Chromatin Accessibility Dynamics Research Articles

Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans.

Chromatin accessibility, a crucial component of genome regulation, has primarily been studied in homogeneous and simple systems, such as isolated cell populations or early-development models. Whether chromatin accessibility can be assessed in complex, dynamic systems in vivo with high sensitivity remains largely unexplored. In this study, we use ATAC-seq to identify chromatin accessibility changes in a whole animal, the model organism Caenorhabditis elegans, from embryogenesis to adulthood. Chromatin accessibility changes between developmental stages are highly reproducible, recapitulate histone modification changes, and reveal key regulatory aspects of the epigenomic landscape throughout organismal development. We find that over 5000 distal noncoding regions exhibit dynamic changes in chromatin accessibility between developmental stages and could thereby represent putative enhancers. When tested in vivo, several of these putative enhancers indeed drive novel cell-type- and temporal-specific patterns of expression. Finally, by integrating transcription factor binding motifs in a machine learning framework, we identify EOR-1 as a unique transcription factor that may regulate chromatin dynamics during development. Our study provides a unique resource for C. elegans, a system in which the prevalence and importance of enhancers remains poorly characterized, and demonstrates the power of using whole organism chromatin accessibility to identify novel regulatory regions in complex systems.

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration

Organoid techniques provide unique platforms to model brain development and neurological disorders. Whereas several methods for recapitulating corticogenesis have been described, a system modeling human medial ganglionic eminence (MGE) development, a critical ventral brain domain producing cortical interneurons and related lineages, has been lacking until recently. Here, we describe the generation of MGE and cortex-specific organoids from human pluripotent stem cells that recapitulate the development of MGE and cortex domains, respectively. Population and single-cell RNA sequencing (RNA-seq) profiling combined with bulk assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses revealed transcriptional and chromatin accessibility dynamics and lineage relationships during MGE and cortical organoid development. Furthermore, MGE and cortical organoids generated physiologically functional neurons and neuronal networks. Finally, fusing region-specific organoids followed by live imaging enabled analysis of human interneuron migration and integration. Together, our study provides a platform for generating domain-specific brain organoids and modeling human interneuron migration and offers deeper insight into molecular dynamics during human brain development.

Dynamics of chromatin accessibility during TGF-\u03b2-induced EMT of Ras-transformed mammary gland epithelial cells

Epithelial-mesenchymal transition (EMT) is induced by transforming growth factor (TGF)-β and facilitates tumor progression. We here performed global mapping of accessible chromatin in the mouse mammary gland epithelial EpH4 cell line and its Ras-transformed derivative (EpRas) using formaldehyde-assisted isolation of regulatory element (FAIRE)-sequencing. TGF-β and Ras altered chromatin accessibility either cooperatively or independently, and AP1, ETS, and RUNX binding motifs were enriched in the accessible chromatin regions of EpH4 and EpRas cells. Etv4, an ETS family oncogenic transcription factor, was strongly expressed and bound to more than one-third of the accessible chromatin regions in EpRas cells treated with TGF-β. While knockdown of Etv4 and another ETS family member Etv5 showed limited effects on the decrease in the E-cadherin abundance and stress fiber formation by TGF-β, gene ontology analysis showed that genes encoding extracellular proteins were most strongly down-regulated by Etv4 and Etv5 siRNAs. Accordingly, TGF-β-induced expression of Mmp13 and cell invasiveness were suppressed by Etv4 and Etv5 siRNAs, which were accompanied by the reduced chromatin accessibility at an enhancer region of Mmp13 gene. These findings suggest a mechanism of transcriptional regulation during Ras- and TGF-β-induced EMT that involves alterations of accessible chromatin, which are partly regulated by Etv4 and Etv5.

Single-cell epigenomics: techniques and emerging applications.

Epigenomics is the study of the physical modifications, associations and conformations of genomic DNA sequences, with the aim of linking these with epigenetic memory, cellular identity and tissue-specific functions. While current techniques in the field are characterizing the average epigenomic features across large cell ensembles, the increasing interest in the epigenetics within complex and heterogeneous tissues is driving the development of single-cell epigenomics. We review emerging single-cell methods for capturing DNA methylation, chromatin accessibility, histone modifications, chromosome conformation and replication dynamics. Together, these techniques are rapidly becoming a powerful tool in studies of cellular plasticity and diversity, as seen in stem cells and cancer.

Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells.

Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.

Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing.

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ±50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.

Dynamics of chromatin accessibility and epigenetic state in response to UV damage.

Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship.

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

BackgroundDevelopment of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.ResultsWe characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes.ConclusionsOur findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility.

Integrated analyses of chromatin accessibility and gene expression data for elucidating the transcriptional regulatory mechanisms during early hematopoietic development in mouse

The regulation of gene expression is a key factor influencing cell fate decisions during differentiation. The molecular mechanisms underlying gene regulation are multi-layered, with regulatory inputs traversing both within and across different layers, thus providing a very difficult system to decipher. Although the gene expression patterns are cell-type specific, the regulatory inputs follow similar fundamental guidelines. One basic and easily interpretable transcriptional control layer is chromatin accessibility which is a consequence of combinatorial binding of transcription factors (TFs) and various chromatin modifications. Hematopoiesis is a well-characterized system for understanding transcriptional control mechanisms. The differentiation of hemogenic endothelium (HE) to hematopoietic progenitor cells (HP) forms a crucial step in hematopoiesis as it marks the exclusive commitment of bi-potent stem cells capable of following both hematopoietic and endothelial lineages, to hematopoietic lineages. In this work, we have performed a genome-wide analysis of chromatin accessibility dynamics in the differentiation of HE to HP and subsequently correlated them to gene expression dynamics for comprehending the basic regulatory themes. Chromatin accessibility was examined by global DNasel Hypersensitivity Sites (DHS) mapping during the differentiation, in addition to measuring gene expression by RNA-Seq for HE and HP. Accessible regions unique to HE and HP, and common peaks were identified. In general, there was a direct correlation between the DHS fold change and the expression fold change of the nearest genes for the corresponding peaks, with peaks near promoter regions showing a considerably higher degree of correlation than the rest of the peak populations. In further analyses, we applied a range of bioinformatics approaches such as peak overlap analysis, de novo motif discovery, and gene ontology analysis with the ultimate aim to identify the factors influencing lineage commitment. To this end, we have formulated a novel cluster-based approach for studying motif co-occurrence to obtain potential combinatorial TF binding regions, and by correlating those with the expression of the TFs we have systematically explored the relationship between the changes in chromatin accessibility and gene expression dynamics. We are presently developing mathematical models that integrate chromatin accessibility, chromatin modifications and transcription factors to predict gene expression patterns specific for a given cellular differentiation state.

Differentiation Dependent Dynamics of Histone Modifications during Myelopoiesis.

Covalent modifications on histones are epigenetic changes that play critical roles in control of gene expression. Most studies correlating specific histone modifications with transcriptional activity have been performed in yeast, and little is known about their dynamics during hematopoietic differentiation. We examined the dynamics of histone modifications and chromatin accessibility during all-trans retinoic acid (ATRA) induced differentiation of promyelocytes down the neutrophil lineage. As a model system, we used the human promyelocytic NB4 cell line, which undergoes neutrophil differentiation in response to ATRA. Using chromatin immunoprecipitation (ChIP) and quantitative PCR, we measured changes in dimethyl K4 (2MeH3K4), trimethyl K4 (3MeH3K4) and acetyl lysine 9 (Ac9H3K9) of histone H3 in the promoters of 3 genes that undergo transcriptional activation (Defensin-a, C/EBP-b and RAR-b), 1 gene that undergoes transcriptional downregulation (Myeloperoxidase), one gene that is constitutively active (GAPDH) and 1 gene that is silent (Albumin) during ATRA-induced differentiation. We correlated the changes in histone modifications with the gene expression pattern of these genes. AcH3K9 levels correlated with active gene transcription. At time 0, levels of AcH3K9 were enriched 50-fold and 100-fold over input in the MPO and GAPDH promoters, respectively, but only 5-fold on the C/EBP-b, Def-a and RAR-b promoters. Consistent with this finding, levels of AcH3K9 increased to 40-fold over input within 24h of differentiation in the Def-a and C/EBP-b promoters. On the silent albumin promoter, AcH3K9 levels never increased over input after ATRA. For the methylation patterns on H3K4, however, the findings were quite revealing. As expected, on the active MPO promoter, 2MeH3K4 was enriched 60-fold. However, 2MeH3K4was also present at high levels (15-30-fold) on the promoters of unexpressed Def-a, C/EBP-b and RAR-b suggesting that silent genes that are “primed” for activation are enriched for 2MeH3K4, consistent with previous data in yeast. After differentiation with ATRA, 2MeH3K4 went up only 3-fold for Def-a and C/EBP-b. The most surprising changes were found in 3MeH3K4 levels and in chromatin modification at the RAR-b promoter. Consistent with previous data showing that 3MeH3K4 is associated with gene activation, time 0 levels of 3MeH3K4 were enriched 80-fold and 150-fold over input in the MPO and GAPDH promoters, respectively, and were at background levels in the C/EBP-b, Def-a, and RAR-b promoters. After differentiation, however, although there was a significant increase in 3MeH3K4 levels within 12 hours in the Def-a promoter, 3MeH3K4 never was present on the C/EBP-b promoter, despite a huge increase in gene transcription, as well as significant and rapid increases in AcH3K9 and 2MeH3K4. Perhaps the most significant findings, however, were at the promoter of the RAR-b gene. RAR-b is unique amongst the genes studies in that it is directly bound by the PML-RAR-a fusion protein. This promoter was not detectable by PCR in ChIP assays after ATRA addition. To test the hypothesis that histones were lost on the RAR-b promoter during ATRA-induced differentiation, we used MNase digestion of chromatin. Nucleosome loss was confirmed by a decrease in precipitated RAR-b promoter DNA within 24 hours of ATRA addition. In contrast, exon1 of RAR-b was modified in a manner similar to Def-a and C/EBP-b. These findings indicate that despite the seemingly coordinately regulated increase in transcription of multiple genes upon myeloid differentiation, the chromatin modifications on the promoters of these genes are regulated quite differently.