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The enhancer-promoter looping model, in which enhancers activate their target genes via physical contact, has long dominated the field of gene regulation. However, the ubiquity of this model has been questioned due to evidence of alternative mechanisms and the lack of its systematic validation, primarily owing to the absence of suitable experimental techniques. In this study, we present a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. By applying MChIP-C to study H3K4me3 promoter-centered interactions in K562 cells, we found that it had greatly improved resolution and sensitivity compared to restriction endonuclease-based C-methods. This allowed us to identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions. Finally, leveraging data from published CRISPRi screens, we found that most functionally-verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.

The study investigated methylation of each cytosine within a fragment of the YJEFN3 gene located at one of the index loci of schizophrenia risk with the aim to determine whether methylation of this fragment is associated with the level of cognitive deficit in patients and to what extent it depends on the risk haplotype. Peripheral blood samples from 70 schizophrenia spectrum patients and 72 healthy subjects who underwent cognitive testing were examined using single-molecule real-time bisulfite sequencing. A negative correlation was found between methylation of the CpG site at chr19:19532203, hg38 (Illumina ID: cg08623644) and the cognitive index in patients. Methylation at cg08623644 was associated with the risk haplotype in the pooled sample and in healthy subjects. The patients showed a weakening of this association. The findings suggest that methylation at cg08623644 may be a peripheral marker of molecular processes associated with the severity of the disease, which deserves further investigation.

Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings. Supplementary Information The online version contains supplementary material available at 10.1186/s13072-021-00411-w.

Liquid–liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

Chromatin loops represent one of the major levels of hierarchical folding of the genome. Although the situation is evolving, current methods have various difficulties with the accurate mapping of loops even in mammalian Hi-C data, and most of them fail to identify chromatin loops in animal species with substantially different genome architecture. This paper presents the loop and significant contact annotation (LASCA) pipeline, which uses Weibull distribution-based modeling to effectively identify loops and enhancer–promoter interactions in Hi-C data from evolutionarily distant species: from yeast and worms to mammals. Available at: https://github.com/ArtemLuzhin/LASCA_pipeline.

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA–DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. Analysis of the RNA–DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

Scientifically interesting as well as practically important phenotypes often belong to the realm of complex traits. To the extent that these traits are hereditary, they are usually ‘highly polygenic’. The study of such traits presents a challenge for researchers, as the complex genetic architecture of such traits makes it nearly impossible to utilise many of the usual methods of reverse genetics, which often focus on specific genes. In recent years, thousands of genome-wide association studies (GWAS) were undertaken to explore the relationships between complex traits and a large number of genetic factors, most of which are characterised by tiny effects. In this review, we aim to familiarise ‘wet biologists’ with approaches for the interpretation of GWAS results, to clarify some issues that may seem counterintuitive and to assess the possibility of using GWAS results in experiments on various complex traits.

Objectives. To identify relationships between the rs7341475 polymorphism and methylation of the reelin gene on the one hand and the severity of schizophrenia symptomatology and the neuropsychological indicator – semantic verbal fluency (VF) – on the other. Materials and methods. Genotypes at the rs7341475 locus were determined in 556 patients with schizophrenia spectrum disorders. PANSS evaluations were obtained for 549 of these patients and 221 were tested for semantic VF. The relationship between methylation of the reelin 5’ region and promotor and the severity of PANSS syndromes and VF was assessed using data from 35 patients using the five-factor PANSS model. Results. A relationship was found between genotype at the rs7341475 locus and PANSS measures (a sex-genotype interaction effect: F = 2.70, p = 0.020). Male homozygotes for the common G allele showed the lowest levels of positive syndrome. Verbal fluency was linked with the characteristics of the methylation of the reelin gene promoter. Conclusions. The results obtained here suggest that the rs7341475 polymorphism may be related to variation in positive symptomatology in men. At the same time, the pattern of methylation of the reelin gene is important for retention of VF – with higher levels of methylation in the transcription initiation site and low levels in the distal part of the promoter.

The largest genome-wide association studies have identified schizophrenia and intelligence associated variants in the MIR137HG locus containing genes encoding microRNA-137 and microRNA-2682. In the present study, we investigated DNA methylation in the MIR137HG intragenic CpG island (CGI) in the peripheral blood of 44 patients with schizophrenia and 50 healthy controls. The CGI included the entire MIR137 gene and the region adjacent to the 5'-end of MIR2682. The aim of the study was to examine the relationship of the CGI methylation with schizophrenia and cognitive functioning. The methylation level of 91 CpG located in the selected region was established for each participant by means of single-molecule real-time bisulfite sequencing. All subjects completed the battery of neuropsychological tests. We found that the CGI was hypomethylated in both groups, except for one site—CpG (chr1: 98 511 049), with significant interindividual variability in methylation. A higher level of methylation of this CpG was seen in male patients and was associated with a decrease in the cognitive index in the combined sample of patients and controls. Our data suggest that further investigation of mechanisms that regulate the MIR137 and MIR2682 genes expression might help to understand the molecular basis of cognitive deficits in schizophrenia.

Genetic variation may impact on local DNA methylation patterns. Therefore, information about allele-specific DNA methylation (ASM) within disease-related loci has been proposed to be useful for the interpretation of GWAS results. To explore mechanisms that may underlie associations between Alzheimer's disease (AD) and schizophrenia risk CLU gene and verbal memory, one of the most affected cognitive domains in both conditions, we studied DNA methylation in a region between AD-associated SNPs rs9331888 and rs9331896 in 72 healthy individuals and 73 schizophrenia patients. Using single-molecule real-time bisulfite sequencing we assessed the haplotype-dependent ASM in this region. We then investigated whether its methylation could influence episodic verbal memory measured with the Rey Auditory Verbal Learning Test in these two cohorts. The region showed a complex methylation pattern, which was similar in healthy and schizophrenia individuals and unrelated to haplotypes. The pattern predicted memory scores in controls. The results suggest that epigenetic modifications within the CLU locus may play a role in memory variation, independent of ASM.

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome nanodomains and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, but increased further mixing of the chromatin compartments and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

Large-scale epigenomic projects have mapped hundreds of thousands of potential regulatory sites in the human genome, but only a small proportion of these elements are proximal to transcription start sites. It is believed that the majority of these sequences are remote promoter-activating genomic sites scattered within several hundreds of kilobases from their cognate promoters and referred to as enhancers. It is still unclear what principles, aside from relative closeness in the linear genome, determine which promoter(s) is controlled by a given enhancer; however, this understanding is of great fundamental and clinical relevance. In recent years, C-methods (chromosome conformation capture-based methods) have become a powerful tool for the identification of enhancer–promoter spatial contacts that, in most cases, reflect their functional link. Here, we describe a new hybridisation-based promoter Capture-C protocol that makes use of biotinylated dsDNA probes generated by PCR from a custom pool of long oligonucleotides. The described protocol allows high-resolution promoter interactome description, providing a flexible and cost-effective alternative to the existing promoter Capture-C modifications. Based on the obtained data, we propose several tips on probe design that could potentially improve the results of future experiments.

Interrogating DNA methylation within schizophrenia risk loci holds promise to identify mechanisms by which genes influence the disease. Based on the hypothesis that allele specific methylation (ASM) of a single CpG, or perhaps CpH, might mediate or mark the effects of genetic variants on disease risk and phenotypes, we explored haplotype specific methylation levels of individual cytosines within a genomic region harbouring the BAG5, APOPT1 and KLC1 genes in peripheral blood of schizophrenia patients and healthy controls. Three DNA fragments located in promoter, intronic and intergenic areas were studied by single-molecule real-time bisulfite sequencing enabling the analysis of long reads of DNA with base-pair resolution and the determination of haplotypes directly from sequencing data. Among 1,012 cytosines studied, we did not find any site where methylation correlated with the disease or cognitive deficits after correction for multiple testing. At the same time, we determined the methylation profile associated with the schizophrenia risk haplotype within the KLC1 fourth intron and confirmed ASM for cytosines located in the vicinity of rs67899457. These genetically associated DNA methylation variations may be related to the pathophysiological mechanism differentiating the risk and non-risk haplotypes and merit further investigation.

Recent advances in psychiatric genetics have led to the discovery of dozens of genomic loci associated with schizophrenia. However, a gap exists between the detection of genetic associations and understanding the underlying molecular mechanisms. This review describes the basic approaches used in the so-called post-GWAS studies to generate biological interpretation of the existing population genetic data, including both molecular (creation and analysis of knockout animals, exploration of the transcriptional effects of common variants in human brain cells) and computational (fine-mapping of causal variability, gene set enrichment analysis, partitioned heritability analysis) methods. The results of the crucial studies, in which these approaches were used to uncover the molecular and neurobiological basis of the disease, are also reported.

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA–DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. We found two microRNAs—MIR3648 and MIR3687 transcribed from the rRNA locus—that are associated with inactive chromatin genome wide. These miRNAs favor bulk heterochromatin over Polycomb-repressed chromatin and interact preferentially with late-replicating genomic regions. Analysis of the RNA–DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

—There is a decrease in the expression of the reelin gene (RELN) in the brain of schizophrenia patients, which can underlie observed cognitive abnormalities. It is suggested that this decrease is caused by the hypermethylation of the RELN promoter. The aim of the study was to investigate methylation of the RELN promoter in the peripheral blood of schizophrenia patients and its association with their cognitive deficits. A modified SMRT-BS (single-molecule real-time bisulfite sequencing) was used. We determined the methylation rate of 170 CpG sites within a 1465 bp DNA region containing the entire CpG island in the RELN promoter in 51 schizophrenia patients and 52 healthy controls. All subjects completed a battery of neuropsychological tests. There were no DNA methylation changes associated with schizophrenia. Most CpGs sites were unmethylated in both groups. At the same time, there was a variability in the methylation level of different regions within the promoter. The methylation level in the area from –258 to –151 bp relative to RELN transcription start site was a significant predictor of the index of patients’ cognitive functioning if sex, age, smoking, education, and polymorphism rs1858815 had been considered. The positive correlation between the methylation rate in this region and cognitive index suggests that the hypomethylation of the RELN promoter could contribute to the development of cognitive deficits in schizophrenia.

Background In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. Results We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. Conclusions Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0142-4) contains supplementary material, which is available to authorized users.

Recently we characterized a class of anti-cancer agents (curaxins) that disturbs DNA/histone interactions within nucleosomes. Here, using a combination of genomic and in vitro approaches, we demonstrate that curaxins strongly affect spatial genome organization and compromise enhancer-promoter communication, which is necessary for the expression of several oncogenes, including MYC. We further show that curaxins selectively inhibit enhancer-regulated transcription of chromatinized templates in cell-free conditions. Genomic studies also suggest that curaxins induce partial depletion of CTCF from its binding sites, which contributes to the observed changes in genome topology. Thus, curaxins can be classified as epigenetic drugs that target the 3D genome organization., Curaxins are a recently discovered class of anti-cancer agents that disturbs DNA/histone interactions within. Here the authors provide evidence that curaxins affect the spatial genome organization and compromise enhancer-promoter communication necessary for expression of several oncogenes, including MYC.

Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods.

To study the role of polymorphism rs7341475 and methylation of the reelin gene in symptoms of schizophrenia and semantic verbal fluency.Genotypes at the locus rs7341475 were identified in 556 patients with schizophrenic disorders. PANSS scores were obtained for 549 patients and 221 patients performed a test for semantic verbal fluency. The association of the reelin promoter methylation with the PANSS and verbal fluency measures was evaluated in 35 patients. A five-factor model of the PANSS was used.The interaction effect of sex with genotype on the PANSS scores was found (F=2.70, p=0.020). Schizophrenic men homozygous for a common allele G had the lowest scores of the positive syndrome. Verbal fluency was related to the reelin promoter methylation.The results suggest that polymorphism rs7341475 may be associated with the variability of positive symptomatology in schizophrenic men. At the same time, the reelin gene methylation pattern, which consists of a higher methylation level in the region of the transcription start site and a lower one in the distal region of the promoter, may be beneficial for verbal fluency.Цель исследования. Выявление связи полиморфизма rs7341475 и метилирования гена рилина с выраженностью симптоматики шизофрении и нейропсихологическим признаком - семантической вербальной беглостью (ВБ). Материал и методы. Генотипы по локусу rs7341475 были определены у 556 пациентов с расстройствами шизофренического спектра. Из них для 549 больных были получены оценки PANSS и 221 больной выполнил пробу на семантическую ВБ. Связь метилирования 5'-области и промотора рилина с выраженностью синдромов PANSS и показателем ВБ оценивали на материале 35 больных. Использовали пятифакторную модель PANSS. Результаты. Выявлена связь между генотипом по локусу rs7341475 и показателями PANSS (эффект взаимодействия пол-генотип: F=2,70, p=0,020). У мужчин-гомозигот по распространенному аллелю G наблюдались наиболее низкие оценки позитивного синдрома. Показатель вербальной беглости был связан с особенностями метилирования промотора гена рилина. Заключение. Результаты позволяют предположить, что полиморфизм rs7341475 может быть связан с вариативностью позитивной симптоматики у мужчин. В то же время для сохранной ВБ важен паттерн метилирования гена рилина - с более высоким уровнем метилирования в регионе сайта инициации транскрипции и низким в дистальной области промотора.

The eukaryotic nucleus harbors genomic DNA, which is tens of thousands of times greater in linear size than the nuclear diameter. Its high condensation is due to DNA packaging in chromatin, and DNA wrapping around nucleosomal globules is a key step in the process. A histone octamer, which forms the nucleosomal globule, interacts with DNA via electrostatic contacts. DNA–histone interactions are rather tight and prevent nucleosomal DNA from being accessed by various enzymes and transcription factors. At the same time, nucleosomes do not prevent transcription and other processes related to the genetic function of DNA. The review considers the structure and diversity of nucleosomes and the central role they play in regulating transcription. Special emphasis is placed on how internucleosomal interactions contribute to genome accessibility to transcription machinery and how nucleosomes are removed from regulatory elements and transcription units in a controlled manner during transcription elongation. Ядра евкаріотних клітин містять геномну ДНК, лінійні розміри якої у десятки тисяч разів перевищують їхній діаметр. Багато в чому такий високий ступінь компактизації забезпечується упаковкою ДНК у хроматин, ключовим етапом якої є намотування ДНК на нуклеосомні глобули. Октамер гістонів, які складають нуклеосомну глобулу, взаємодіє з ДНК за посередництвом електростатичних контактів. ДНК-гістонові взаємодії достатньо міцні і утруднюють доступ до нуклеосомної ДНК багатьох ферментів і транскрипційних факторів. У той же час наявність нуклеосом не перешкоджає проходженню транскрипції та інших процесів, пов’язаних з реалізацією генетичних функцій ДНК. В огляді розглянуто структуру і розмаїття нуклеосом та їхню центральну роль у регуляції транскрипції. Особливу увагу приділено значенню міжнуклеосомних взаємодій у забезпеченні доступності геному для транскрипційної машинерії, а також регульованому видаленню нуклеосом з регуляторних елементів і транскрипційних одиниць в процесі елонгації транскрипції. Ядра эукариотических клеток содержат геномную ДНК, линейные размеры которой в десятки тысяч раз превышают их диаметр. Во многом такая высокая степень компактизации обеспечивается упаковкой ДНК в хроматин, ключевым этапом которой является наматывание ДНК на нуклеосомные глобулы. Октамер гистонов, составляющих нуклеосомную глобулу, взаимодействует с ДНК посредством электростатических контактов. ДНК-гистоновые взаимодействия достаточно прочны и затрудняют доступ к нуклеосомной ДНК многих ферментов и транскрипционных факторов. В то же время наличие нуклеосом не препятствует прохождению транскрипции и других процессов, связанных с реализацией генетических функций ДНК. В настоящем обзоре рассмотрены структура и многообразие нуклеосом и их центральная роль в регуляции транскрипции. Особое внимание уделено значению межнуклеосомных взаимодействий в обеспечении доступности генома для транскрипционной машинерии и регулируемому удалению нуклеосом с регуляторных элементов и транскрипционных единиц в процессе элонгации транскрипции.

The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements.

Chromosome conformation capture (3C) and derivative experimental procedures are used to estimate the spatial proximity between different genomic elements, thus providing information about the 3D organization of genomic domains and whole genomes within the nucleus. All C-methods are based on the proximity ligation-the preferential ligation of joined DNA fragments obtained upon restriction enzyme digestion of in vivo cross-linked chromatin. Here, using the mouse beta-globin genes in erythroid cells as a model, we estimated the actual frequencies of ligation between the fragments bearing the promoter of the major beta-globin gene and its distant enhancers and showed that the number of ligation products produced does not exceed 1% of all fragments subjected to the ligation. Although this low yield of 3C ligation products may be explained entirely by technical issues, it may as well reflect a low frequency of interaction between DNA regulatory elements in vivo.