SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology.

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

Clock-dependent chromatin topology modulates circadian transcription and behavior.

The circadian clock in animals orchestrates widespread oscillatory gene expression programs, which underlie 24-h rhythms in behavior and physiology. Several studies have shown the possible roles of transcription factors and chromatin marks in controlling cyclic gene expression. However, how daily active enhancers modulate rhythmic gene transcription in mammalian tissues is not known. Using circular chromosome conformation capture (4C) combined with sequencing (4C-seq), we discovered oscillatory promoter-enhancer interactions along the 24-h cycle in the mouse liver and kidney. Rhythms in chromatin interactions were abolished in arrhythmic Bmal1 knockout mice. Deleting a contacted intronic enhancer element in the Cryptochrome 1 (Cry1) gene was sufficient to compromise the rhythmic chromatin contacts in tissues. Moreover, the deletion reduced the daily dynamics of Cry1 transcriptional burst frequency and, remarkably, shortened the circadian period of locomotor activity rhythms. Our results establish oscillating and clock-controlled promoter-enhancer looping as a regulatory layer underlying circadian transcription and behavior.

A day in the life of chromatin: how enhancer-promoter loops shape daily behavior.

Each spring, we get out of bed 1 h ahead of our biological wake-up time due to the misalignment of internal clocks with the light-dark cycle. Genetic discoveries revealed that clock genes encode transcription factors that are expressed throughout many tissues, yet a gap has remained in understanding the temporal dynamics of transcription. Two groups now apply circular chromosome conformation capture and high-throughput sequencing to dissect how "time of day"-dependent changes in chromatin drive core clock oscillations. A surprise is the finding that disruption of enhancer-promoter contacts within chromatin leads to an advance in the "wake-up" time of mice. Furthermore, the assembly of transcriptionally active domains of chromatin requires the ordered recruitment of core clock transcription factors each day. These studies show that waking up involves highly dynamic changes in the three-dimensional positioning of genes within the cell.

Developmental and evolutionary comparative analysis of a regulatory landscape in mouse and chicken.

Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these non-coding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences.

Reshaping Chromatin Architecture around DNA Breaks.

DNA double-strand breaks (DSBs) elicit major chromatin changes. Using super-resolution microscopy in human cells, Ochs et al. unveil that the DSB response protein 53BP1 and its effector RIF1 organize DSB-flanking chromatin into circular micro-domains. These structures control the spatial distribution of DSB repair factors safeguarding genome integrity.

Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner.

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.

Deregulation of enhancer structure, function, and dynamics in acute lymphoblastic leukemia.

Enhancers control dynamic changes in gene expression and orchestrate the tightly controlled transcriptional circuitries that direct and coordinate cell growth, proliferation, survival, lineage commitment, and differentiation during lymphoid development. Enhancer hijacking and neoenhancer formation at oncogene loci, as well as aberrant activation of oncogene-associated enhancers, can induce constitutive activation of self-perpetuating oncogenic transcriptional circuitries, and contribute to the malignant transformation of immature lymphoid progenitors in acute lymphoblastic leukemia (ALL). In this review, we present recent discoveries of the role of enhancer dynamics in mouse and human lymphoid development, and discuss how genetic and epigenetic alterations of enhancer function can promote leukemogenesis, and potential strategies for targeting the enhancer machinery in the treatment of ALL.

Chromatin topology, condensates and gene regulation: shifting paradigms or just a phase?

In the past decade, two major advances in our understanding of nuclear organization have taken the field of gene regulation by storm. First, technologies that can analyze the three-dimensional conformation of chromatin have revealed how the genome is organized and have provided novel insights into how regulatory regions in the genome interact. Second, the recognition that many proteins can form membraneless compartments through liquid-liquid phase separation (LLPS) has challenged long-standing notions of how proteins within the nucleus are organized and has offered a tantalizing general mechanism by which many aspects of nuclear function may be regulated. However, the functional roles of chromatin topology and LLPS in regulating gene expression remain poorly understood. These topics were discussed with great fervor during an open discussion held at a recent workshop titled 'Chromatin-based regulation of development' organized by The Company of Biologists. Here, we summarize the major points covered during this debate and discuss how they tie into current thinking in the field of gene regulation.

To be or not be (in the LAD): emerging roles of lamin proteins in transcriptional regulation.

Lamins are components of the nuclear lamina, a protein meshwork that underlies the nuclear membrane. Lamins interact with chromatin in transcriptionally silent regions defined as lamina-associated-domains (LADs). However, recent studies have shown that lamins regulate active transcription inside LADs. In addition, ChIP-seq analysis has shown that lamins interact with lamin-dependent promoters and enhancers located in the interior of the nucleus. Moreover, functional studies suggest that lamins regulate transcription at associated-promoters and long-range chromatin interactions of key developmental gene programs. This review will discuss emerging, non-canonical functions of lamins in controlling non-silent genes located both inside and outside of LADs, focusing on transcriptional regulation and chromatin organization in Drosophila and mammals as metazoan model organisms.

CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing.

Clustered regularly interspaced short palindromic repeat (CRISPR) and Cas9-associated protein systems provide a powerful genetic manipulation tool that can drive plant research forward. Nuclease-dead Cas9 (dCas9) is an enzymatically inactive mutant of Cas9 in which its endonuclease activity is non-functional. The applications of CRISPR/dCas9 have expanded and diversified in recent years. Originally, dCas9 was used as a CRISPR/Cas9 re-engineering tool that enables targeted expression of any gene or multiple genes through recruitment of transcriptional effector domains without introducing irreversible DNA-damaging mutations. Subsequent applications have made use of its ability to recruit modifying enzymes and reporter proteins to DNA target sites. In this paper, the most recent progress in the applications of CRISPR/dCas9 in plants, which include gene activation and repression, epigenome editing, modulation of chromatin topology, live-cell chromatin imaging and DNA-free genetic modification, will be reviewed. The associated strategies for exploiting the CRISPR/dCas9 system for crop improvement with a dimer of the future of the CRISPR/dCas9 system in the functional genomics of crops and the development of traits will be briefly discussed.

Spatial Distribution of Heterochromatin Bodies in the Nuclei of Triatoma infestans (Klug).

Constitutive heterochromatin typically exhibits low gene density and is commonly found adjacent or close to the nuclear periphery, in contrast to transcriptionally active genes concentrated in the innermost nuclear region. In Triatoma infestans cells, conspicuous constitutive heterochromatin forms deeply stained structures named chromocenters. However, to the best of our knowledge, no information exists regarding whether these chromocenters acquire a precise topology in the cell nuclei or whether their 18S rDNA, which is important for ribosome function, faces the nuclear center preferentially. In this work, the spatial distribution of fluorescent Feulgen-stained chromocenters and the distribution of their 18S rDNA was analyzed in Malpighian tubule cells of T. infestans using confocal microscopy. The chromocenters were shown to be spatially positioned relatively close to the nuclear periphery, though not adjacent to it. The variable distance between the chromocenters and the nuclear periphery suggests mobility of these bodies within the cell nuclei. The distribution of 18S rDNA at the edge of the chromocenters was not found to face the nuclear interior exclusively. Because the genome regions containing 18S rDNA in the chromocenters also face the nuclear periphery, the proximity of the chromocenters to this nuclear region is not assumed to be associated with overall gene silencing.

Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast.

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.

A role for chromatin topology in imprinted domain regulation.

Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.

Deciphering the Epigenetic Code in Embryonic and Dental Pulp Stem Cells.

A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors.

Neutrophil nucleus: shaping the past and the future.

Neutrophils are innate immune cells that are key to protecting the host against infection and maintaining body homeostasis. However, if dysregulated, they can contribute to disease, such as in cancer or chronic autoinflammatory disorders. Recent studies have highlighted the heterogeneity in the neutrophil compartment and identified the presence of immature neutrophils and their precursors in these pathologies. Therefore, understanding neutrophil maturity and the mechanisms through which they contribute to disease is critical. Neutrophils were first characterized morphologically by Ehrlich in 1879 using microscopy, and since then, different technologies have been used to assess neutrophil maturity. The advances in the imaging field, including state-of-the-art microscopy and machine learning algorithms for image analysis, reinforce the use of neutrophil nuclear morphology as a fundamental marker of maturity, applicable for objective classification in clinical diagnostics. New emerging approaches, such as the capture of changes in chromatin topology, will provide mechanistic links between the nuclear shape, chromatin organization, and transcriptional regulation during neutrophil maturation.

Neural pericytes: A genomic archival state programmed by CHromatin topology.

Accumulating evidence suggests that mural pericytes, apart from stabilizing the associated microvessels, play additional roles in regeneration of local cellular elements. Herein, the mechanistic basis for such diverse and at times contradictory roles adopted by pericytes in the brain is reviewed. Core concepts of an emerging model are discussed wherein mural pericytes reside in a metastable "archival state" that conceals a neural progenitor identity. Upon angiogenic remodeling, a selected subpopulation of pericytes reclaim the progenitor state during transdifferentiation and contribute to neural regeneration. The genomic basis for neural transdifferentiation of pericytes is reviewed with reference to the extant literature.

Bacon: a comprehensive computational benchmarking framework for evaluating targeted chromatin conformation capture-specific methodologies.

Chromatin conformation capture (3C)-based technologies have enabled the accurate detection of topological genomic interactions, and the adoption of ChIP techniques to 3C-based protocols makes it possible to identify long-range interactions. To analyze these large and complex datasets, computational methods are undergoing rapid and expansive evolution. Thus, a thorough evaluation of these analytical pipelines is necessary to identify which commonly used algorithms and processing pipelines need to be improved. Here we present a comprehensive benchmark framework, Bacon, to evaluate the performance of several computational methods. Finally, we provide practical recommendations for users working with HiChIP and/or ChIA-PET analyses.

3D-Epigenomic Regulation of Gene Transcription in Hepatocellular Carcinoma.

The fundamental cause of transcription dysregulation in hepatocellular carcinoma (HCC) remains elusive. To investigate the underlying mechanisms, comprehensive 3D-epigenomic analyses are performed in cellular models of THLE2 (a normal hepatocytes cell line) and HepG2 (a hepatocellular carcinoma cell line) using integrative approaches for chromatin topology, genomic and epigenomic variation, and transcriptional output. Comparing the 3D-epigenomes in THLE2 and HepG2 reveal that most HCC-associated genes are organized in complex chromatin interactions mediated by RNA polymerase II (RNAPII). Incorporation of genome-wide association studies (GWAS) data enables the identification of non-coding genetic variants that are enriched in distal enhancers connecting to the promoters of HCC-associated genes via long-range chromatin interactions, highlighting their functional roles. Interestingly, CTCF binding and looping proximal to HCC-associated genes appear to form chromatin architectures that overarch RNAPII-mediated chromatin interactions. It is further demonstrated that epigenetic variants by DNA hypomethylation at a subset of CTCF motifs proximal to HCC-associated genes can modify chromatin topological configuration, which in turn alter RNAPII-mediated chromatin interactions and lead to dysregulation of transcription. Together, the 3D-epigenomic analyses provide novel insights of multifaceted interplays involving genetics, epigenetics, and chromatin topology in HCC cells.

3D chromatin architecture and epigenetic regulation in cancer stem cells.

Dedifferentiation of cell identity to a progenitor-like or stem cell-like state with increased cellular plasticity is frequently observed in cancer formation. During this process, a subpopulation of cells in tumours acquires a stem cell-like state partially resembling to naturally occurring pluripotent stem cells that are temporarily present during early embryogenesis. Such characteristics allow these cancer stem cells (CSCs) to give rise to the whole tumour with its entire cellular heterogeneity and thereby support metastases formation while being resistant to current cancer therapeutics. Cancer development and progression are demarcated by transcriptional dysregulation. In this article, we explore the epigenetic mechanisms shaping gene expression during tumorigenesis and cancer stem cell formation, with an emphasis on 3D chromatin architecture. Comparing the pluripotent stem cell state and epigenetic reprogramming to dedifferentiation in cellular transformation provides intriguing insight to chromatin dynamics. We suggest that the 3D chromatin architecture could be used as a target for re-sensitizing cancer stem cells to therapeutics.