Local Chromatin Organization Research Articles

Protamine-Mediated Tangles Produce Extreme Deoxyribonucleic Acid Compaction.

In sperm cells, protamine replaces histones to compact DNA 10-20 times more than in somatic cells. To characterize the extreme compaction, we employed confocal microscopy and optical tweezers to determine the conformations and stability of protamine-bound λ-DNA. Confocal images show increasing compaction of λ-DNA at increasing protamine concentration. In the presence of protamine, single λ-DNA molecules form tangles that withstand forces strong enough (∼55 pN) for strand separation and shorten the contour length by up to 40% even at high forces, as well as bends and loops that rupture at 10-40 pN forces. Strand separation nucleates tangles, implicating protamine interactions with DNA bases. Molecular dynamics simulations show that Arg sidechains of protamine each form hydrogen bonds with multiple bases, frequently in the form of a wedge between the two strands of DNA. Protamine may participate in both local and higher-order chromatin organization, leading to extreme compaction and global transcription silencing.

RNA polymerase II pausing regulates chromatin organization in erythrocytes.

Chicken erythrocytes are nucleated cells often considered to be transcriptionally inactive, although the epigenetic changes and chromatin remodeling that would mediate transcriptional repression and the extent of gene silencing during avian terminal erythroid differentiation are not fully understood. Here, we characterize the changes in gene expression, chromatin accessibility, genome organization and chromatin nuclear disposition during the terminal stages of erythropoiesis in chicken and uncover complex chromatin reorganization at different genomic scales. We observe a robust decrease in transcription in erythrocytes, but a set of genes maintains their expression, including genes involved in RNA polymerase II (Pol II) promoter-proximal pausing. Erythrocytes exhibit a reoriented nuclear architecture, with accessible chromatin positioned towards the nuclear periphery together with the paused RNA Pol II. In erythrocytes, chromatin domains are partially lost genome-wide, except at minidomains retained around paused promoters. Our results suggest that promoter-proximal pausing of RNA Pol II contributes to the transcriptional regulation of the erythroid genome and highlight the role of RNA polymerase in the maintenance of local chromatin organization.

Maintenance and dynamic reprogramming of chromatin organization during development.

Controlled transcription of genes is critical for cell differentiation and development. Gene expression regulation therefore involves a multilayered control from nucleosome composition in histone variants and their post-translational modifications to higher-order folding of chromatin fibers and chromatin interactions in nuclear space. Recent technological advances have allowed gaining insight into these mechanisms, the interplay between local and higher-order chromatin organization, and the dynamic changes that occur during stress response and developmental transitions. In this review, we will discuss chromatin organization from the nucleosome to its three-dimensional structure in the nucleus, and consider how these different layers of organization are maintained during the cell cycle or rapidly reprogrammed during development.

Multiscale 3D genome reorganization during skeletal muscle stem cell lineage progression and aging.

Little is known about three-dimensional (3D) genome organization in skeletal muscle stem cells [also called satellite cells (SCs)]. Here, we comprehensively map the 3D genome topology reorganization during mouse SC lineage progression. Specifically, rewiring at the compartment level is most pronounced when SCs become activated. Marked loss in topologically associating domain (TAD) border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can form TAD clusters and super-enhancer-containing TAD clusters orchestrate stage-specific gene expression. Furthermore, we uncover that transcription factor PAX7 is pivotal in enhancer-promoter (E-P) loop formation. We also identify cis-regulatory elements that are crucial for local chromatin organization at Pax7 locus and Pax7 expression. Lastly, we unveil that geriatric SC displays a prominent gain in long-range contacts and loss of TAD border insulation. Together, our results uncover that 3D chromatin extensively reorganizes at multiple architectural levels and underpins the transcriptome remodeling during SC lineage development and SC aging.

Polymorphic inverted repeats near coding genes impact chromatin topology and phenotypic traits in Arabidopsis thaliana

Transposons are mobile elements that are commonly silenced to protect eukaryotic genome integrity. In plants, transposable element (TE)-derived inverted repeats (IRs) are commonly found near genes, where they affect host gene expression. However, the molecular mechanisms of such regulation are unclear in most cases. Expression of these IRs is associated with production of 24-nt small RNAs, methylation of the IRs, and drastic changes in local 3D chromatin organization. Notably, many of these IRs differ between Arabidopsis thaliana accessions, causing variation in short-range chromatin interactions and gene expression. CRISPR-Cas9-mediated disruption of two IRs leads to a switch in genome topology and gene expression with phenotypic consequences. Our data show that insertion of an IR near a gene provides an anchor point for chromatin interactions that profoundly impact the activity of neighboring loci. This turns IRs into powerful evolutionary agents that can contribute to rapid adaptation.

Photo-Cross-Linking To Delineate Epigenetic Interactome.

Covalent modifications of DNA and histones are key cellular epigenetic marks to regulate gene functions. Most of these epigenetic marks are added or removed by corresponding enzymes known as writers and erasers, whose catalytic activities normally rely on the presence of cellular metabolites as cofactors. Epigenetic marks can either directly alter the chromatin structure and dynamics through changing the intra-/internucleosomal histone-histone and histone-DNA interactions or recruit readers that further bring in other proteins with chromatin-modifying/remodeling activities to reshape the local and regional chromatin organization. In these two ways, epigenetic modifications modulate diverse DNA-templated processes, such as gene transcription, DNA replication, and DNA damage repair. Therefore, elucidation of the regulatory mechanisms and biological significance of epigenetic marks requires the identification and characterization of the protein-protein, protein-nucleic acid, and protein-small molecule interactions that control the underlying epigenetic processes. Here, we review the recent advances in using photo-cross-linking strategies to interrogate the epigenetic interactome, focusing on the protein-protein interactions mediated by epigenetic marks in histone tails. We also discuss future directions of developing photo-cross-linking-based tools and methods toward the investigation of the binding events in nucleosomal/chromatinic contexts, and toward the in situ capture of the epigenetic interactome in live cells or even organisms.

DNA Damage Drives DNA Methylation and 3D Chromatin Organization Alterations in Glioblastoma

<h3>Purpose/Objective(s)</h3> The objective of this study is to determine how the epigenome is locally altered after DNA damage and investigate the impact of these changes on 3D chromatin organization, gene regulation and treatment resistance. In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome is largely unknown. Since epigenetic alterations such as DNA-methylation can impact the binding of factors involved in chromatin folding, and transcription factors that bind regulatory elements, the downstream impact on gene regulation could lead to the emergence of treatment resistance. Using newly developed tools to enable these studies, we hypothesize that genome-wide DNA damage leads to local alterations in DNA-methylation, genome organization, and results in persistent gene-expression alterations near sites of repaired damage. <h3>Materials/Methods</h3> We use patient-derived human glioblastoma stem-like cells (GSCs) as a model. DNA breaks are induced using (i) irradiation or (ii) a novel "multi-cut" CRISPR-Cas9 DNA break system. Genomic profiling methods include HiC (genome-wide chromosome conformation capture), DNA-methylation profiling (bisulfite sequencing), targeting DNA-seq and RNA-seq. <h3>Results</h3> With radiation, we find significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. It is difficult to study local alterations around sites of radiation induced damage because breaks are introduced at different sites in each cell, resulting in stochastic DNA methylation alterations. To circumvent this issue, we developed a multi-cut CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. Induction of pre-mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation. To assess repair efficiency (i.e., deletions, translocations), we employed a custom panel of DNA probes that flank the 142 or 483 sites to allow for high coverage sequencing at each site following cut induction. To understand how DNA damage may lead to local epigenetic alterations and 3D chromatin organization changes, we performed HiC (genome-wide chromosome conformation capture), before and after cut induction, and subjected cultures to further downstream analysis. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites, enrichment of DNA methylation alterations and altered gene-expression. <h3>Conclusion</h3> The multi-cut CRISPR-Cas9 DNA break system, which we develop and characterize here, can be rapidly employed in cell lines or animal models to study pre-mapped genome-wide DNA damage, and has widespread applicability in the radiobiology and DNA damage and repair fields. The findings here provide a mechanistic view of the interplay between genome-wide DNA damage, DNA methylation and genome re-organization, and have wide-ranging implications for the effect of DNA damage on the epigenome.

Abstract 3622: DNA damage drives DNA methylation and 3D chromatin organization alterations in glioblastoma

Abstract In glioblastoma, treatment with radiation and chemotherapy leads to DNA-damage and most DNA breaks are faithfully repaired, but the impact on the epigenome has not been thoroughly investigated. Since epigenetic alterations in DNA-methylation can impact the binding of factors involved in chromatin folding, such as CTCF as well as transcription factors that bind regulatory elements, the downstream impact on gene regulation could lead to the emergence of treatment resistance. Here, using radiation, we show there are significant and wide-spread alterations in DNA-methylation after treating multiple glioblastoma cultures. However, it is difficult to study these alterations because breaks are introduced at different sites in each cell, resulting in irreproducible DNA methylation alterations between replicates and cell lines. To circumvent this issue, we have developed a “multi-cut” CRISPR-Cas9 DNA break system that targets 142 or 483 pre-defined loci. We find that induction of mapped genome-wide cuts reproduces a similar level of toxicity as standard doses of radiation and we characterize the kinetics of overall cellular resolution of DNA damage using immunofluorescence markers and COMET assays. In order to assess repair efficiency (i.e., deletions, translocations, complete repair), we have designed a custom panel of DNA probes that flank the 142 or 483 sites to allow for high coverage sequencing of each cut site. To understand how DNA damage may lead to local epigenetic alterations and 3D chromatin organization changes, we performed HiC (genome-wide chromosome conformation capture), before and after cut induction, and subject cultures to further downstream analysis. Our findings show significant mega-base scale alterations in chromatin contacts centered around cut sites. To understand how these alterations may contribute to treatment resistance after DNA damage, we show these alternating chromatin contacts result in new promoter and enhancer interactions, which may drive gene-expression alterations after DNA damage at nearby loci. These results provide a mechanistic view of the interplay between DNA damage, DNA methylation and genome re-organization in glioblastoma. Importantly, these studies shed light on how these alterations evolve and their effect on local gene expression and ensuing treatment resistance. Citation Format: Aram S. Modrek, Catherine Do, Zeyan Zhang, Yingwen Deng, Jerome Karp, Ravesanker Ezhilarasan, Giulia Cova, Matija Snuderl, Aristotelis Tsirigos, Jane Skok, Erik P. Sulman. DNA damage drives DNA methylation and 3D chromatin organization alterations in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3622.

A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila.

ABSTRACTSingle-molecule localization microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues because of high background from out-of-focus emitters and optical aberrations. Here, we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-point accumulation for imaging in nanoscale topography (PAINT) routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to what is seen in cultured cells. Lamin Dm0 shows a complementary localization to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C causes the nuclear pores to distribute more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Given that nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organization and contribute to the disease phenotypes caused by human lamin A/C laminopathies.

Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer.

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5' capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA-DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

Retrotransposons as Drivers of Mammalian Brain Evolution

Retrotransposons, a large and diverse class of transposable elements that are still active in humans, represent a remarkable force of genomic innovation underlying mammalian evolution. Among the features distinguishing mammals from all other vertebrates, the presence of a neocortex with a peculiar neuronal organization, composition and connectivity is perhaps the one that, by affecting the cognitive abilities of mammals, contributed mostly to their evolutionary success. Among mammals, hominids and especially humans display an extraordinarily expanded cortical volume, an enrichment of the repertoire of neural cell types and more elaborate patterns of neuronal connectivity. Retrotransposon-derived sequences have recently been implicated in multiple layers of gene regulation in the brain, from transcriptional and post-transcriptional control to both local and large-scale three-dimensional chromatin organization. Accordingly, an increasing variety of neurodevelopmental and neurodegenerative conditions are being recognized to be associated with retrotransposon dysregulation. We review here a large body of recent studies lending support to the idea that retrotransposon-dependent evolutionary novelties were crucial for the emergence of mammalian, primate and human peculiarities of brain morphology and function.

High-resolution three-dimensional chromatin profiling of the Chinese hamster ovary cell genome.

Chinese hamster ovary (CHO) cell lines are the pillars of a multibillion‐dollar biopharmaceutical industry producing recombinant therapeutic proteins. The effects of local chromatin organization and epigenetic repression within these cell lines result in unpredictable and unstable transgene expression following random integration. Limited knowledge of the CHO genome and its higher order chromatin organization has thus far impeded functional genomics approaches required to tackle these issues. Here, we present an integrative three‐dimensional (3D) map of genome organization within the CHOK1SV® 10E9 cell line in conjunction with an improved, less fragmented CHOK1SV 10E9 genome assembly. Using our high‐resolution chromatin conformation datasets, we have assigned ≈90% of sequence to a chromosome‐scale genome assembly. Our genome‐wide 3D map identifies higher order chromatin structures such as topologically associated domains, incorporates our chromatin accessibility data to enhance the identification of active cis‐regulatory elements, and importantly links these cis‐regulatory elements to target promoters in a 3D promoter interactome. We demonstrate the power of our improved functional annotation by evaluating the 3D landscape of a transgene integration site and two phenotypically different cell lines. Our work opens up further novel genome engineering targets, has the potential to inform vital improvements for industrial biotherapeutic production, and represents a significant advancement for CHO cell line development.

Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale.

Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries, and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements, and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states, and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.

A unified view of the sequence and functional organization of the human RNA polymerase II promoter.

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5′ and 3′ ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

Negative regulation of Ifng expression by MLL4 through CNS-28

Abstract Interferon-g (IFN-g) is a key cytokine produced by type 1 T helper (Th1) cells in response to viral or intracellular bacterial infections in mammals. While a number of positive regulatory elements have been well studied, no negative regulatory elements at the Ifng locus for its expression have been identified. Here we report a negative regulatory mechanism of Ifng expression by MLL4, a histone methyltransferase responsible for H3K4me1. Deletion of Mll4 resulted in loss of H3K4me1 signals at the previously unrecognized CNS-28 site and led to enhanced IFN-g expression during Th1 differentiation. GATA3 bound to the CNS-28 site and deletion of Gata3 resulted in a similar upregulation of IFN-g with Mll4 deletion. Furthermore, deletion of Mll4 compromised GATA3 binding to CNS-28 and led to a substantial reorganization of the 3D chromatin structure at the Ifng genomic locus in both naïve CD4+ T cells and Th1 cells. Thus, we conclude that MLL4 negatively regulates Ifng expression by facilitating GATA3 binding to the CNS-28 site and maintaining a special local chromatin organization.

Characterizing the interplay between gene nucleotide composition bias and splicing

BackgroundNucleotide composition bias plays an important role in the 1D and 3D organization of the human genome. Here, we investigate the potential interplay between nucleotide composition bias and the regulation of exon recognition during splicing.ResultsBy analyzing dozens of RNA-seq datasets, we identify two groups of splicing factors that activate either about 3200 GC-rich exons or about 4000 AT-rich exons. We show that splicing factor–dependent GC-rich exons have predicted RNA secondary structures at 5′ ss and are dependent on U1 snRNP–associated proteins. In contrast, splicing factor–dependent AT-rich exons have a large number of decoy branch points, SF1- or U2AF2-binding sites and are dependent on U2 snRNP–associated proteins. Nucleotide composition bias also influences local chromatin organization, with consequences for exon recognition during splicing. Interestingly, the GC content of exons correlates with that of their hosting genes, isochores, and topologically associated domains.ConclusionsWe propose that regional nucleotide composition bias over several dozens of kilobase pairs leaves a local footprint at the exon level and induces constraints during splicing that can be alleviated by local chromatin organization at the DNA level and recruitment of specific splicing factors at the RNA level. Therefore, nucleotide composition bias establishes a direct link between genome organization and local regulatory processes, like alternative splicing.

Geometrical Heterogeneity Dominates Thermal Fluctuations in Facilitating Chromatin Contacts.

Within a living cell, the myriad of proteins that bind DNA introduce heterogeneously spaced kinks into an otherwise semiflexible DNA double helix. To investigate the effects of heterogeneous nucleosome binding on chromatin organization, we extend the wormlike chain model to include statistically spaced, rigid kinks. On timescales where nucleosome positions are fixed, we find that the probability of chromatin loop formation can vary by up to six orders of magnitude between two sets of nucleosome positions drawn from the same distribution. On longer timescales, we show that continuous rerandomization due to nucleosome turnover results in chromatin tracing out an effective WLC with a dramatically smaller Kuhn length than bare DNA. Together, these observations demonstrate that nucleosome spacing acts as the primary source of the structural heterogeneity that dominates local and global chromatin organization.

Integrative characterization of G-Quadruplexes in the three-dimensional chromatin structure

ABSTRACTDNA molecules are highly compacted in the eukaryotic nucleus where distal regulatory elements reach their targets through three-dimensional chromosomal interactions. G-quadruplexes, stable four-stranded non-canonical DNA structures, can change local chromatin organization through the exclusion of nucleosomes. However, the relationship between G-quadruplexes and higher-order genome organization remains unknown. Here, we found that G-quadruplexes are significantly enriched at boundaries of topological associated domains (TADs). Architectural protein occupancy, which plays critical roles in the formation of TADs, was highly correlated with the content of G-quadruplexes at TAD boundaries. Moreover, adjacent boundaries containing G-quadruplexes frequently interacted with each other because of the high enrichment of architectural protein binding sites. Similar to CCCTC-binding factor (CTCF) binding sites, G-quadruplexes also showed strong insulation ability in the separation of adjacent regions. Additionally, the insulation ability of CTCF binding sites and TAD boundaries was significantly reinforced by G-quadruplexes. Furthermore, G-quadruplex motifs on different strands were associated with the orientation of CTCF binding sites. These findings suggest a potential role for G-quadruplexes in loop extrusion. The enrichment of transcription factor binding sites (TFBSs) around regulatory elements containing G-quadruplexes led to frequent interactions between regulatory elements containing G-quadruplexes. Intriguingly, more than 99% of G-quadruplexes overlapped with TFBSs. The binding sites of CTCF and cohesin proteins were preferentially located surrounding G-quadruplexes. Accordingly, we proposed a new mechanism of long-distance gene regulation in which G-quadruplexes are involved in distal interactions between enhancers and promoters.

Chromatin three-dimensional interactions mediate genetic effects on gene expression.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

ATAC-seq reveals regional differences in enhancer accessibility during the establishment of spatial coordinates in the Drosophila blastoderm.

Establishment of spatial coordinates during Drosophila embryogenesis relies on differential regulatory activity of axis patterning enhancers. Concentration gradients of activator and repressor transcription factors (TFs) provide positional information to each enhancer, which in turn promotes transcription of a target gene in a specific spatial pattern. However, the interplay between an enhancer regulatory activity and its accessibility as determined by local chromatin organization is not well understood. We profiled chromatin accessibility with ATAC-seq in narrow, genetically tagged domains along the antero-posterior axis in the Drosophila blastoderm. We demonstrate that one-quarter of the accessible genome displays significant regional variation in its ATAC-seq signal immediately after zygotic genome activation. Axis patterning enhancers are enriched among the most variable intervals, and their accessibility changes correlate with their regulatory activity. In an embryonic domain where an enhancer receives a net activating TF input and promotes transcription, it displays elevated accessibility in comparison to a domain where it receives a net repressive input. We propose that differential accessibility is a signature of patterning cis-regulatory elements in the Drosophila blastoderm and discuss potential mechanisms by which accessibility of enhancers may be modulated by activator and repressor TFs.