Single-cell epigenomics uncovers heterochromatin instability and transcription factor dysfunction during mouse brain aging.

Nuclear Repositioning Precedes Promoter Accessibility and Is Linked to the Switching Frequency of a Plasmodium falciparum Invasion Gene

Measurements of Brain Age May Help Target Therapy

Backround and aimsComplex host-virus interactions account for adaptive and innate immunity dysfunctions and viral cccDNA mini-chromosome persistence, key features of HBV chronicity and challenges for HBV cure. The extent of HBV direct impact on liver transcriptome remains controversial. Transcriptional activation in eukaryotic cells is tightly linked with disruption of nucleosome organization at accessible genomic sites of remodeled chromatin. We sought to investigate the impact of HBV on chromatin accessibility and transcription.MethodsWe used ATAC-seq (Assay for Transposase Accessible Chromatin followed by high throughput sequencing) to detect early changes in chromatin accessibility coupled with RNA-seq in HBV-infected Primary Human Hepatocytes (PHHs).ResultsAn increasing number of genomic sites change their nucleosome organization over time after HBV infection, with a prevalent, but not exclusive, reduction of chromatin accessibility at specific sites that is partially prevented by inhibiting HBV transcription and replication. ATAC-seq and RNA-seq integration showed that HBV infection impacts on liver fatty acids, bile acids, iron metabolism and liver cancer pathways. The upregulation of iron uptake genes leads to a significant increase of iron content in HBV-infected PHHs whereas iron chelation inhibits cccDNA transcription and viral replication. The chromatin accessibility and transcriptional changes imposed by HBV early after infection persist, as an epigenetic scar, in chronic HBV (CHB) patients and in HBV-related HCCs. These changes are to a large extent independent from viral replication levels and disease activity.ConclusionsAltogether our results show that HBV infection impacts on host cell chromatin landscape and specific transcriptional programs including liver metabolism and liver cancer pathways. Re-wiring of iron metabolism boosts viral replication early after infection. The modulation of genes involved in cancer-related pathways may favor the development or the selection of a pro-neoplastic phenotype and persists in HBV-related HCCs.

Animal studies have shown that virus infection causes changes in host chromatin accessibility, but little is known about changes in chromatin accessibility of plants infected by viruses and its potential impact. Here, rice infected by rice stripe virus (RSV) was used to investigate virus-induced changes in chromatin accessibility. Our analysis identified a total of 6462 open- and 3587 closed-differentially accessible chromatin regions (DACRs) in rice under RSV infection by ATAC-seq. Additionally, by integrating ATAC-seq and RNA-seq, 349 up-regulated genes in open-DACRs and 126 down-regulated genes in closed-DACRs were identified, of which 34 transcription factors (TFs) were further identified by search of upstream motifs. Transcription levels of eight of these TFs were validated by reverse transcription-PCR. Importantly, four of these TFs (OsWRKY77, OsWRKY28, OsZFP12 and OsERF91) interacted with RSV proteins and are therefore predicted to play important roles in RSV infection. This is the first application of ATAC-seq and RNA-seq techniques to analyse changes in rice chromatin accessibility caused by RSV infection. Integrating ATAC-seq and RNA-seq provides a new approach to select candidate TFs in response to virus infection.

BackgroundGene expression changes in response to developmental and environmental cues rely on cis-regulatory sequence elements (cREs). BRG1/BRM-Associated Factors (BAF) chromatin remodeling complexes maintain chromatin accessibility at many cREs, enabling binding by transcription factors (TFs). However, cREs exhibit a broad range of sensitivity to loss of BAF function, and the basis of this variability remains unknown.ResultsTo identify the characteristics of BAF-dependent cREs, we mapped chromatin accessibility changes following acute pharmacologic BAF inhibition in GM12878 lymphoblastoid cells. We integrated these results with over 100 TF and histone modification ChIP-seq datasets and used machine learning to identify features that predict chromatin accessibility changes. We found that Activator Protein 1 (AP-1) factors and lymphoid lineage-defining TFs including RUNX3 and PU.1 predicted BAF-dependence. Strikingly, we found that cREs bearing the chromatin signature of “primed” enhancers – enriched for H3K4me1 but lacking H3K27ac – were significantly more sensitive to BAF inhibition than typical active enhancers. As primed enhancers are known to facilitate transcriptional responses to stimuli, we tested the requirement of BAF activity in these responses. Acute BAF inhibition was sufficient to prevent both chromatin and transcriptional responses to interferon gamma and dexamethasone. cREs which normally gained accessibility in response to stimulation failed to do so with BAF inhibition, and these cREs were linked to genes with suppressed transcriptional induction.ConclusionsCollectively, our results demonstrate a requirement for continuous BAF activity to enable stimulus response and suggest that defective signal responsiveness may be a pathogenic mechanism in disease states caused by loss-of-function mutations in BAF subunits.

BackgroundDecoding the genetic mechanisms of late‐onset Alzheimer’s disease (LOAD) is a major challenge in the post‐GWAS era, since the majority of the LOAD associated SNPs are in noncoding regions. Noncoding disease‐associated loci have been shown to be enriched for regulatory elements in tissues and cells relevant to the disease. Thus, post‐GWAS research requires an in‐depth characterization of cell‐type‐specific DNA regulatory elements and gene expression patterns.MethodsWe conducted ATAC‐seq profiling using NeuN sorted‐nuclei from 40 frozen brain tissues to determine LOAD‐specific changes in chromatin accessibility landscape in a cell‐type specific manner. We complemented these experiments by generating single‐nuclei(sn)RNA‐seq data from a subset group of the same brain samples.ResultsThe study has five major findings for the field of LOAD epigenetics. First, we generated a map of LOAD‐associated cell‐type specific chromatin accessibility sites. Second, we identified 211 LOAD‐specific differential chromatin accessibility sites in neuronal‐nuclei. Third, we provided a catalogue of 842 female‐specific disease‐associated chromatin accessibility sites in non‐neuronal cells. Forth, we showed that the LOAD‐associated cell‐type‐specific changes in chromatin accessibility overlapped with ∼25% of the LOAD‐GWAS regions. Thus, suggesting a noncoding regulatory mechanism, namely chromatin accessibility, by which several LOAD‐GWAS loci may exert their pathogenic effect. Fifth, we functionally validated the ATAC‐seq findings using snRNA‐seq datasets. Data integration identified overlaps between the LOAD‐specific ATAC‐seq signals with the transcriptome profiles of known and new LOAD loci and demonstrated that LOAD‐associated changes in chromatin accessibility can result in gene dysregulation.ConclusionTo our knowledge, this study represents the first and most comprehensive systematic interrogation of the chromatin accessibility landscape in LOAD and the functional effect on gene dysregulation. Moreover, the outcomes unveiled how cell‐type‐specific and sex‐dependent alterations in chromatin structure and transcription programs are associated with LOAD. These findings enhance the interpretation of LOAD‐GWAS discoveries towards translating association to causation, provide potential pathomechanisms, and suggest novel LOAD‐loci. Furthermore, our results convey mechanistic insights into sex differences in LOAD risk and clinicopathology.

Functional and structural age-associated changes in the blood-brain barrier (BBB) may affect the neurovascular unit and contribute to the onset and progression of age-associated neurodegenerative pathologies, including Alzheimer’s disease. The current study interrogated the RNA profile of the BBB in an ageing human autopsy brain cohort and an ageing mouse model using combined laser capture microdissection and expression profiling. Only 12 overlapping genes were altered in the same direction in the BBB of both ageing human and mouse cohorts. These included genes with roles in regulating vascular tone, tight junction protein expression and cell adhesion, all processes prone to dysregulation with advancing age. Integrated mRNA and miRNA network and pathway enrichment analysis of the datasets identified 15 overlapping miRNAs that showed altered expression. In addition to targeting genes related to DNA binding and/or autophagy, many of the miRNAs identified play a role in age-relevant processes, including BBB dysfunction and regulating the neuroinflammatory response. Future studies have the potential to develop targeted therapeutic approaches against these candidates to prevent vascular dysfunction in the ageing brain.

Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.

Background: The neonatal mammalian heart has a transient capacity for regeneration, which is lost shortly after birth. A series of critical developmental transitions including a switch from hyperplastic to hypertrophic growth occur during this postnatal regenerative window, preparing the heart for the increased contractile demands of postnatal life. Postnatal cardiomyocyte maturation and loss of regenerative capacity are associated with expression alterations of thousands of genes embedded within tightly controlled transcriptional networks, which remain poorly understood. Interestingly, although mitogenic stimulation of neonatal cardiomyocytes results in proliferation, the same stimuli induce hypertrophy in adult cardiomyocytes by activating different transcriptional pathways, indicating that cardiomyocyte maturation may result from epigenetic modifications during development. Notably, DNA methylation and chromatin compaction are both important epigenetic modifications associated with a decrease in transcription factor (TF) accessibility to DNA. However, the role of both DNA methylation and chromatin compaction during postnatal cardiac maturation remain largely unknown.Hypothesis: Postnatal changes in DNA methylation and chromatin compaction silence transcriptional networks required for cardiomyocyte proliferation during postnatal heart development.Aims: This PhD Thesis focuses on characterising the role of DNA methylation and chromatin dynamics during postnatal cardiac development and maturation using in vitro and in vivo model systems. Three major Aims are addressed:• Aim 1: To characterize transcription and DNA methylation dynamics during postnatal rodent cardiac development (Chapter 3).• Aim 2: To determine the role of DNA methylation in regulating the expression of cell cycle related genes in mouse and human cardiomyocytes (Chapter 4).• Aim 3: To understand the relationship between chromatin accessibility and transcription during mouse and human cardiomyocyte development (Chapter 5).Results: In Aim 1, global DNA methylation changes were characterized during postnatal development. Marked changes in the expression levels of key DNA methylation enzymes were observed during the first month of postnatal heart development. Postnatal inhibition of DNA methylation using 5-aza-2’-deoxycytidine (5aza-dC; 1mg/kg/day) reduced global DNA methylation levels in the heart in vivo and was associated with a marked increase in cardiomyocyte proliferation (~3-fold) and ~50% reduction in binucleated cardiomyocytes compared to saline-treated controls, suggesting DNA methylation is required for postnatal cardiomyocyte cell cycle arrest. Nextgeneration mRNA sequencing (RNA-seq) and genome-wide sequencing of methylated DNA (methyl-CpG binding domain enrichment and sequencing (MBD-seq)) identified dynamic changes in the cardiac methylome during postnatal development (2545 differentially methylated regions (DMRs) from postnatal day (P) 1 to P14 in the mouse). ~80% of DMRs were hypermethylated and these DMRs were associated with transcriptional shut down of important developmental signalling pathways.In Aim 2, a specific subset of cell cycle genes displaying differentially regulated gene expression and DNA methylation patterns were profiled to understand how DNA methylation is involved in regulating cardiomyocyte proliferation. Gene expression profiling of these cell cycle genes during development and following 5aza-dC treatment in mouse hearts, as well as 3D human cardiac organoids (hCOs), verified that DNA methylation was required for transcriptional regulation of a small group of cell cycle genes. Interestingly, 5aza-dC (10µM) also induced ~2-fold increase in cardiomyocyte proliferation in hCOs. These findings suggest that DNA methylation has a direct role in the regulation of a subset of genes required for cardiomyocyte proliferation in both mice and humans.Chromatin accessibility results from the integrated action of multiple epigenetic marks. In Aim 3, cardiomyocyte nuclei were isolated from both human and mouse developmental heart samples for RNA-seq and for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to profile changes in transcription and chromatin accessibility during cardiomyocyte development in mice (P1 to P56) and humans (foetal (14-19 weeks (wks)), 0-10 years (yrs), 10-30 yrs and 30+ yrs). RNA-seq confirmed that cardiomyocyte cell cycle arrest occurs between P1 and P14 in the mouse. Moreover, ATAC-seq identified open chromatin signatures at various genomic features, including CG islands, transcription start sites (TSSs), enhancers, TF binding sites and protein coding regions, while intergenic regions were associated with compact chromatin. Integration of RNA-seq and ATAC-seq data demonstrated a strong correlation between transcription and chromatin accessibility at the different stages of cardiomyocyte development. TF motif analysis identified E2F transcription factor 4 (E2f4) and Forkhead box M1 (Foxm1) sites as being transcriptionally repressed and undergoing chromatin compaction from P1 to P56. E2f4 and Foxm1 are well known TFs that regulate cell cycle, thus suggesting that cell cycle genes are epigenetically silenced during cardiomyocyte maturation.Conclusion: This PhD Thesis provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation and chromatin compaction. Together, this PhD Thesis provides new insights into the role of DNA methylation and chromatin dynamics during cardiac development. This Thesis provides a new framework for the epigenetic regulation of transcription during postnatal cardiomyocyte maturation and points towards an epigenetic mechanism for cardiomyocyte terminal differentiation during the neonatal period.

YAP, the key protein effector of the Hippo pathway, is a transcriptional co-activator that controls the expression of cell cycle genes, promotes cell growth and proliferation and regulates organ size. YAP modulates gene transcription by binding to distal enhancers, but the mechanisms of gene regulation by YAP-bound enhancers remain poorly understood. Here we show that constitutive active YAP5SA leads to widespread changes in chromatin accessibility in untransformed MCF10A cells. Newly accessible regions include YAP-bound enhancers that mediate activation of cycle genes regulated by the Myb-MuvB (MMB) complex. By CRISPR-interference we identify a role for YAP-bound enhancers in phosphorylation of Pol II at Ser5 at MMB-regulated promoters, extending previously published studies that suggested YAP primarily regulates the pause-release step and transcriptional elongation. YAP5SA also leads to less accessible ‘closed’ chromatin regions, which are not directly YAP-bound but which contain binding motifs for the p53 family of transcription factors. Diminished accessibility at these regions is, at least in part, a consequence of reduced expression and chromatin-binding of the p53 family member ΔNp63 resulting in downregulation of ΔNp63-target genes and promoting YAP-mediated cell migration. In summary, our studies uncover changes in chromatin accessibility and activity that contribute to the oncogenic activities of YAP.

Aging represents the multifactorial decline in physiological function of every living organism. Over the past decades, several hallmarks of aging have been defined, including epigenetic deregulation. Indeed, multiple epigenetic events were found altered across different species during aging. Epigenetic changes directly contributing to aging and aging-related diseases include the accumulation of histone variants, changes in chromatin accessibility, loss of histones and heterochromatin, aberrant histone modifications, and deregulated expression/activity of miRNAs. As a consequence, cellular processes are affected, which results in the development or progression of several human pathologies, including cancer, diabetes, osteoporosis, and neurodegenerative disorders. In this review, we focus on epigenetic mechanisms underlying aging-related processes in various species and describe how these deregulations contribute to human diseases.

Environmental factors affect not only paternal condition but may translate into the following generations where sperm-mediated small RNAs (sRNAs) can contribute to the transmission of paternal effects. sRNAs play a key role in the male germ line in genome maintenance and repair, and particularly in response to environmental stress and the resulting increase in transposable element (TE) activity. Here, we investigated how the social environment (high competition, low competition) of male zebrafish Danio rerio affects sRNAs in sperm and how these are linked to gene expression and TE activity in their offspring. In a first experiment, we collected sperm samples after exposing males to each social environment for 2 weeks to test for differentially expressed sperm micro- (miRNA) and piwi-interacting RNAs (piRNA). In a separate experiment, we performed in vitro fertilisations after one 2-week period using a split-clutch design to control for maternal effects and collected embryos at 24 h to test for differentially expressed genes and TEs. We developed new computational prediction tools to link sperm sRNAs with differentially expressed TEs and genes in the embryos. Our results support the idea that the molecular stress response in the male germ line has significant down-stream effects on the molecular pathways, and we provide a direct link between sRNAs, TEs and gene expression.

Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.

Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for β-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of γ-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.

We have investigated changes in chromosome conformation, nuclear organization, and transcription during differentiation and maturation of control and mutant motor neurons harboring hexanucleotide expansions in the C9orf72 gene that cause amyotrophic lateral sclerosis (ALS). Using an in vitro reprogramming, differentiation and neural maturation protocol, we obtained highly purified populations of post-mitotic motor neurons for both normal and diseased cells. As expected, as fibroblasts are reprogrammed into iPSCs, and as iPSCs differentiate into motor neurons, chromatin accessibility, chromosome conformation, and nuclear organization change along with large-scale alterations in transcriptional profiles. We find that the transcriptome changes extensively during the first three weeks of post-mitotic neuronal maturation, with thousands of genes changing expression, but then is relatively stable for the next three weeks. In contrast, chromosome conformation and nuclear organization continue to change over the entire 6-week maturation period: chromosome territoriality increases, long-range interactions along chromosomes decrease, compartmentalization strength increases, and centromeres and telomeres increasingly cluster. In motor neurons derived from ALS patients such changes in chromosome conformation were much reduced. Chromatin accessibility changes also showed delayed maturation. The transcriptome in these cells matured relatively normally but with notable changes in expression of genes involved in lipid, sterol and mitochondrial function. We conclude that neural maturation is associated with large scale post-mitotic changes in gene expression, chromosome conformation and nuclear organization, and that these processes are defective in motor neurons derived from ALS patients carrying C9orf72 hexanucleotide repeat expansions.