B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity.

The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.

Interactome maps of mouse gene regulatory domains reveal basic principles of transcriptional regulation.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner.

Ep300 sequestration to functionally distinct glucocorticoid receptor binding loci underlie rapid gene activation and repression.

The rapid transcriptional response to the transcription factor, glucocorticoid receptor (GR), including gene activation or repression, is mediated by the spatial association of genes with multiple GR binding sites (GBSs) over large genomic distances. However, only a minority of the GBSs have independent GR-mediated activating capacity, and GBSs with independent repressive activity were rarely reported. To understand the positive and negative effects of GR we mapped the regulatory environment of its gene targets. We show that the chromatin interaction networks of GR-activated and repressed genes are spatially separated and vary in the features and configuration of their GBS and other non-GBS regulatory elements. The convergence of the KLF4 pathway in GR-activated domains and the STAT6 pathway in GR-repressed domains, impose opposite transcriptional effects to GR, independent of hormone application. Moreover, the ROR and Rev-erb transcription factors serve as positive and negative regulators, respectively, of GR-mediated gene activation. We found that the spatial crosstalk between GBSs and non-GBSs provides a physical platform for sequestering the Ep300 co-activator from non-GR regulatory loci in both GR-activated and -repressed gene compartments. While this allows rapid gene repression, Ep300 recruitment to GBSs is productive specifically in the activated compartments, thus providing the basis for gene induction.

Dose-Dependent Transcriptional Response to Ionizing Radiation Is Orchestrated with DNA Repair within the Nuclear Space.

Radiation therapy is commonly used to treat glioblastoma multiforme (GBM) brain tumors. Ionizing radiation (IR) induces dose-specific variations in transcriptional programs, implicating that they are tightly regulated and critical components in the tumor response and survival. Yet, our understanding of the downstream molecular events triggered by effective vs. non-effective IR doses is limited. Herein, we report that variations in the genetic programs are positively and functionally correlated with the exposure to effective or non-effective IR doses. Genome architecture analysis revealed that gene regulation is spatially and temporally coordinated with DNA repair kinetics. The radiation-activated genes were pre-positioned in active sub-nuclear compartments and were upregulated following the DNA damage response, while the DNA repair activity shifted to the inactive heterochromatic spatial compartments. The IR dose affected the levels of DNA damage repair and transcription modulation, but not the order of the events, which was linked to their spatial nuclear positioning. Thus, the distinct coordinated temporal dynamics of DNA damage repair and transcription reprogramming in the active and inactive sub-nuclear compartments highlight the importance of high-order genome organization in synchronizing the molecular events following IR.

Chromatin dynamics enable transcriptional rhythms in the cnidarian Nematostella vectensis.

In animals, circadian rhythms are driven by oscillations in transcription, translation, and proteasomal degradation of highly conserved genes, resulting in diel cycles in the expression of numerous clock-regulated genes. Transcription is largely regulated through the binding of transcription factors to cis-regulatory elements within accessible regions of the chromatin. Chromatin remodeling is linked to circadian regulation in mammals, but it is unknown whether cycles in chromatin accessibility are a general feature of clock-regulated genes throughout evolution. To assess this, we applied an ATAC-seq approach using Nematostella vectensis, grown under two separate light regimes (light:dark (LD) and constant darkness (DD)). Based on previously identified N. vectensis circadian genes, our results show the coupling of chromatin accessibility and circadian transcription rhythmicity under LD conditions. Out of 180 known circadian genes, we were able to list 139 gene promoters that were highly accessible compared to common promoters. Furthermore, under LD conditions, we identified 259 active enhancers as opposed to 333 active enhancers under DD conditions, with 171 enhancers shared between the two treatments. The development of a highly reproducible ATAC-seq protocol integrated with published RNA-seq and ChIP-seq databases revealed the enrichment of transcription factor binding sites (such as C/EBP, homeobox, and MYB), which have not been previously associated with circadian signaling in cnidarians. These results provide new insight into the regulation of cnidarian circadian machinery. Broadly speaking, this supports the notion that the association between chromatin remodeling and circadian regulation arose early in animal evolution as reflected in this non-bilaterian lineage.

Early Th1 cell differentiation is marked by a Tfh cell-like transition.

Follicular helper T (Tfh) cells comprise an important subset of helper T cells; however, their relationship with other helper lineages is incompletely understood. Herein, we showed interleukin-12 acting via the transcription factor STAT4 induced both Il21 and Bcl6 genes, generating cells with features of both Tfh and Th1 cells. However, STAT4 also induced the transcription factor T-bet. With ChIP-seq, we defined the genome-wide targets of T-bet and found that it repressed Bcl6 and other markers of Tfh cells, thereby attenuating the nascent Tfh cell-like phenotype in the late phase of Th1 cell specification. Tfh-like cells were rapidly generated after Toxoplasma gondii infection in mice, but T-bet constrained Tfh cell expansion and consequent germinal center formation and antibody production. Our data argue that Tfh and Th1 cells share a transitional stage through the signal mediated by STAT4, which promotes both phenotypes. However, T-bet represses Tfh cell functionalities, promoting full Th1 cell differentiation.

The Position and Complex Genomic Architecture of Plant T-DNA Insertions Revealed by 4SEE.

The integration of T-DNA in plant genomes is widely used for basic research and agriculture. The high heterogeneity in the number of integration events per genome, their configuration, and their impact on genome integrity highlight the critical need to detect the genomic locations of T-DNA insertions and their associated chromosomal rearrangements, and the great challenge in doing so. Here, we present 4SEE, a circular chromosome conformation capture (4C)-based method for robust, rapid, and cost-efficient detection of the entire scope of T-DNA locations. Moreover, by measuring the chromosomal architecture of the plant genome flanking the T-DNA insertions, 4SEE outlines their associated complex chromosomal aberrations. Applying 4SEE to a collection of confirmed T-DNA lines revealed previously unmapped T-DNA insertions and chromosomal rearrangements such as inversions and translocations. Uncovering such events in a feasible, robust, and cost-effective manner by 4SEE in any plant of interest has implications for accurate annotation and phenotypic characterization of T-DNA insertion mutants and transgene expression in basic science applications as well as for plant biotechnology.

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.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes.

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

Epigenetic targeting of activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Igκ, and Igλ). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment.

Spatial congregation of STAT binding directs selective nuclear architecture during T-cell functional differentiation.

Higher-order genome organization shows tissue-specific patterns. However, functional relevance and the mechanisms shaping the genome architecture are poorly understood. Here we report a profound shift from promiscuous to highly selective genome organization that accompanies the effector lineage choice of differentiating T cells. As multipotent naive cells receive antigenic signals and commit to a T helper (Th) pathway, the genome-wide contacts of a lineage-specific cytokine locus are preferentially enriched for functionally relevant genes. Despite the establishment of divergent interactomes and global reprogramming of transcription in Th1 versus Th2, the overall expression status of the contact genes is surprisingly similar between the two lineages. Importantly, during differentiation, the genomic contacts are retained and strengthened precisely at DNA binding sites of the specific lineage-determining STAT transcription factor. In cells from the specific STAT knock-out mouse, the signature cytokine locus is unable to shed the promiscuous contacts established in the naive T cells, indicating the importance of genomic STAT binding. Altogether, the global aggregation of STAT binding loci from genic and nongenic regions highlights a new role for differentiation-promoting transcription factors in direct specification of higher-order nuclear architecture through interacting with regulatory regions. Such subnuclear environments have significant implications for efficient functioning of the mature effector lymphocytes.

FIE, a nuclear PRC2 protein, forms cytoplasmic complexes in Arabidopsis thaliana.

Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers that regulate developmental pathways in plants. PcGs form nuclear multi-subunit Polycomb Repressive Complexes (PRCs). The PRC2 complex mediates gene repression via methylation of lysine 27 on histone H3, which consequently leads to chromatin condensation. In Arabidopsis thaliana, several PRC2 complexes with different compositions were identified, each controlling a particular developmental program.The core subunit FIE is crucial for PRC2 function throughout the plant life cycle, yet accurate information on its spatial and temporal localization was absent. This study focused on identifying FIE accumulation patterns, using microscopy and biochemical approaches. Analysing endogenous FIE and transgenic gFIE-green fluorescent protein fusion protein (gFIE-GFP) showed that FIE accumulates in the nuclei of every cell type examined. Interestingly, gFIE-GFP, as well as the endogenous FIE, also localized to the cytoplasm in all examined tissues. In both vegetative and reproductive organs, FIE formed cytoplasmic high-molecular-mass complexes, in parallel to the nuclear PRC2 complexes. Moreover, size-exclusion chromatography and bimolecular fluorescence complementation assays indicated that in inflorescences FIE formed a cytoplasmic complex with MEA, a PRC2 histone methyltransferase subunit. In contrast, CLF and SWN histone methyltransferases were strictly nuclear. Presence of PRC2 subunits in cytoplasmic complexes has not been previously described in plants. Our findings are in agreement with accumulating evidence demonstrating cytoplasmic localization and function of PcGs in metazoa. The cytosolic accumulation of PRC2 components in plants supports the model that PcGs have alternative non-nuclear functions that go beyond chromatin methylation.

Mouse model of endemic Burkitt translocations reveals the long-range boundaries of Ig-mediated oncogene deregulation.

Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5' proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil N-glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to ∼350 kb upstream of Myc or the related oncogene Mycn. A comprehensive analysis of epigenetic marks, PolII recruitment, and transcription in tumor cells demonstrates that the 3' Igh enhancer (Eα) vastly remodels ∼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this long-range epigenetic reprogramming is directly proportional to the physical interaction of Eα with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3' enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic-like chromosomal rearrangements.

Diverse gene reprogramming events occur in the same spatial clusters of distal regulatory elements.

The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation.

Rapid DNA-FISH in Arabidopsis thaliana Somatic Cells.

Fluorescence in situ hybridization (FISH) technique has been widely used to detect and localize specific DNA and RNA sequences in interphase nuclei and chromosomes in animals and plants. Here, we present a protocol for localization of genomic loci in nuclei of the model plant Arabidopsis thaliana. This protocol includes several advances and adaptations to A. thaliana, including preparation of nuclei and chromosomes without the use of liquid nitrogen, and an in situ hybridization procedure that preserves chromatin structure without the use of paraformaldehyde and formamide. Simultaneous denaturation of the BAC (bacterial artificial chromosome) probe and nuclei followed by annealing at high temperature allows hybridization in less than an hour. These hybridization conditions also provide high signal to noise ratio by a small number of washes. Thus, this simplified in situ hybridization procedure is completed in one working day.

Genomic retargeting of p53 and CTCF is associated with transcriptional changes during oncogenic HRas-induced transformation.

Gene transcription is regulated by distant regulatory elements via combinatorial binding of transcription factors. It is increasingly recognized that alterations in chromatin state and transcription factor binding in these distant regulatory elements may have key roles in cancer development. Here we focused on the first stages of oncogene-induced carcinogenic transformation, and characterized the regulatory network underlying transcriptional changes associated with this process. Using Hi-C data, we observe spatial coupling between differentially expressed genes and their differentially accessible regulatory elements and reveal two candidate transcription factors, p53 and CTCF, as determinants of transcriptional alterations at the early stages of oncogenic HRas-induced transformation in human mammary epithelial cells. Strikingly, the malignant transcriptional reprograming is promoted by redistribution of chromatin binding of these factors without major variation in their expression level. Our results demonstrate that alterations in the regulatory landscape have a major role in driving oncogene-induced transcriptional reprogramming.

Regulatory chromatin landscape in Arabidopsis thaliana roots uncovered by coupling INTACT and ATAC-seq.

BACKGROUND: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever-growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors (TFs) to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus, the accessibility of these loci for TF binding is highly regulated and determines where and when TFs bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana and define organ-specific regulatory sites and binding motifs of TFs at these sites. RESULTS: We coupled the recently described isolation of nuclei tagged in specific cell types (INTACT) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions but are correlated with root-specific expression of distant genes. Importantly, these gene-distant sites are enriched for binding motifs of TFs important for root development as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel gene-distant regulatory elements. CONCLUSIONS: By coupling INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root-specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.

Genomic effects of glucocorticoids.

Glucocorticoids and their receptor (GR) have been an important area of research because of their pleiotropic physiological functions and extensive use in the clinic. In addition, the association between GR and glucocorticoids, which is highly specific, leads to rapid nuclear translocation where GR associates with chromatin to regulate gene transcription. This simplified model system has been instrumental for studying the complexity of transcription regulation processes occurring at chromatin. In this review we discuss our current understanding of GR action that has been enhanced by recent developments in genome wide measurements of chromatin accessibility, histone marks, chromatin remodeling and 3D chromatin structure in various cell types responding to glucocorticoids.