Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair.

Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties.

BACKGROUND: Super-enhancers (SEs), which activate genes involved in cell-type specificity, have mainly been defined as genomic regions with top-ranked enrichment(s) of histone H3 with acetylated K27 (H3K27ac) and/or transcription coactivator(s) including a bromodomain and extra-terminal domain (BET) family protein, BRD4. However, BRD4 preferentially binds to multi-acetylated histone H4, typically with acetylated K5 and K8 (H4K5acK8ac), leading us to hypothesize that SEs should be defined by high H4K5acK8ac enrichment at least as well as by that of H3K27ac. RESULTS: Here, we conducted genome-wide profiling of H4K5acK8ac and H3K27ac, BRD4 binding, and the transcriptome by using a BET inhibitor, JQ1, in three human glial cell lines. When SEs were defined as having the top ranks for H4K5acK8ac or H3K27ac signal, 43% of H4K5acK8ac-ranked SEs were distinct from H3K27ac-ranked SEs in a glioblastoma stem-like cell (GSC) line. CRISPR-Cas9-mediated deletion of the H4K5acK8ac-preferred SEs associated with MYCN and NFIC decreased the stem-like properties in GSCs. CONCLUSIONS: Collectively, our data highlights H4K5acK8ac's utility for identifying genes regulating cell-type specificity.

Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians.

Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.

Comparative analysis of metazoan chromatin organization.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Impact of sequencing depth in ChIP-seq experiments.

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40-50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data.

Identification of chromatin marks at TERRA promoter and encoding region.

TERRA is a long non-coding RNA that is essential for telomere integrity. Although it is transcribed from subtelomeres and telomeres, how it is expressed in heterochromatic region is currently unknown. In this study, we focused our analysis on TERRA-encoding region TelBam3.4 and TelBam3.4-like sequences, and determined their transcription start sites, as well as enrichment of RNA polymerase II and histone modifications. We found that H3K4me3 and H3K9me3 are present at TERRA promoters, whereas H3K27ac and H3K9me3 are present at telomeric repeats. Consistently, we show that presence of active histone modifications H3K4me3 and H3K27ac are correlated to TERRA expression. These results mark an important step towards understanding telomere maintenance and transcription.

Nature and function of insulator protein binding sites in the Drosophila genome.

Chromatin insulator elements and associated proteins have been proposed to partition eukaryotic genomes into sets of independently regulated domains. Here we test this hypothesis by quantitative genome-wide analysis of insulator protein binding to Drosophila chromatin. We find distinct combinatorial binding of insulator proteins to different classes of sites and uncover a novel type of insulator element that binds CP190 but not any other known insulator proteins. Functional characterization of different classes of binding sites indicates that only a small fraction act as robust insulators in standard enhancer-blocking assays. We show that insulators restrict the spreading of the H3K27me3 mark but only at a small number of Polycomb target regions and only to prevent repressive histone methylation within adjacent genes that are already transcriptionally inactive. RNAi knockdown of insulator proteins in cultured cells does not lead to major alterations in genome expression. Taken together, these observations argue against the concept of a genome partitioned by specialized boundary elements and suggest that insulators are reserved for specific regulation of selected genes.

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

Enrichment of HP1a on Drosophila chromosome 4 genes creates an alternate chromatin structure critical for regulation in this heterochromatic domain.

Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP-chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low T(m) downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.

Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

A lipidome landscape of aging in mice.

Understanding the molecular mechanisms of aging is crucial for enhancing healthy longevity. We conducted untargeted lipidomics across 13 biological samples from mice at various life stages (2, 12, 19 and 24 months) to explore the potential link between aging and lipid metabolism, considering sex (male or female) and microbiome (specific pathogen-free or germ-free) dependencies. By analyzing 2,704 molecules from 109 lipid subclasses, we characterized common and tissue-specific lipidome alterations associated with aging. For example, the levels of bis(monoacylglycero)phosphate containing polyunsaturated fatty acids increased in various organs during aging, whereas the levels of other phospholipids containing saturated and monounsaturated fatty acids decreased. In addition, we discovered age-dependent sulfonolipid accumulation, absent in germ-free mice, correlating with Alistipes abundance determined by 16S ribosomal RNA gene amplicon sequencing. In the male kidney, glycolipids such as galactosylceramides, galabiosylceramides (Gal2Cer), trihexosylceramides (Hex3Cer), and mono- and digalactosyldiacylglycerols were detected, with two lipid classes-Gal2Cer and Hex3Cer-being significantly enriched in aged mice. Integrated analysis of the kidney transcriptome revealed uridine diphosphate galactosyltransferase 8A (UGT8a), alkylglycerone phosphate synthase and fatty acyl-coenzyme A reductase 1 as potential enzymes responsible for the male-specific glycolipid biosynthesis in vivo, which would be relevant to sex dependency in kidney diseases. Inhibiting UGT8 reduced the levels of these glycolipids and the expression of inflammatory cytokines in the kidney. Our study provides a valuable resource for clarifying potential links between lipid metabolism and aging.

Keratinocytes of the Upper Epidermis and Isthmus of Hair Follicles Express Hemoglobin mRNA and Protein.

The epidermis, the keratinized stratified squamous epithelium surrounding the body surface, offers a valuable framework to investigate how terrestrial animals overcome environmental stresses. However, the mechanisms underlying epidermal barrier function remain nebulous. In this study, we examined genes highly expressed in the human and mouse upper epidermis, the outer frontier that induces various barrier-related genes. Transcriptome analysis revealed that the messenger RNA level of hemoglobin α (HBA), an oxygen carrier in erythroid cells, was enriched in the upper epidermis compared with that in the whole epidermis. Immunostaining analysis confirmed HBA protein expression in human and mouse keratinocytes (KCs) of the stratum spinosum and stratum granulosum. HBA was also expressed in hair follicle KCs in the isthmus region; its expression levels were more prominent than those in interfollicular KCs. HBA expression was not observed in noncutaneous keratinized stratified squamous epithelia of mice, for example, the vagina, esophagus, and forestomach. HBA expression was upregulated in human epidermal KC cultures after UV irradiation, a major cause of skin-specific oxidative stress. Furthermore, HBA knockdown increased UV-induced production of ROS in primary KCs. Our findings suggest that epidermal HBA expression is induced by oxidative stress and acts as an antioxidant, contributing to skin barrier function.

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells.

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.

Microenvironmental modulation in tandem with human stem cell transplantation enhances functional recovery after chronic complete spinal cord injury.

While rapid advancements in regenerative medicine strategies for spinal cord injury (SCI) have been made, most research in this field has focused on the early stages of incomplete injury. However, the majority of patients experience chronic severe injury; therefore, treatments for these situations are fundamentally important. Here, we hypothesized that environmental modulation via a clinically relevant hepatocyte growth factor (HGF)-releasing scaffold and human iPS cell-derived neural stem/progenitor cells (hNS/PCs) transplantation contributes to functional recovery after chronic complete transection SCI. Effective release of HGF from a collagen scaffold induced progressive axonal elongation and increased grafted cell viability by activating microglia/macrophages and meningeal cells, inhibiting inflammation, reducing scar formation, and enhancing vascularization. Furthermore, hNS/PCs transplantation enhanced endogenous neuronal regrowth, the extension of graft axons, and the formation of circuits around the lesion and lumbar enlargement between host and graft neurons, resulting in the restoration of locomotor and urinary function. This study presents an effective therapeutic strategy for severe chronic SCI and provides evidence for the feasibility of regenerative medicine strategies using clinically relevant materials.

Activation of ILC2s through constitutive IFNγ signaling reduction leads to spontaneous pulmonary fibrosis.

Pulmonary fibrosis (PF), a condition characterized by inflammation and collagen deposition in the alveolar interstitium, causes dyspnea and fatal outcomes. Although the bleomycin-induced PF mouse model has improved our understanding of exogenous factor-induced fibrosis, the mechanism governing endogenous factor-induced fibrosis remains unknown. Here, we find that Ifngr1-/-Rag2-/- mice, which lack the critical suppression factor for group 2 innate lymphoid cells (ILC2), develop PF spontaneously. The onset phase of fibrosis includes ILC2 subpopulations with a high Il1rl1 (IL-33 receptor) expression, and fibrosis does not develop in ILC-deficient or IL-33-deficient mice. Although ILC2s are normally localized near bronchioles and blood vessels, ILC2s are increased in fibrotic areas along with IL-33 positive fibroblasts during fibrosis. Co-culture analysis shows that activated-ILC2s directly induce collagen production from fibroblasts. Furthermore, increased IL1RL1 and decreased IFNGR1 expressions are confirmed in ILC2s from individuals with idiopathic PF, highlighting the applicability of Ifngr1-/-Rag2-/- mice as a mouse model for fibrosis research.

A flexible cross-platform single-cell data processing pipeline.

Single-cell RNA-sequencing analysis to quantify the RNA molecules in individual cells has become popular, as it can obtain a large amount of information from each experiment. We introduce UniverSC ( https://github.com/minoda-lab/universc ), a universal single-cell RNA-seq data processing tool that supports any unique molecular identifier-based platform. Our command-line tool, docker image, and containerised graphical application enables consistent and comprehensive integration, comparison, and evaluation across data generated from a wide range of platforms. We also provide a cross-platform application to run UniverSC via a graphical user interface, available for macOS, Windows, and Linux Ubuntu, negating one of the bottlenecks with single-cell RNA-seq analysis that is data processing for researchers who are not bioinformatically proficient.

Maternal Renal Dysfunction in Late Pregnancy in Twin and Singleton Pregnancies: Retrospective Study.

This study aimed to evaluate the differences in the impact on maternal renal function between singleton and twin pregnancies in the second half of pregnancy. It retrospectively enrolled 1711 pregnant women consisting of 1547 singleton pregnancies and 164 twin pregnancies from Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital from January 2019 to June 2021. Patients underwent renal function tests (serum blood urea nitrogen, creatinine, and estimated glomerular filtration rate (eGFR)) at least one month before delivery. The main outcome measure was maternal renal dysfunction, defined as a serum creatinine level above 0.8 mg/dL. The serum creatinine level was significantly higher and the eGFR was significantly lower in twin than in singleton pregnancies (p < 0.001). In addition, the rate of renal dysfunction was significantly higher in twin than in singleton pregnancies (7.9% vs. 2.6%; p < 0.01). Multivariate analysis revealed that twin pregnancy (odds ratio (OR) 3.38), nulliparity (OR 2.31), and preeclampsia (OR 3.64) were significant risk factors for maternal renal dysfunction. Maternal renal dysfunction was observed in 13 twin pregnancies, all of which recovered to within normal limits during the early months of the postpartum period. Twin pregnancy is a significant risk factor for maternal renal dysfunction; renal function should be carefully monitored in twin pregnancies.

Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator.

Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by the transcriptional regulator, AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase-accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules, CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire+mTEC precursors during the Aire+mTEC differentiation process of the postnatal thymus.

A simple ATAC-seq protocol for population epigenetics.

We describe here a protocol for the generation of sequence-ready libraries for population epigenomics studies. The protocol is a streamlined version of the Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) that provides a positive display of accessible, presumably euchromatic regions. The protocol is straightforward and can be used with small individuals such as daphnia and schistosome worms, and probably many other biological samples of comparable size, and it requires little molecular biology handling expertise.