Gene-to-gene coordinated regulation of transcription and alternative splicing by 3D chromatin remodeling upon NF-κB activation.

The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.

The NF-κB pathway is essential for coordinating gene expression in response to various stimuli, including viral infections. Most studies have focused on the role of NF-κB in transcriptional regulation. In the present study, the impact of the potent NF-κB activator HTLV-1 Tax oncoprotein on the three-dimensional organization of the genome was investigated. Tax-mediated NF-κB activation was found to restructure the 3D genomic landscape in cells and to bring genes together in multigene complexes that are coordinately regulated either transcriptionally or through alternative splicing by NF-κB. Induced coordinate changes in transcription and alternative splicing included the two master genes of NF-κB pathway NFKBIA and RELA. The findings have significant implications for understanding cell fate determination and disease development associated with HTLV-1 infection, as well as chronic NF-κB activation in various human inflammatory diseases and cancer.

Transcription induces context-dependent remodeling of chromatin architecture during differentiation.

Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts.

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the spatiotemporal arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find a close link between chromatin mobility and transcriptional status: active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

RNA polymerase II CTD is dispensable for transcription and required for termination in human cells.

The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C-terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD-Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD-Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long-distance interactions and CTCF-binding patterns in CTD-Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination.

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

Short tandem repeats are important contributors to silencer elements in T cells.

The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements.

Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

Drought Tolerance Strategies and Autophagy in Resilient Wheat Genotypes.

Drought resiliency strategies combine developmental, physiological, cellular, and molecular mechanisms. Here, we compare drought responses in two resilient spring wheat (Triticum aestivum) genotypes: a well-studied drought-resilient Drysdale and a resilient genotype from the US Pacific North-West Hollis. While both genotypes utilize higher water use efficiency through the reduction of stomatal conductance, other mechanisms differ. First, Hollis deploys the drought escape mechanism to a greater extent than Drysdale by accelerating the flowering time and reducing root growth. Second, Drysdale uses physiological mechanisms such as non-photochemical quenching (NPQ) to dissipate the excess of harvested light energy and sustain higher Fv/Fm and ϕPSII, whereas Hollis maintains constant NPQ but lower Fv/Fm and ϕPSII values. Furthermore, more electron donors of the electron transport chain are in the oxidized state in Hollis than in Drysdale. Third, many ROS homeostasis parameters, including peroxisome abundance, transcription of peroxisome biogenesis genes PEX11 and CAT, catalase protein level, and enzymatic activity, are higher in Hollis than in Drysdale. Fourth, transcription of autophagy flux marker ATG8.4 is upregulated to a greater degree in Hollis than in Drysdale under drought, whereas relative ATG8 protein abundance under drought stress is lower in Hollis than in Drysdale. These data demonstrate the activation of autophagy in both genotypes and a greater autophagic flux in Hollis. In conclusion, wheat varieties utilize different drought tolerance mechanisms. Combining these mechanisms within one genotype offers a promising strategy to advance crop resiliency.

Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes.

The spatiotemporal organization of chromatin influences many nuclear processes: from chromosome segregation to transcriptional regulation. To get a deeper understanding of these processes, it is essential to go beyond static viewpoints of chromosome structures, to accurately characterize chromatin's diffusion properties. We present GP-FBM: a computational framework based on Gaussian processes and fractional Brownian motion to extract diffusion properties from stochastic trajectories of labeled chromatin loci. GP-FBM uses higher-order temporal correlations present in the data, therefore, outperforming existing methods. Furthermore, GP-FBM allows to interpolate incomplete trajectories and account for substrate movement when two or more particles are present. Using our method, we show that average chromatin diffusion properties are surprisingly similar in interphase and mitosis in mouse embryonic stem cells. We observe surprising heterogeneity in local chromatin dynamics, correlating with potential regulatory activity. We also present GP-Tool, a user-friendly graphical interface to facilitate usage of GP-FBM by the research community.

Assessment of 3D Interactions Between Promoters and Distal Regulatory Elements with Promoter Capture Hi-C (PCHi-C).

Chromosome conformation capture and its variants interrogate population-average chromatin structure at a higher resolution and throughput than microscopic methods. Capture Hi-C is a variant tailored for the simultaneous assessment of all interactions with thousands of specific bait sequences, so is particularly suited to genome-wide studies of promoter interactions with distal regulatory elements, such as enhancers. We present the principles and methods for Promoter Capture Hi-C (PCHi-C), from experimental design to data analysis.

Myod1 and GR coordinate myofiber-specific transcriptional enhancers.

Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.

4C-Seq: Interrogating Chromatin Looping with Circular Chromosome Conformation Capture.

Chromosome conformation capture and its variants have allowed chromatin topology to be interrogated at a superior resolution and throughput than by microscopic methods. Among the method derivatives, 4C-seq (circular chromosome conformation capture, coupled to high-throughput sequencing) is a versatile, cost-effective means of assessing all chromatin interactions with a specific genomic region of interest, making it particularly suitable for interrogating chromatin looping events. We present the principles and procedures for designing and implementing successful 4C-seq experiments.

Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene.

The Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-throughput enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus.

Defining Functionally Relevant Spatial Chromatin Domains: It is a TAD Complicated.

Chromosome conformation capture and orthologous methods uncovered the spatial organization of metazoan chromosomes into autonomously folded substructures, often termed topologically associated domains (TADs). There is a striking correlation between TAD organization and hallmarks of genome function, such as histone modifications or gene expression, and disruptions of specific TAD structures have been associated with pathological misexpression of underlying genes. However, complete disruption of TADs seems to have mild effects on the transcriptome, raising questions as to the importance of chromatin topology in regulating the expression of most genes. Furthermore, despite a growing number of genetic perturbation studies, it is still largely unclear how TAD-like domains are defined, maintained, or potentially reorganized. This perspective article discusses the recent work exploring the complexity of the relationship between TADs and transcription, arguing that it is not satisfactorily explained by any of the "rules" that have been previously described.

Dissecting Gene Repression: Not Just Location, Location, Location.

Large heterochromatic domains are found tethered to the lamina. But, is this nuclear environment repressive per se, or just the 'ground state' of inactive chromatin? Elegant studies from the van Steensel group (Leemans et al., Cell, 2019) recently demonstrated that the lamina is indeed repressive, but that intrinsic promoter properties also dictate gene activity.

ChiCMaxima: a robust and simple pipeline for detection and visualization of chromatin looping in Capture Hi-C.

Capture Hi-C (CHi-C) is a new technique for assessing genome organization based on chromosome conformation capture coupled to oligonucleotide capture of regions of interest, such as gene promoters. Chromatin loop detection is challenging because existing Hi-C/4C-like tools, which make different assumptions about the technical biases presented, are often unsuitable. We describe a new approach, ChiCMaxima, which uses local maxima combined with limited filtering to detect DNA looping interactions, integrating information from biological replicates. ChiCMaxima shows more stringency and robustness compared to previously developed tools. The tool includes a GUI browser for flexible visualization of CHi-C profiles alongside epigenomic tracks.

4See: A Flexible Browser to Explore 4C Data.

It is established that transcription of many metazoan genes is regulated by distal regulatory sequences beyond the promoter. Enhancers have been identified at up to megabase distances from their regulated genes, and/or proximal to or within the introns of unregulated genes. The unambiguous identification of the target genes of newly identified regulatory elements can thus be challenging. Well-studied enhancers have been found to come into direct physical proximity with regulated genes, presumably by the formation of chromatin loops. Chromosome conformation capture (3C) derivatives that assess the frequency of proximity between different genetic elements is thus a popular method for exploring gene regulation by distal regulatory elements. For studies of chromatin loops and promoter-enhancer communication, 4C (circular chromosome conformation capture) is one of the methods of choice, optimizing cost (required sequencing depth), throughput, and resolution. For ease of visual inspection of 4C data we present 4See, a versatile and user-friendly browser. 4See allows 4C profiles from the same bait to be flexibly plotted together, allowing biological replicates to either be compared, or pooled for comparisons between different cell types or experimental conditions. 4C profiles can be integrated with gene tracks, linear epigenomic profiles, and annotated regions of interest, such as called significant interactions, allowing rapid data exploration with limited computational resources or bioinformatics expertise.

TADs are 3D structural units of higher-order chromosome organization in Drosophila.

Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.

Chromosome topology guides the Drosophila Dosage Compensation Complex for target gene activation.

X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.

Detecting Spatial Chromatin Organization by Chromosome Conformation Capture II: Genome-Wide Profiling by Hi-C.

The chromosome conformation capture (3C) method has been invaluable in studying chromatin interactions in a population of cells at a resolution surpassing that of light microscopy, for example in the detection of functional contacts between enhancers and promoters. Recent developments in sequencing-based chromosomal contact mapping (Hi-C, 5C and 4C-Seq) have allowed researchers to interrogate pairwise chromatin interactions on a wider scale, shedding light on the three-dimensional organization of chromosomes. These methods present significant technical and bioinformatic challenges to consider at the start of the project. Here, we describe two alternative methods for Hi-C, depending on the size of the genome, and discuss the major computational approaches to convert the raw sequencing data into meaningful models of how genomes are organized.