, H3K27me3 is essential for regulating gene expression during development, and chromatin bearing this mark is generally considered transcriptionally inert. The PRC2 complex has also been linked to programmed DNA elimination during development in ciliates such as Paramecium. Due to a lack of mechanistic insight, a direct involvement has been questioned as most eliminated DNA segments in Paramecium are shorter than the size of a nucleosome. Here, we identify two sets of histone methylation readers essential for PRC2-mediated DNA elimination in Paramecium: Firefly1/2 and Mayfly1-4. The chromodomain proteins Firefly1/2 act in tight association with TFIIS4, a transcription elongation factor required for noncoding RNA transcription. These noncoding transcripts act as scaffolds for sequence-specific targeting by PIWI-bound sRNAs, resulting in local nucleosome depletion and DNA elimination. Our findings elucidate the molecular mechanism underlying the role of PRC2 in PIWI-mediated DNA elimination and suggest that its role in IES elimination may be to activate rather than repress transcription., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers., Competing Interests: Declaration of interests A.B.S. is a co-inventor on a patent relating to the Fiber-seq method (US17/995,058)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. Although transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by functional genomics, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. This study provides clear evidence for Swi/Snf playing a direct role in gene repression via a cis transcriptional interference mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mammalian RNA polymerase II preinitiation complexes assemble adjacent to a nucleosome whose proximal edge (NPE) is typically 40 to 50 bp downstream of the transcription start site. At active promoters, that +1 nucleosome is universally modified by trimethylation on lysine 4 of histone H3 (H3K4me3). The Pol II preinitiation complex only extends 35 bp beyond the transcription start site, but nucleosomal templates with an NPE at +51 are nearly inactive in vitro with promoters that lack a TATA element and thus depend on TFIID for promoter recognition. Significantly, this inhibition is relieved when the +1 nucleosome contains H3K4me3, which can interact with TFIID subunits. Here, we show that H3K4me3 templates with both TATA and TATA-less promoters are active with +35 NPEs when transcription is driven by TFIID. Templates with +20 NPE are also active but at reduced levels compared to +35 and +51 NPEs, consistent with a general inhibition of promoter function when the proximal nucleosome encroaches on the preinitiation complex. Remarkably, dinucleosome templates support transcription when H3K4me3 is only present in the distal nucleosome, suggesting that TFIID-H3K4me3 interaction does not require modification of the +1 nucleosome. Transcription reactions performed with an alternative protocol retaining most nuclear factors results primarily in early termination, with a minority of complexes successfully traversing the first nucleosome. In such reactions, the +1 nucleosome does not substantially affect the level of termination even with an NPE of +20, indicating that a nucleosome barrier is not a major driver of early termination by Pol II., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa , de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin., Competing Interests: Competing interests statement:The authors declare no competing interest.

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design., Competing Interests: Competing interests statement:The authors declare no competing interest.

Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin., (© 2024 Sahrhage et al.)

N6-Methyladenosine (m6A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m6A sites is notably asymmetric along mRNAs, with a strong preference toward the 3' terminus of the transcript. How m6A regional preference is shaped remains incompletely understood. In this study, by performing m6A-seq on chromatin-associated RNAs, we found that m6A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m6A sites, suggesting an intricate interplay between m6A methylation and nucleosome-mediated transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m6A sites. In addition, inhibiting Pol-II movement increases nearby m6A methylation levels. By analyzing massively parallel assays for m6A, we found that RNA secondary structures inhibit m6A methylation. Remarkably, the m6A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m6A motifs to the methyltransferase complex and subsequent m6A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m6A regional preference., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many factors control the elongation phase of transcription by RNA polymerase II (Pol II), a process that plays an essential role in regulating gene expression. We utilized cells expressing degradation tagged subunits of NELFB, PAF1 and RTF1 to probe the effects of depletion of the factors on nascent transcripts using PRO-Seq and on chromatin architecture using DFF-ChIP. Although NELF is involved in promoter proximal pausing, depletion of NELFB had only a minimal effect on the level of paused transcripts and almost no effect on control of productive elongation. Instead, NELF depletion increased the utilization of downstream transcription start sites and caused a dramatic, genome-wide loss of H3K4me3 marked nucleosomes. Depletion of PAF1 and RTF1 both had major effects on productive transcript elongation in gene bodies and also caused initiation site changes like those seen with NELFB depletion. Our study confirmed that the first nucleosome encountered during initiation and early elongation is highly positioned with respect to the major TSS. In contrast, the positions of H3K4me3 marked nucleosomes in promoter regions are heterogeneous and are influenced by transcription. We propose a model defining NELF function and a general role of the H3K4me3 modification in blocking transcription initiation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Several covalent modifications are found associated with the transcriptionally active chromatin regions constituted by the genes transcribed by RNA polymerase (pol) II. Pol III-transcribed genes code for the small, stable RNA species, which participate in many cellular processes, essential for survival. Pol III transcription is repressed under most of the stress conditions by its negative regulator Maf1. We found that most of the histone acetylations increase with starvation-induced repression on several genes transcribed by the yeast pol III. On one of these genes, SNR6 (coding for the U6snRNA), a strongly positioned nucleosome in the gene upstream region plays regulatory role under repression. On this nucleosome, the changes in H3K9 and H3K14 acetylations show different dynamics. During repression, acetylation levels on H3K9 show steady increase whereas H3K14 acetylation increases with a peak at 40 min after which levels reduce. Both the levels settle by 2 hr to a level higher than the active state, which revert to normal levels with nutrient repletion. The increase in H3 acetylations is seen in the mutants reported to show reduced SNR6 transcription but not in the maf1Δ cells. This increase on a regulatory nucleosome may be part of the signaling mechanisms, which prepare cells for the stress-related quick repression as well as reactivation. The contrasting association of the histone acetylations with pol II and pol III transcription may be an important consideration to make in research studies focused on drug developments targeting histone modifications., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aneeshkumar Gopalakrishnan Arimbasseri reports financial support was provided by Council of Scientific & Industrial Research. Ashutosh Shukla reports financial support was provided by Council of Scientific & Industrial Research. Ashis Kumar Pradhan reports financial support was provided by Council of Scientific & Industrial Research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Eukaryotic genome is packaged into chromatin. Thus, transcription takes place in the context of chromatin that is an array of nucleosomes. Nucleosome poses a barrier for the gene regulatory factors to access DNA for transcription to occur. Fortunately, eukaryotic cells have evolved mechanisms of nucleosomal disassembly and reassembly for transcription through chromatin. Such nucleosomal alteration in controlling transcription is governed by a heterodimeric chromatin remodeling factor, FACT (facilitates chromatin transcription), which is evolutionarily conserved from yeast to humans. FACT facilitates chromatin disassembly at the promoter and reassembly at the open reading frame. Such chromatin regulatory functions of FACT promote transcription. Likewise, other DNA transacting processes such as DNA replication and repair are also regulated by FACT via modulation of chromatin dynamics. Intriguingly, FACT is found to be upregulated in breast and other cancers with oncogenic potential. Thus, FACT and/or its upstream regulatory pathways/factors can be employed for cancer prognosis and targeted for an effective cancer therapy. Further, FACT is found to be downregulated and/or mutated in various cancers including breast cancer. Here, we describe FACT and its involvement in breast and other cancers with prognostic and targeted therapeutic implications., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Proper maintenance of epigenetic information after replication is dependent on the rapid assembly and maturation of chromatin. Chromatin Assembly Complex 1 (CAF-1) is a conserved histone chaperone that deposits (H3-H4) 2 tetramers as part of the replication-dependent chromatin assembly process. Loss of CAF-1 leads to a delay in chromatin maturation, albeit with minimal impact on steady-state chromatin structure. However, the mechanisms by which CAF-1 mediates the deposition of (H3-H4) 2 tetramers and the phenotypic consequences of CAF-1-associated assembly defects are not well understood. We used nascent chromatin occupancy profiling to track the spatiotemporal kinetics of chromatin maturation in both wild-type (WT) and CAF-1 mutant yeast cells. Our results show that loss of CAF-1 leads to a heterogeneous rate of nucleosome assembly, with some nucleosomes maturing at near WT kinetics and others showing significantly slower maturation kinetics. The slow-to-mature nucleosomes are enriched in intergenic and poorly transcribed regions, suggesting that transcription-dependent assembly mechanisms can reset the slow-to-mature nucleosomes following replication. Nucleosomes with slow maturation kinetics are also associated with poly(dA:dT) sequences, which implies that CAF-1 deposits histones in a manner that counteracts resistance from the inflexible DNA sequence, promoting the formation of histone octamers as well as ordered nucleosome arrays. In addition, we show that the delay in chromatin maturation is accompanied by a transient and S-phase-specific loss of gene silencing and transcriptional regulation, revealing that the DNA replication program can directly shape the chromatin landscape and modulate gene expression through the process of chromatin maturation., (© 2023 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)

Centromeres are no longer considered to be silent. Both centromeric and pericentric transcription have been discovered, and their RNA transcripts have been characterized and probed for functions in numerous monocentric model organisms recently. Here, we will discuss the challenges in centromere transcription studies due to the repetitive nature and sequence similarity in centromeric and pericentric regions. Various technological breakthroughs have helped to tackle these challenges and reveal unique features of the centromeres and pericentromeres. We will briefly introduce these techniques, including third-generation long-read DNA and RNA sequencing, protein-DNA and RNA-DNA interaction detection methods, and epigenomic and nucleosomal mapping techniques. Interestingly, some newly analyzed repeat-based holocentromeres also resemble the architecture and the transcription behavior of monocentromeres. We will summarize evidences that support the functions of the transcription process and stalling, and those that support the functions of the centromeric and pericentric RNAs. The processing of centromeric and pericentric RNAs into multiple variants and their diverse structures may also provide clues to their functions. How future studies may address the separation of functions of specific centromeric transcription steps, processing pathways, and the transcripts themselves will also be discussed., (© 2023. The Author(s).)

In this issue of Molecular Cell, Abril-Garrido et al. 1 used cryo-EM to uncover that the +1 nucleosome inhibits transcription by interfering with the function of the TFIIH translocase via mechanisms that depend on its position relative to the transcription start site., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The directionality of gene promoters-the ratio of protein-coding over divergent noncoding transcription-is highly variable. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription relative to protein-coding transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus to a decrease in promoter directionality. We find that RSC has a modest effect on nucleosome positioning upstream in promoters at the sites of divergent transcription. These promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 DNA-binding protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription relative to coding direction transcription. We propose that any DNA-binding factor, when stably associated with cryptic transcription start sites, forms a barrier which represses divergent transcription, thereby promoting promoter directionality., (© 2022 Wu et al.)

Nucleosomes are disrupted transiently during eukaryotic transcription, yet the displaced histones must be retained and redeposited onto DNA, to preserve nucleosome density and associated histone modifications. Here, we show that the essential Spt5 processivity factor of RNA polymerase II (Pol II) plays a direct role in this process in budding yeast. Functional orthologues of eukaryotic Spt5 are present in archaea and bacteria, reflecting its universal role in RNA polymerase processivity. However, eukaryotic Spt5 is unique in having an acidic amino terminal tail (Spt5N) that is sandwiched between the downstream nucleosome and the upstream DNA that emerges from Pol II. We show that Spt5N contains a histone-binding motif that is required for viability in yeast cells and prevents loss of nucleosomal histones within actively transcribed regions. These findings indicate that eukaryotic Spt5 combines two essential activities, which together couple processive transcription to the efficient capture and re-deposition of nucleosomal histones., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases., Competing Interests: Declaration of interests The others declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The most basic level of eukaryotic gene regulation is the presence or absence of nucleosomes on DNA regulatory elements. In an effort to elucidate in vivo nucleosome patterns, in vitro studies are frequently used. In vitro, short DNA fragments are more favorable for nucleosome formation, increasing the likelihood of nucleosome occupancy. This may in part result from the fact that nucleosomes prefer to form on the terminal ends of linear DNA. This phenomenon has the potential to bias in vitro reconstituted nucleosomes and skew results. If the ends of DNA fragments are known, the reads falling close to the ends are typically discarded. In this study we confirm the phenomenon of end bias of in vitro nucleosomes. We describe a method in which nearly identical libraries, with different known ends, are used to recover nucleosomes which form towards the terminal ends of fragmented DNA. Finally, we illustrate that although nucleosomes prefer to form on DNA ends, it does not appear to skew results or the interpretation thereof., Competing Interests: Two authors, DAB and CEB, are brothers. CEB owns and operates the software consulting company, Qubit Software LLC. The contributions of Qubit Software LLC were strictly pro bono, and the custom codes written are publicly available. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Thus the authors declare no conflict of interest.

High Mobility Group Box (HMGB) proteins are small architectural DNA binding proteins that regulate multiple genomic processes such as DNA damage repair, nucleosome sliding, telomere homeostasis, and transcription. In doing so they control both normal cellular functions and impact a myriad of disease states, including cancers and autoimmune diseases. HMGB proteins bind to DNA and nucleosomes to modulate the local chromatin environment, which facilitates the binding of regulatory protein factors to the genome and modulates higher order chromosomal organization. Numerous studies over the years have characterized the structure and function of interactions between HMGB proteins and DNA, both biochemically and inside cells, providing valuable mechanistic insight as well as evidence these interactions influence pathological processes. This review highlights recent studies supporting the roles of HMGB1 and HMGB2 in global organization of the genome, as well as roles in transcriptional regulation and telomere maintenance via interactions with G-quadruplex structures. Moreover, emerging models for how HMGB proteins function as RNA binding proteins are presented. Nuclear HMGB proteins have broad regulatory potential to impact numerous aspects of cellular metabolism in normal and disease states.

Purpose of Review: Comprehensive sequencing studies aimed at determining the genetic landscape of myeloid neoplasms have identified epigenetic regulators to be among the most commonly mutated genes. Detailed studies have also revealed a number of epigenetic vulnerabilities. The purpose of this review is to outline these vulnerabilities and to discuss the new generation of drugs that exploit them., Recent Findings: In addition to deoxyribonucleic acid-methylation, novel epigenetic dependencies have recently been discovered in various myeloid neoplasms and many of them can be targeted pharmacologically. These include not only chromatin writers, readers, and erasers but also chromatin movers that shift nucleosomes to allow access for transcription. Inhibitors of protein-protein interactions represent a novel promising class of drugs that allow disassembly of oncogenic multiprotein complexes., Summary: An improved understanding of disease-specific epigenetic vulnerabilities has led to the development of second-generation mechanism-based epigenetic drugs against myeloid neoplasms. Many of these drugs have been introduced into clinical trials and synergistic drug combination regimens have been shown to enhance efficacy and potentially prevent drug resistance., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The 'open' and 'compact' regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Quiescence is a reversible G 0 state essential for differentiation, regeneration, stem-cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state., Competing Interests: CC, RD, KB, TT No competing interests declared, (© 2021, Cucinotta et al.)

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions., (© 2021 Viktorovskaya et al.; Published by Cold Spring Harbor Laboratory Press.)

The intrinsic dynamic nature of chromosomes is emerging as a fundamental component in regulating DNA transcription, replication, and damage-repair among other nuclear functions. With this increased awareness, reinforced over the last ten years, many new experimental techniques, mainly based on microscopy and chromosome conformation capture, have been introduced to study the genome in space and time. Owing to the increasing complexity of these cutting-edge techniques, computational approaches have become of paramount importance to interpret, contextualize, and complement such experiments with new insights. Hence, it is becoming crucial for experimental biologists to have a clear understanding of the diverse theoretical modeling approaches available and the biological information each of them can provide., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A-H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA.

Nucleosome positioning is crucial for the genome's function. Though the role of DNA sequence in positioning nucleosomes is well understood, a detailed mechanistic understanding on the impact of transcription remains lacking. Using numerical simulations, we investigated the dependence of nucleosome density profiles on transcription level across multiple species. We found that the low nucleosome affinity of yeast, but not mouse, promoters contributes to the formation of phased nucleosomes arrays for inactive genes. For the active genes, a heterogeneous distribution of +1 nucleosomes, caused by a tug-of-war between two types of remodeling enzymes, is essential for reproducing their density profiles. In particular, while positioning enzymes are known to remodel the +1 nucleosome and align it toward the transcription start site (TSS), spacer enzymes that use a pair of nucleosomes as their substrate can shift the nucleosome array away from the TSS. Competition between these enzymes results in two types of nucleosome density profiles with well- and ill-positioned +1 nucleosome. Finally, we showed that Pol II assisted histone exchange, if occurring at a fast speed, can abolish the impact of remodeling enzymes. By elucidating the role of individual factors, our study reconciles the seemingly conflicting results on the overall impact of transcription in positioning nucleosomes across species., Competing Interests: The authors have declared that no competing interests exist.

Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ∼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The hominidae-specific histone variant H4G is expressed in breast cancer patients in a stage-dependent manner. H4G localizes primarily in the nucleoli via its interaction with nucleophosmin (NPM1). H4G is involved in rDNA transcription and ribosome biogenesis, which facilitates breast cancer cell proliferation. However, the molecular mechanism underlying this process remains unknown. Here, we show that H4G is not stably incorporated into nucleolar chromatin, even with the chaperoning assistance of NPM1. H4G likely form transient nucleosome-like-structure that undergoes rapid dissociation. In addition, the nucleolar chromatin in H4GKO cells is more compact than WT cells. Altogether, our results suggest that H4G relaxes the nucleolar chromatin and enhances rRNA transcription by forming destabilized nucleosome in breast cancer cells., (© 2020 Wiley Periodicals LLC.)

The incorporation of histone variants into nucleosomes has important functional consequences in all aspects of eukaryotic chromatin biology. H2A.Z is a conserved histone variant found in all eukaryotes from yeast to mammals. Recent studies in yeast have shed light on the questions of where and how nucleosomes containing this variant are situated at promoters and in relation to genes, and what its specificity implies with regard to transcription. In yeast, H2A.Z appears to be primarily incorporated into the first nucleosome in the direction of transcription initiation, either of an mRNA transcript or a divergently transcribed upstream antisense non-coding RNA. This specificity of H2A.Z is due in part to the localization at promoters of SWR1, the ATP-dependent chromatin remodeler that incorporates H2A.Z into nucleosomes. Replacement of H2A.Z with canonical H2A is dependent on the function of the transcription pre-initiation complex. The recent studies summarized in this review reveal that the directionality of H2A.Z occupancy in relation to transcription thus reflects a balance of incorporation and eviction activities, which likely have varying contributions at distinct sets of genes across the genome.

Protein-DNA interactions are dynamic, and these dynamics are an important aspect of chromatin-associated processes such as transcription or replication. Due to a lack of methods to study on- and off-rates across entire genomes, protein-DNA interaction dynamics have not been studied extensively. Here, we determine in vivo off-rates for the Saccharomyces cerevisiae chromatin organizing factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide range, varying between 4.2 and 33 min. Sites with different off-rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome-free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off-rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off-Rates by SEQuencing) is a meaningful way of investigating protein-DNA binding dynamics genome-wide., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing., (Published by Oxford University Press on behalf of Nucleic Acids Research 2020.)

Negative cofactor 2 (NC2), including two subunits NC2α and NC2β, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α-NC2β interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2β is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Compared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. We performed a genetic screen in Saccharomyces cerevisiae to identify histone mutants that exhibit transcriptional readthrough of terminators. Amino acid substitutions identified by the screen map to the nucleosome DNA entry-exit site. The strongest H3 mutants revealed widespread genomic changes, including increased sense-strand transcription upstream and downstream of genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3' ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to transcription and highlight the importance of the DNA entry-exit site in broadly maintaining the integrity of the transcriptome., Competing Interests: AH, ME, AF, JS, LL, KA No competing interests declared, (© 2020, Hildreth et al.)

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing., Competing Interests: Competing interest statement: During the middle of the project, H.L. became an employee of NB Life Laboratory, LLC. P.P. was a scientist from Nano Temper Technologies, Inc. G.Z. holds equity in NB Life Laboratory LLC.

The nucleosome is a small unit of chromatin, which is dynamic in eukaryotes. Chromatin conformation and post-translational modifications affect nucleosome dynamics under certain conditions, playing an important role in the epigenetic regulation of transcription, replication and reprogramming. The Snf2 remodeling family is one of the crucial remodeling complexes that tightly regulate chromatin structure and affect nucleosome dynamics. This family alters nucleosome positioning, exchanges histone variants, and assembles and disassembles nucleosomes at certain locations. Moreover, the Snf2 family, in conjunction with other co-factors, regulates gene expression in Saccharomyces cerevisiae. Here we first review recent findings on the Snf2 family remodeling complexes and then use some examples to illustrate the cooperation between different members of Snf2 family, and the cooperation between Snf2 family and other co-factors in gene regulation especially during transcription initiation.

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation.

RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity., (© 2020 Merkl et al.)

The nucleosome presents a formidable barrier to DNA-templated transcription by the RNA polymerase II machinery. Overcoming this transcriptional barrier in a locus-specific manner requires sequence-specific recognition of nucleosomal DNA by 'pioneer' transcription factors (TFs). Cell fate decisions, in turn, depend on the coordinated action of pioneer TFs at cell lineage-specific gene regulatory elements. Although it is already appreciated that pioneer factors play a critical role in cell differentiation, our understanding of the structural and biochemical mechanisms by which they act is still rapidly expanding. Recent research has revealed novel insight into modes of nucleosome-TF binding and uncovered kinetic principles by which nucleosomal DNA compaction affects both TF binding and residence time. Here, we review progress and argue that these structural and kinetic studies suggest new models of gene regulation by pioneer TFs.

In eukaryotes, the nucleosome is the basic unit of chromatin. Since the genomic DNA is tightly wrapped around the histone octamer in the nucleosome, its function is severely restricted in chromatin. To overcome the nucleosome barrier, the nucleosome structure must dynamically change during genomic DNA functions. Recent structural studies revealed that chromatin remodelers and RNA polymerase II drastically alter the nucleosome structures during the chromatin remodeling and transcription processes. These results provide important information for understanding how genes are properly regulated in eukaryotes. Here, we review the recent structural studies of nucleosome versatility and dynamics during chromatin remodeling and transcription by RNA polymerase II., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

RNA polymerase II (RNAP II) non-coding transcription is now known to cover almost the entire eukaryotic genome, a phenomenon referred to as pervasive transcription. As a consequence, regions previously thought to be non-transcribed are subject to the passage of RNAP II and its associated proteins for histone modification. This is the case for the nucleosome-depleted regions (NDRs), which provide key sites of entry into the chromatin for proteins required for the initiation of coding gene transcription and DNA replication. In this review, recent data on the effects of pervasive transcription through NDRs are summarized and a model is proposed to explain how RNAP II-driven transcription is able to modify the nucleosomes flanking the NDRs, leading to nucleosome repositioning and NDR closure. Even though much of the mechanistic detail underlying these events remains to be elucidated, such a model provides a basis to explain how non-coding transcription through NDRs can regulate the initiation of coding gene expression and DNA replication., (© 2019 WILEY Periodicals, Inc.)

Nucleotide excision repair (NER) consists of global genomic NER (GG-NER) and transcription coupled NER (TC-NER) subpathways. In eukaryotic cells, genomic DNA is wrapped around histone octamers (an H3-H4 tetramer and two H2A-H2B dimers) to form nucleosomes, which are well known to profoundly inhibit the access of NER proteins. Through unbiased screening of histone H4 residues in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain, we identified 24 mutations that enhance or decrease UV sensitivity of Saccharomyces cerevisiae cells. The histone H4 H75E mutation, which is largely embedded in the nucleosome and interacts with histone H2B, significantly attenuates GG-NER and Rad26-independent TC-NER but does not affect TC-NER in the presence of Rad26. All the other histone H4 mutations, except for T73F and T73Y that mildly attenuate GG-NER, do not substantially affect GG-NER or TC-NER. The attenuation of GG-NER and Rad26-independent TC-NER by the H4H75E mutation is not due to decreased chromatin accessibility, impaired methylation of histone H3 K79 that is at the center of the LRS domain, or lowered expression of NER proteins. Instead, the attenuation is at least in part due to impaired recruitment of Rad4, the key lesion recognition and verification protein, to chromatin following induction of DNA lesions., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)