Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes.

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.

Single-cell profiling of transcriptome and histone modifications with EpiDamID.

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, but the combined detection of histone post-translational modifications and transcription at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins. Using these, we render the DamID technology compatible with the genome-wide identification of histone post-translational modifications. Importantly, this includes the possibility to jointly measure chromatin marks and transcription at the single-cell level. We use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. In addition, we map H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specific to notochord. Overall, EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes.

Interplay between CTCF boundaries and a super enhancer controls cohesin extrusion trajectories and gene expression.

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.

Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies.

We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromatin conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. By contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long-molecule sequencing on an Oxford Nanopore or Pacific Biosciences platform. Each concatemer of proximity ligation products represents a snapshot topology of a different individual allele, revealing its multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox is able to generate background models and three-way interaction profiles from the same dataset. These profiles can be used to distinguish whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously occurring at chromatin hubs. The entire procedure can be completed in 2 w, and requires standard molecular biology and data analysis skills and equipment, plus access to a third-generation sequencing platform.

DamC reveals principles of chromatin folding in vivo without crosslinking and ligation.

Current understanding of chromosome folding is largely reliant on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after chromatin crosslinking. To measure chromosome structure in vivo, quantitatively and without crosslinking and ligation, we implemented a modified version of DNA adenine methyltransferase identification (DamID) named DamC, which combines DNA methylation-based detection of chromosomal interactions with next-generation sequencing and biophysical modeling of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of topologically associating domains (TADs), CTCF loops and confirms 3C-based measurements of the scaling of contact probabilities. Combining DamC with transposon-mediated genomic engineering shows that new loops can be formed between ectopic and endogenous CTCF sites, which redistributes physical interactions within TADs. DamC provides the first crosslinking- and ligation-free demonstration of the existence of key structural features of chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.

Inheritance of the Golgi Apparatus and Cytokinesis Are Controlled by Degradation of GBF1.

Although much is known about how chromosome segregation is coupled to cell division, how intracellular organelles partition during mitotic division is poorly understood. We report that the phosphorylation-dependent degradation of the ARFGEF GBF1 regulates organelle trafficking during cell division. We show that, in mitosis, GBF1 is phosphorylated on Ser292 and Ser297 by casein kinase-2 allowing recognition by the F-box protein ßTrCP. GBF1 interaction with ßTrCP recruits GBF1 to the SCFßTrCP ubiquitin ligase complex, triggering its degradation. Phosphorylation and degradation of GBF1 occur along microtubules at the intercellular bridge of telophase cells and are required for Golgi membrane positioning and postmitotic Golgi reformation. Indeed, expression of a non-degradable GBF1 mutant inhibits the transport of the Golgi cluster adjacent to the midbody toward the Golgi twin positioned next to the centrosome and results in defective Golgi reassembly and cytokinesis failure. These findings define a mechanism that controls postmitotic Golgi reassembly and inheritance.

Dimorphic DNA methylation during temperature-dependent sex determination in the sea turtle Lepidochelys olivacea.

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination.

Cause and Consequence of Tethering a SubTAD to Different Nuclear Compartments.

Detailed genomic contact maps have revealed that chromosomes are structurally organized in megabase-sized topologically associated domains (TADs) that encompass smaller subTADs. These domains segregate in the nuclear space to form active and inactive nuclear compartments, but cause and consequence of compartmentalization are largely unknown. Here, we combined lacO/lacR binding platforms with allele-specific 4C technologies to track their precise position in the three-dimensional genome upon recruitment of NANOG, SUV39H1, or EZH2. We observed locked genomic loci resistant to spatial repositioning and unlocked loci that could be repositioned to different nuclear subcompartments with distinct chromatin signatures. Focal protein recruitment caused the entire subTAD, but not surrounding regions, to engage in new genomic contacts. Compartment switching was found uncoupled from transcription changes, and the enzymatic modification of histones per se was insufficient for repositioning. Collectively, this suggests that trans-associated factors influence three-dimensional compartmentalization independent of their cis effect on local chromatin composition and activity.

CTCF Binding Polarity Determines Chromatin Looping.

CCCTC-binding factor (CTCF) is an architectural protein involved in the three-dimensional (3D) organization of chromatin. In this study, we assayed the 3D genomic contact profiles of a large number of CTCF binding sites with high-resolution 4C-seq. As recently reported, our data also suggest that chromatin loops preferentially form between CTCF binding sites oriented in a convergent manner. To directly test this, we used CRISPR/Cas9 genome editing to delete core CTCF binding sites in three loci, including the CTCF site in the Sox2 super-enhancer. In all instances, CTCF and cohesin recruitment were lost, and chromatin loops with distal, convergent CTCF sites were disrupted or destabilized. Re-insertion of oppositely oriented CTCF recognition sequences restored CTCF and cohesin recruitment, but did not re-establish chromatin loops. We conclude that CTCF binding polarity plays a functional role in the formation of higher-order chromatin structure.

YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers.

The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.

Age-dependent increment of hydroxymethylation in the brain cortex in the triple-transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a complex disorder whose etiology is associated with environmental and genetic factors. Recently there have been several attempts to analyze the role of epigenetic alterations in the origin and progression of this neurodegenerative condition. To evaluate the potential participation of the methylation status of the genome that may contribute to AD progression, we have studied the levels and distribution of the 5-methylcytosine and 5-hydroxymethylcytosine in different brain regions at different ages. We analyzed and quantified the immunosignal of these two epigenetic marks in young versus old wild-type mice and in the triple-transgenic mouse model of AD (3xTg-AD). The results show a decline in global 5-methylcytosine mark over time in all studied brain regions concomitant with a significant and widespread increase in 5-hydroxymethylcytosine mark in the aged transgenic mice in contrast to the age-matched controls. These differences in the methylation pattern of brain DNA in the 3xTg-AD that accumulates along age indicates abnormal formation of permissive chromatin structure associated with the increase in AD-related markers.

A long non-coding RNA promotes full activation of adult gene expression in the chicken α-globin domain.

Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult α(D) globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult α(D) gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the α(D) adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure.

CTCF demarcates chicken embryonic α-globin gene autonomous silencing and contributes to adult stage-specific gene expression.

Genomic loci composed of more than one gene are frequently subjected to differential gene expression, with the chicken α-globin domain being a clear example. In the present study we aim to understand the globin switching mechanisms responsible for the epigenetic silencing of the embryonic π gene and the transcriptional activation of the adult α(D) and α(A) genes at the genomic domain level. In early stages, we describe a physical contact between the embryonic π gene and the distal 3' enhancer that is lost later during development. We show that such a level of regulation is achieved through the establishment of a DNA hypermethylation sub-domain that includes the embryonic gene and the adjacent genomic sequences. The multifunctional CCCTCC-binding factor (CTCF), which is located upstream of the α(D) gene promoter, delimits this sub-domain and creates a transition between the inactive sub-domain and the active sub-domain, which includes the adult α(D) gene. In avian-transformed erythroblast HD3 cells that are induced to differentiate, we found active DNA demethylation of the adult α(D) promoter, coincident with the incorporation of 5-hydroxymethylcytosine (5hmC) and concomitant with adult gene transcriptional activation. These results suggest that autonomous silencing of the embryonic π gene is needed to facilitate an optimal topological conformation of the domain. This model proposes that CTCF is contributing to a specific chromatin configuration that is necessary for differential α-globin gene expression during development.

Robust 4C-seq data analysis to screen for regulatory DNA interactions.

Regulatory DNA elements can control the expression of distant genes via physical interactions. Here we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq). Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation.

Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter.

BACKGROUND: Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. METHODS: To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. RESULTS: We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. CONCLUSIONS: This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.

Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes.

Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor-encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1.

Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes.

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC.

Globin genes transcriptional switching, chromatin structure and linked lessons to epigenetics in cancer: a comparative overview.

At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.