Search

Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes., (© 2021. The Author(s).)

The epigenome is multidimensional, with individual molecular components operating on different levels to control transcriptional output. Techniques that combine measurements of these features can reveal their precise correspondence in genomic space, or temporal connectivity, to better understand how they jointly regulate genes. ATAC-Me is an integrated method to probe DNA methylation and chromatin accessibility from a single DNA fragment library. Intact nuclei undergo Tn5 transposition to isolate DNA fragments within nucleosome-free regions. Isolated fragments are exposed to sodium bisulfite before library amplification and sequencing. A typical ATAC-Me experiment detects ~60,000-75,000 peak regions (P < 0.05), covering ~3-4 million CpG sites with at least 5× coverage. These sites display a range of methylation values depending on the cellular and genomic context. The approach is well suited for time course studies that aim to capture chromatin and DNA methylation dynamics in tandem during cellular differentiation. The protocol is completed in 2 d with standard molecular biology equipment and expertise. Analysis of resulting data uses publicly available software requiring basic bioinformatics skills to interpret results., (© 2021. Springer Nature Limited.)

Tumour progression within the tissue microenvironment is accompanied by complex biomechanical alterations of the extracellular environment. While histopathology images provide robust biochemical markers for tumor progression in clinical settings, a quantitative single cell score using nuclear morphology and chromatin organization integrated with the long range mechanical coupling within the tumor microenvironment is missing. We propose that the spatial chromatin organization in individual nuclei characterises the cell state and their alterations during tumor progression. In this paper, we first built an image analysis pipeline and implemented it to classify nuclei from patient derived breast tissue biopsies of various cancer stages based on their nuclear and chromatin features. Replacing H&E with DNA binding dyes such as Hoescht stained tissue biopsies, we improved the classification accuracy. Using the nuclear morphology and chromatin organization features, we constructed a pseudo-time model to identify the chromatin state changes that occur during tumour progression. This enabled us to build a single-cell mechano-genomic score that characterises the cell state during tumor progression from a normal to a metastatic state. To gain further insights into the alterations in the local tissue microenvironments, we also used the nuclear orientations to identify spatial neighbourhoods that have been posited to drive tumor progression. Collectively, we demonstrate that image-based single cell chromatin and nuclear features are important single cell biomarkers for phenotypic mapping of tumor progression., (© 2021. The Author(s).)

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. We present a refined solution structure of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. We show that the conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and that most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89 we find strong evidence for an elevated pKa of 5.3. Our data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo., (© 2021. The Author(s).)

Nuclear bodies are membraneless condensates that may form via liquid-liquid phase separation. The viscoelastic chromatin network could impact their stability and may hold the key for understanding experimental observations that defy predictions of classical theories. However, quantitative studies on the role of the chromatin network in phase separation have remained challenging. Using a diploid human genome model parameterized with chromosome conformation capture (Hi-C) data, we study the thermodynamics and kinetics of nucleoli formation. Dynamical simulations predict the formation of multiple droplets for nucleolar particles that experience specific interactions with nucleolus-associated domains (NADs). Coarsening dynamics, surface tension, and coalescence kinetics of the simulated droplets are all in quantitative agreement with experimental measurements for nucleoli. Free energy calculations further support that a two-droplet state, often observed for nucleoli in somatic cells, is metastable and separated from the single-droplet state with an entropic barrier. Our study suggests that nucleoli-chromatin interactions facilitate droplets' nucleation but hinder their coarsening due to the coupled motion between droplets and the chromatin network: as droplets coalesce, the chromatin network becomes increasingly constrained. Therefore, the chromatin network supports a nucleation and arrest mechanism to stabilize the multi-droplet state for nucleoli and possibly for other nuclear bodies., (© 2021. The Author(s).)

Single-cell multi-omics sequencing technology can infer cell heterogeneity and reveal relationships across molecular layers. Combining single-cell RNA sequencing, DNA methylation, and chromatin accessibility allows a multimodal understanding of cell function and epigenetic regulation within individual cells. Here, we offer a protocol to perform scChaRM-seq (single-cell chromatin accessibility, RNA barcoding, and DNA methylation sequencing), which has been applied to study de novo DNA methylation and its relationship with transcription and chromatin accessibility in single human oocytes. For complete details on the use and execution of this protocol, please refer to Yan et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here, we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell-type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different chromatin proteins., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells (ESCs) to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin., Competing Interests: Declaration of interests D.R. is a cofounder of Constellation and Fulcrum Pharmaceuticals., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Construction of chromosomes 3D models based on single cell Hi-C data constitute an important challenge. We present a reconstruction approach, DPDchrom, that incorporates basic knowledge whether the reconstructed conformation should be coil-like or globular and spring relaxation at contact sites. In contrast to previously published protocols, DPDchrom can naturally form globular conformation due to the presence of explicit solvent. Benchmarking of this and several other methods on artificial polymer models reveals similar reconstruction accuracy at high contact density and DPDchrom advantage at low contact density. To compare 3D structures insensitively to spatial orientation and scale, we propose the Modified Jaccard Index. We analyzed two sources of the contact dropout: contact radius change and random contact sampling. We found that the reconstruction accuracy exponentially depends on the number of contacts per genomic bin allowing to estimate the reconstruction accuracy in advance. We applied DPDchrom to model chromosome configurations based on single-cell Hi-C data of mouse oocytes and found that these configurations differ significantly from a random one, that is consistent with other studies., Competing Interests: The authors have declared that no competing interests exist.

Recent studies of transcription have revealed an advanced set of overarching principles that govern vitamin D action on a genome-wide scale. These tenets of vitamin D transcription have emerged as a result of the application of now well-established techniques of chromatin immunoprecipitation coupled to next-generation DNA sequencing that have now been linked directly to CRISPR-Cas9 genomic editing in culture cells and in mouse tissues in vivo. Accordingly, these techniques have established that the vitamin D hormone modulates sets of cell-type specific genes via an initial action that involves rapid binding of the VDR-ligand complex to multiple enhancer elements at open chromatin sites that drive the expression of individual genes. Importantly, a sequential set of downstream events follows this initial binding that results in rapid histone acetylation at these sites, the recruitment of additional histone modifiers across the gene locus, and in many cases, the appearance of H3K36me3 and RNA polymerase II across gene bodies. The measured recruitment of these factors and/or activities and their presence at specific regions in the gene locus correlate with the emerging presence of cognate transcripts, thereby highlighting sequential molecular events that occur during activation of most genes both in vitro and in vivo. These features provide a novel approach to the study of vitamin D analogs and their actions in vivo and suggest that they can be used for synthetic compound evaluation and to select for novel tissue- and gene-specific features. This may be particularly useful for ligand activation of nuclear receptors given the targeting of these factors directly to genetic sites in the nucleus.

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains., Results: Here we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components and transcription mostly do not affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains., Conclusions: In sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components., (© 2021. The Author(s).)

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The accumulation of large epigenomics data consortiums provides us with the opportunity to extrapolate existing knowledge to new cell types and conditions. We propose Epitome, a deep neural network that learns similarities of chromatin accessibility between well characterized reference cell types and a query cellular context, and copies over signal of transcription factor binding and modification of histones from reference cell types when chromatin profiles are similar to the query. Epitome achieves state-of-the-art accuracy when predicting transcription factor binding sites on novel cellular contexts and can further improve predictions as more epigenetic signals are collected from both reference cell types and the query cellular context of interest., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Over the past decade, genome-wide assays for chromatin interactions in single cells have enabled the study of individual nuclei at unprecedented resolution and throughput. Current chromosome conformation capture techniques survey contacts for up to tens of thousands of individual cells, improving our understanding of genome function in 3D. However, these methods recover a small fraction of all contacts in single cells, requiring specialised processing of sparse interactome data. In this review, we highlight recent advances in methods for the interpretation of single-cell genomic contacts. After discussing the strengths and limitations of these methods, we outline frontiers for future development in this rapidly moving field., (© The Author(s) 2021. Published by Oxford University Press.)

Hi-C is a genome-wide assay based on Chromosome Conformation Capture and high-throughput sequencing to decipher 3D chromatin organization in the nucleus. However, computational methods to detect functional interactions utilizing Hi-C data face challenges including the correction for various sources of biases and the identification of functional interactions with low counts of interacting fragments. We present Chrom-Lasso, a lasso linear regression model that removes complex biases assumption-free and identifies functional interacting loci with increased power by combining information of local reads distribution surrounding the area of interest. We showed that interacting regions identified by Chrom-Lasso are more enriched for 5C validated interactions and functional GWAS hits than that of GOTHiC and Fit-Hi-C. To further demonstrate the ability of Chrom-Lasso to detect interactions of functional importance, we performed time-series Hi-C and RNA-seq during T cell activation and exhaustion. We showed that the dynamic changes in gene expression and chromatin interactions identified by Chrom-Lasso were largely concordant with each other. Finally, we experimentally confirmed Chrom-Lasso's finding that Erbb3 was co-regulated with distinct neighboring genes at different states during T cell activation. Our results highlight Chrom-Lasso's utility in detecting weak functional interaction between cis-regulatory elements, such as promoters and enhancers., (© The Author(s) 2021. Published by Oxford University Press.)

Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0-1 and ±3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes., Competing Interests: Declaration of interests H.F. is affiliated with Graduate School of Medical Sciences, Weill Cornell Medicine, and the Cell Biology Program at the Sloan Kettering Institute. The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromatin fibers must fold or coil in the process of chromosome condensation. Patterns of coiling have been demonstrated for reconstituted chromatin, but the actual trajectories of fibers in condensed states of chromosomes could not be visualized because of the high density of the material. We have exploited partial decondensation of mitotic chromosomes to reveal their internal structure at sub-nucleosomal resolution by cryo-electron tomography, without the use of stains, fixatives, milling, or sectioning. DNA gyres around nucleosomes were visible, allowing the nucleosomes to be identified and their orientations to be determined. Linker DNA regions were traced, revealing the trajectories of the chromatin fibers. The trajectories were irregular, with almost no evidence of coiling and no short- or long-range order of the chromosomal material. The 146-bp core particle, long known as a product of nuclease digestion, is identified as the native state of the nucleosome, with no regular spacing along the chromatin fibers., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Upon replication stress, budding yeast checkpoint kinase Mec1 ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas., (© 2021. The Author(s).)

The recent development of experimental methods for measuring chromatin state at single-cell resolution has created a need for computational tools capable of analyzing these datasets. Here we developed Signac, a comprehensive toolkit for the analysis of single-cell chromatin data. Signac enables an end-to-end analysis of single-cell chromatin data, including peak calling, quantification, quality control, dimension reduction, clustering, integration with single-cell gene expression datasets, DNA motif analysis and interactive visualization. Through its seamless compatibility with the Seurat package, Signac facilitates the analysis of diverse multimodal single-cell chromatin data, including datasets that co-assay DNA accessibility with gene expression, protein abundance and mitochondrial genotype. We demonstrate scaling of the Signac framework to analyze datasets containing over 700,000 cells., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function 1-3 . Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi 4-6 . However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation 7 , are invisible with such approaches 8 . Here we developed immunoGAM, an extension of genome architecture mapping (GAM) 2,9 , to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation 2,10 . We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions., (© 2021. The Author(s).)

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation., (© 2021. The Author(s).)

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In cytological examination, suspicious cells are evaluated regarding malignancy and cancer type. To assist this, we previously proposed an automated method based on supervised learning that classifies cells in lung cytological images as benign or malignant. However, it is often difficult to label all cells. In this study, we developed a weakly supervised method for the classification of benign and malignant lung cells in cytological images using attention-based deep multiple instance learning (AD MIL). Images of lung cytological specimens were divided into small patch images and stored in bags. Each bag was then labeled as benign or malignant, and classification was conducted using AD MIL. The distribution of attention weights was also calculated as a color map to confirm the presence of malignant cells in the image. AD MIL using the AlexNet-like convolutional neural network model showed the best classification performance, with an accuracy of 0.916, which was better than that of supervised learning. In addition, an attention map of the entire image based on the attention weight allowed AD MIL to focus on most malignant cells. Our weakly supervised method automatically classifies cytological images with acceptable accuracy based on supervised learning without complex annotations., (© 2021. The Author(s).)

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G 2 -phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated., (© 2021. The Author(s).)

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Replication timing (RT) associates with genome architecture, while having a mixed relationship to histone marks. By profiling replication at high resolution and assessing broad histone marks across the cell cycle at the resolution of RT with and without genetic perturbation, we address the causal relationship between histone marks and RT. Four primary chromatin states, including an uncharacterized H3K36me2 state, emerge and define 97% of the mappable genome. RT and local replication patterns (e.g., initiation zones) quantitatively associate with chromatin states, histone mark dynamics, and spatial chromatin structure. Manipulation of broad histone marks and enhancer elements by overexpressing the histone H3 lysine 9/36 tri-demethylase KDM4A impacts RT across 11% of the genome. Broad histone modification changes were strong predictors of the observed RT alterations. Lastly, replication within H3K36me2-enriched neighborhoods is sensitive to KDM4A overexpression and is controlled at a megabase scale. These studies establish a role for collective chromatin mark regulation in modulating RT., Competing Interests: Declaration of interests In the past year, J.R.W. is or was serving as a consultant or advisor for Qsonica, Salarius Pharmaceuticals, and Daiichi Sankyo, Inc. The other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the 'gold standard' for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology.

Background: Macrophage polarization has a causal role in the pathogenesis and resolution of various clinical diseases. DNA-binding transcription factors (TFs) have been identified as essential factors during gene transcription. Better insight into the TFs that regulate macrophage polarization could provide novel therapeutic targets., Methods: IFN-γ (50 ng/mL) or IL4 (20 ng/mL) was utilized to stimulate bone marrow-derived macrophages from mice for 24 h for M1- and M2-polarized macrophage model construction, respectively. First, ATAC-seq (Assay for Targeting Accessible-Chromatin with high throughout sequencing) and motif analysis were conducted to identify potential transcription factors (TFs) involved in M1 and M2 macrophage polarization. Second, essential TFs were identified through RNA-seq, after which, their expression was compared between M0-polarized and M1/M2-polarized macrophages. Furthermore, a multiomic analysis of RNA-seq (siRNA knock down of the identified TFs), ChIP-seq and ATAC-seq was utilized to explore the TF-regulated molecular network. GO and KEGG analyses were used to expound the main functions of the TF-regulated molecular network. Finally, the top 5 TF-regulated genes were validated through flow cytometry, ELISA and qPCR. The cut-off values for high-throughput sequencing and qPCR were FDR < 0.05 and P < 0.05, respectively., Results: Compared with M0 macrophages, 10,771 and 4,848 peaks were identified by ATAC-seq during M1 and M2 macrophage polarization, respectively (FDR < 0.05). Fifty and 62 TF binding motifs were identified for the TFs that participate in M1 and M2 macrophage polarization, respectively. The most significantly highly expressed TFs in M1 and M2 macrophages were identified by RNA-seq as Irf1 and Egr1, with LogFC values of 3.2 and 2.8, respectively. Multiomic analyses further found that Irf1 regulated the transcription of 90 genes and that Egr1 regulated the transcription of 116 genes. The Irf1-regulated molecular network played a key role in the inflammatory response and viral defence of M1 macrophages, and 116 Egr1-regulated genes included anti-inflammatory and cell proliferation genes. Validation experiments indicated that IFN-γ-induced Gbp5, Nos2, CD86, Cxcl10 and Cxcl5 expression was significantly downregulated in siIrf1-BMDMs, and IL4-induced Itgax, Nipal1, Bhlhe40, CD206 and Ffar4 expression was significantly downregulated in siEgr1-BMDMs (P < 0.05)., Conclusions: Through multiomic analyses of epigenetic sequencing and RNA-seq with partial validation, the current study found that Irf1- and Egr1-induced transcription plays key roles in M1 and M2 macrophage polarization, respectively., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures 1 . Recent surveys of mouse and human brains with single-cell transcriptomics 2-6 and high-throughput imaging technologies 7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans., (© 2021. The Author(s).)

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing 1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data 3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments 4 . By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum., (© 2021. The Author(s).)

During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape 1 . In the developing brain, cell fate specification and topographic identity are important for defining cell identity 2 and confer selective vulnerabilities to neurodevelopmental disorders 3 . Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development., (© 2021. The Author(s).)

The discovery that overexpressing one or a few critical transcription factors can switch cell state suggests that gene regulatory networks are relatively simple. In contrast, genome-wide association studies (GWAS) point to complex phenotypes being determined by hundreds of loci that rarely encode transcription factors and which individually have small effects. Here, we use computer simulations and a simple fitting-free polymer model of chromosomes to show that spatial correlations arising from 3D genome organisation naturally lead to stochastic and bursty transcription as well as complex small-world regulatory networks (where the transcriptional activity of each genomic region subtly affects almost all others). These effects require factors to be present at sub-saturating levels; increasing levels dramatically simplifies networks as more transcription units are pressed into use. Consequently, results from GWAS can be reconciled with those involving overexpression. We apply this pan-genomic model to predict patterns of transcriptional activity in whole human chromosomes, and, as an example, the effects of the deletion causing the diGeorge syndrome., (© 2021. The Author(s).)

Here, we describe three complementary microscopy-based approaches to quantify morphological changes of chromatin organization in cultured adherent cells: the analysis of the coefficient of variation of DNA, the measurement of DNA-free nuclear areas, and the quantification of chromatin-associated proteins at the nuclear edge. These approaches rely on confocal imaging and stochastic optical reconstruction microscopy and allow a fast and robust quantification of chromatin compaction. These approaches circumvent inter-variability between imaging conditions and apply to every type of adherent cells. For complete details on the use and execution of this protocol, please refer to Neguembor et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: Super-enhancers are clusters of enhancer elements that play critical roles in the maintenance of cell identity. Current investigations on super-enhancers are centered on the established ones in static cell types. How super-enhancers are established during cell differentiation remains obscure., Results: Here, by developing an unbiased approach to systematically analyze the evolving landscape of super-enhancers during cell differentiation in multiple lineages, we discover a general trend where super-enhancers emerge through three distinct temporal patterns: conserved, temporally hierarchical, and de novo. The three types of super-enhancers differ further in association patterns in target gene expression, functional enrichment, and 3D chromatin organization, suggesting they may represent distinct structural and functional subtypes. Furthermore, we dissect the enhancer repertoire within temporally hierarchical super-enhancers, and find enhancers that emerge at early and late stages are enriched with distinct transcription factors, suggesting that the temporal order of establishment of elements within super-enhancers may be directed by underlying DNA sequence. CRISPR-mediated deletion of individual enhancers in differentiated cells shows that both the early- and late-emerged enhancers are indispensable for target gene expression, while in undifferentiated cells early enhancers are involved in the regulation of target genes., Conclusions: In summary, our analysis highlights the heterogeneity of the super-enhancer population and provides new insights to enhancer functions within super-enhancers., (© 2021. The Author(s).)

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia., (© 2021. The Author(s).)

Here, we describe a fractionation protocol optimized to quantify changes in relative abundance of the chromatin-bound proteome (chromatome) by tandem mass tag multiplexing-based tandem mass spectrometry. It has been applied to yeast cells before and after exposure to DNA-damaging drugs to characterize changes in chromatin composition induced by the DNA damage response. We detail steps for stringent chromatin fractionation, sample preparation for mass spectrometry, and its evaluation. For complete details on the use and execution of this protocol, please refer to Challa et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Identifying transcription factors (TFs) whose DNA bindings are altered by genetic variants that regulate susceptibility genes is imperative to understand transcriptional dysregulation in disease etiology. Here, we develop a statistical framework to analyze extensive ChIP-seq and GWAS data and identify 22 breast cancer risk-associated TFs. We find that, by analyzing genetic variations of TF-DNA bindings, the interaction of FOXA1 with co-factors such as ESR1 and E2F1, and the interaction of TFs with chromatin features (i.e., enhancers) play a key role in breast cancer susceptibility. Using genetic variants occupied by the 22 TFs, transcriptome-wide association analyses identify 52 previously unreported breast cancer susceptibility genes, including seven with evidence of essentiality from functional screens in breast relevant cell lines. We show that FOXA1 and co-factors form a core TF-transcriptional network regulating the susceptibility genes. Our findings provide additional insights into genetic variations of TF-DNA bindings (particularly for FOXA1) underlying breast cancer susceptibility., (© 2021. The Author(s).)

MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3 and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The B cells in our body generate protective antibodies by introducing somatic hypermutations (SHM) into the variable region of immunoglobulin genes (IgVs). The mutations are generated by activation induced deaminase (AID) that converts cytosine to uracil in single stranded DNA (ssDNA) generated during transcription. Attempts have been made to correlate SHM with ssDNA using bisulfite to chemically convert cytosines that are accessible in the intact chromatin of mutating B cells. These studies have been complicated by using different definitions of "bisulfite accessible regions" (BARs). Recently, deep-sequencing has provided much larger datasets of such regions but computational methods are needed to enable this analysis. Here we leveraged the deep-sequencing approach with unique molecular identifiers and developed a novel Hidden Markov Model based Bayesian Segmentation algorithm to characterize the ssDNA regions in the IGHV4-34 gene of the human Ramos B cell line. Combining hierarchical clustering and our new Bayesian model, we identified recurrent BARs in certain subregions of both top and bottom strands of this gene. Using this new system, the average size of BARs is about 15 bp. We also identified potential G-quadruplex DNA structures in this gene and found that the BARs co-locate with G-quadruplex structures in the opposite strand. Using various correlation analyses, there is not a direct site-to-site relationship between the bisulfite accessible ssDNA and all sites of SHM but most of the highly AID mutated sites are within 15 bp of a BAR. In summary, we developed a novel platform to study single stranded DNA in chromatin at a base pair resolution that reveals potential relationships among BARs, SHM and G-quadruplexes. This platform could be applied to genome wide studies in the future., Competing Interests: The authors have declared that no competing interests exist.

The mammalian DNA methylome is formed by two antagonizing processes, methylation by DNA methyltransferases (DNMT) and demethylation by ten-eleven translocation (TET) dioxygenases. Although the dynamics of either methylation or demethylation have been intensively studied in the past decade, the direct effects of their interaction on gene expression remain elusive. Here, we quantify the concurrence of DNA methylation and demethylation by the percentage of unmethylated CpGs within a partially methylated read from bisulfite sequencing. After verifying 'methylation concurrence' by its strong association with the co-localization of DNMT and TET enzymes, we observe that methylation concurrence is strongly correlated with gene expression. Notably, elevated methylation concurrence in tumors is associated with the repression of 40~60% of tumor suppressor genes, which cannot be explained by promoter hypermethylation alone. Furthermore, methylation concurrence can be used to stratify large undermethylated regions with negligible differences in average methylation into two subgroups with distinct chromatin accessibility and gene regulation patterns. Together, methylation concurrence represents a unique methylation metric important for transcription regulation and is distinct from conventional metrics, such as average methylation and methylation variation., (© 2021. The Author(s).)