Nuclear morphology is shaped by loop-extrusion programs.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

Uterine cellular leiomyomas are characterized by common HMGA2 aberrations, followed by chromosome 1p deletion and MED12 mutation: morphological, molecular, and immunohistochemical study of 52 cases.

Cellular leiomyoma (CL) represents an uncommon variant of uterine leiomyoma with limited data concerning its immunohistochemical and molecular profile. We performed a comprehensive analysis of 52 CL cases all of which were analyzed immunohistochemically. Molecular analysis was possible in 32 cases with sufficient DNA, and 38 cases with sufficient RNA. The immunohistochemical results showed a high expression of smooth muscle markers (calponin (100%), desmin (100%), smooth muscle actin (98.1%), caldesmon (96.1%), transgelin (96.1%), smooth muscle myosin heavy chain (86.5%), and smoothelin (61.5%)). Concerning markers of endometrial stromal differentiation, the expression of CD10 was observed in 65.4% cases (42.2% with H-score > 50), and IFITM1 in 36.5% cases (1.9% with H-score > 50). 36.5% showed HMGA2 overexpression at the IHC level, associated with increased mRNA expression in 14/14 cases. The rearrangement of the HMGA2 gene was detected in 13.2%. Chromosome 1p deletion was found in 19.3%, while 9.4% of tumors showed a pathogenic mutation in the MED12 gene. In conclusion, CL is immunohistochemically characterized by a high expression of "smooth muscle" markers commonly associated with a co-expression of "endometrial stromal" markers, where IFITM1 shows superior performance compared to CD10 regarding its specificity for differentiation from endometrial stromal tumors. The sensitivity of smoothelin in CL seems rather low, but no data is available to assess its specificity. On a molecular level, the most common mutually exclusive aberration in CL affects HMGA2, followed by chromosome 1p deletions and MED12 mutations.

Prognostic importance of expression of mini-chromosome maintenance proteins (MCMs) in patients with nasopharyngeal cancer treated with curative radiotherapy.

OBJECTIVE: The prognostic importance of minichromosome maintenance complex expression in nasopharyngeal cancer is still unknown. We aimed to find whether minichromosome maintenance complex 2-7 expression may potentially be used to predict the prognosis of nasopharyngeal cancer patients treated with definitive radiotherapy. METHODS: Between April 2007 and July 2020, patients with nasopharyngeal cancer treated with radiotherapy were identified. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissues of cases. A single pathologist analyzed the histologic specimens of all patients. RESULTS: Totally, 67 patients were included. The median followup was 75.3 months. Higher tumor (T) stage was correlated with minichromosome maintenance complex 2 overexpression. Minichromosome maintenance complex s expression was also associated with histopathologic subgroups. According to univariate analysis, AJCC stage, histopathological subgroups, tumor response after treatment, minichromosome maintenance complex 2, 3, 5, 6 and 7 expression were the prognostic factors that predict overall survival. According to multivariate analysis minichromosome maintenance complex 7 expression was the only prognostic marker for both progression-free survival and overall survival. CONCLUSION: The overexpression of minichromosome maintenance complex 2, 3, 5, 6 and 7 indicated bad prognosis. Minichromosome maintenance complex 7 was an independent prognostic factor for survival outcomes in nasopharyngeal cancer and may be a potential therapeutic target for treatment.

Chromosome Folding Promotes Intrachromosomal Aberrations under Radiation- and Nuclease-Induced DNA Breakage.

The long-standing question in radiation and cancer biology is how principles of chromosome organization impact the formation of chromosomal aberrations (CAs). To address this issue, we developed a physical modeling approach and analyzed high-throughput genomic data from chromosome conformation capture (Hi-C) and translocation sequencing (HTGTS) methods. Combining modeling of chromosome structure and of chromosomal aberrations induced by ionizing radiation (IR) and nuclease we made predictions which quantitatively correlated with key experimental findings in mouse chromosomes: chromosome contact maps, high frequency of cis-translocation breakpoints far outside of the site of nuclease-induced DNA double-strand breaks (DSBs), the distinct shape of breakpoint distribution in chromosomes with different 3D organizations. These correlations support the heteropolymer globule principle of chromosome organization in G1-arrested pro-B mouse cells. The joint analysis of Hi-C, HTGTS and physical modeling data offers mechanistic insight into how chromosome structure heterogeneity, globular folding and lesion dynamics drive IR-recurrent CAs. The results provide the biophysical and computational basis for the analysis of chromosome aberration landscape under IR and nuclease-induced DSBs.

A Brownian ratchet model for DNA loop extrusion by the cohesin complex.

The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively, cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities. ATP and DNA binding promote cohesin conformational changes that guide DNA through a kleisin N-gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesin's heads and hinge, are now juxtaposed. Gripping state disassembly, following ATP hydrolysis, triggers unidirectional hinge module movement, which completes topological DNA entry by directing DNA through the ATPase head gate. If head gate passage fails, hinge module motion creates a Brownian ratchet that, instead, drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian motion has important implications for chromosomal cohesin function.

When a cell divides, it has to ensure that each of its daughter cells inherits one copy of its genetic information. It does this by duplicating its chromosomes (the DNA molecules that encode the genome) and distributing one copy of each to its daughter cells. Once a cell duplicates a chromosome, the two identical chromosomes must be held together until the cell is ready to divide in two. A ring-shaped protein complex called cohesin does this by encircling the two chromosomes. Cohesin embraces both chromosome copies, as they emerge from the DNA replicating machinery. The complex is formed of several proteins that bind to a small molecule called ATP, whose arrival and subsequent breakdown release energy. Cohesin also interacts with DNA in a different way: it can create loops of chromatin (the complex formed by DNA and its packaging proteins) that help regulate the activity of genes. Experiments performed on single molecules isolated in the laboratory show that cohesin can form a small loop of DNA that is then enlarged through a process called DNA loop extrusion. However, it is not known whether loop extrusion occurs in the cell. Although both of cohesin's roles have to do with how DNA is organised in the cell, it remains unclear how a single protein complex can engage in two such different activities. To answer this question, Higashi et al. used a structure of cohesin from yeast cells gripping onto DNA to build a model that simulates how the complex interacts with chromosomes and chromatin. This model suggested that when ATP is broken down, the cohesin structure shifts and DNA enters the ring, allowing DNA to be entrapped and chromosomes to be bound together. However, a small change in how DNA is gripped initially could prevent it from entering the ring, creating a ratchet mechanism that forms and enlarges a DNA loop. This molecular model helps explain how cohesin can either encircle DNA or create loops. However, Higashi et al.'s findings also raise the question of whether loop extrusion is possible inside cells, where DNA is densely packed and bound to proteins which could be obstacles to loop extrusion. Further research to engineer cohesin that can only perform one of these roles would help to clarify their individual contributions in the cell.

Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes.

Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Long noncoding RNA IRL regulates NF-κB-mediated immune responses through suppression of miR-27c-3p-dependent IRAK4 downregulation in teleost fish.

Growing pieces of evidence show that the long noncoding RNAs (lncRNAs) as new regulators participate in the regulation of various physiological and pathological processes. The study of lncRNA in lower invertebrates is still unclear compared with that in mammals. Here, we identified a novel lncRNA, termed IRAK4-related lncRNA (IRL), as a key regulator for innate immunity in teleost fish. We find that miR-27c-3p inhibits IRAK4 expression and thus weakens the NF-κB-mediated signaling pathway. Furthermore, the Gram-negative bacterium Vibrio anguillarum and lipopolysaccharide significantly upregulated host lncRNA IRL expression. Results indicate that IRL functions as a competing endogenous RNA for miR-27c-3p to regulate protein abundance of IRAK4; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA IRL can competitively adsorb miRNA to regulate the miR-27c-3p/IRAK4 axis that is widespread in teleost fish.

Genome organization: Tag it, move it, place it.

Chromosomes are selectively organized within the nuclei of interphase cells reflecting the current fate of each cell and are reorganized in response to various physiological cues to maintain homeostasis. Although substantial progress is being made to establish the various patterns of genome architecture, less is understood on how chromosome folding/positioning is achieved. Here, we discuss recent insights into the cellular mechanisms dictating chromatin movements including the use of epigenetic modifications and allosterically regulated transcription factors, as well as a nucleoskeleton system comprised of actin, myosin, and actin-binding proteins. Together, these nuclear factors help coordinate the positioning of both general and cell-specific genomic architectural features.

Inferring chromosome radial organization from Hi-C data.

BACKGROUND: The nonrandom radial organization of eukaryotic chromosome territories (CTs) inside the nucleus plays an important role in nuclear functional compartmentalization. Increasingly, chromosome conformation capture (Hi-C) based approaches are being used to characterize the genome structure of many cell types and conditions. Computational methods to extract 3D arrangements of CTs from this type of pairwise contact data will thus increase our ability to analyze CT organization in a wider variety of biological situations. RESULTS: A number of full-scale polymer models have successfully reconstructed the 3D structure of chromosome territories from Hi-C. To supplement such methods, we explore alternative, direct, and less computationally intensive approaches to capture radial CT organization from Hi-C data. We show that we can infer relative chromosome ordering using PCA on a thresholded inter-chromosomal contact matrix. We simulate an ensemble of possible CT arrangements using a force-directed network layout algorithm and propose an approach to integrate additional chromosome properties into our predictions. Our CT radial organization predictions have a high correlation with microscopy imaging data for various cell nucleus geometries (lymphoblastoid, skin fibroblast, and breast epithelial cells), and we can capture previously documented changes in senescent and progeria cells. CONCLUSIONS: Our analysis approaches provide rapid and modular approaches to screen for alterations in CT organization across widely available Hi-C data. We demonstrate which stages of the approach can extract meaningful information, and also describe limitations of pairwise contacts alone to predict absolute 3D positions.

Statistical mechanics of chromosomes: in vivo and in silico approaches reveal high-level organization and structure arise exclusively through mechanical feedback between loop extruders and chromatin substrate properties.

The revolution in understanding higher order chromosome dynamics and organization derives from treating the chromosome as a chain polymer and adapting appropriate polymer-based physical principles. Using basic principles, such as entropic fluctuations and timescales of relaxation of Rouse polymer chains, one can recapitulate the dominant features of chromatin motion observed in vivo. An emerging challenge is to relate the mechanical properties of chromatin to more nuanced organizational principles such as ubiquitous DNA loops. Toward this goal, we introduce a real-time numerical simulation model of a long chain polymer in the presence of histones and condensin, encoding physical principles of chromosome dynamics with coupled histone and condensin sources of transient loop generation. An exact experimental correlate of the model was obtained through analysis of a model-matching fluorescently labeled circular chromosome in live yeast cells. We show that experimentally observed chromosome compaction and variance in compaction are reproduced only with tandem interactions between histone and condensin, not from either individually. The hierarchical loop structures that emerge upon incorporation of histone and condensin activities significantly impact the dynamic and structural properties of chromatin. Moreover, simulations reveal that tandem condensin-histone activity is responsible for higher order chromosomal structures, including recently observed Z-loops.

DNA-loop extruding condensin complexes can traverse one another.

Condensin, a key component of the structure maintenance of chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA1. It remains unclear, however, how condensin complexes work together to collectively package DNA into chromosomes. Here we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these motor proteins, which, individually, extrude DNA in one direction only are able to dynamically change each other's DNA loop sizes, even when far apart. When they are in close proximity, condensin complexes are able to traverse each other and form a loop structure, which we term a Z-loop-three double-stranded DNA helices aligned in parallel with one condensin at each edge. Z-loops can fill gaps left by single loops and can form symmetric dimer motors that pull in DNA from both sides. These findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.

Hyperosmotic stress: in situ chromatin phase separation.

Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse ("congelation"). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a "global" structural level (µm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an "intermediate" level (sub-µm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 µm, essentially unchanged by hyperosmotic stress. At a "local" structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

Increased Fetal Chromosome Detection with the Use of Operative Hysteroscopy During Evacuation of Products of Conception for Diagnosed Miscarriage.

STUDY OBJECTIVE: To determine whether incorporation of operative hysteroscopy with biopsy of products of conception, in conjunction with a suction curettage for a first trimester missed abortion, affected the rate of maternal cell contamination when chromosomal analysis was performed on the products of conception, and to determine the rates of retained products of conception with incorporation of hysteroscopy after suction curettage. DESIGN: Retrospective chart study. SETTING: Private, minimally invasive surgery and infertility practice with academic-community hospital affiliation. PATIENTS: Infertility patients undergoing evacuation of products of conception for documented first trimester miscarriages between 2006 and 2017. INTERVENTIONS: Suction curettage or hysteroscopic biopsy and suction curettage, followed by chromosomal analysis of products of conception for determination of fetal genetics. MEASUREMENTS AND RESULTS: A total of 264 charts were analyzed. Patients were categorized into 2 groups based on surgical collection of products of conception: group 1 (N = 174), suction curettage only, and group 2 (N = 90), a single procedure consisting of operative hysteroscopy with biopsy of products of conception followed by suction curettage and then diagnostic hysteroscopy to look for retained products. Data for chromosome detection and retained products of conception were available for 246 and 239 patients, respectively. No significant differences were detected between the groups for age, body mass index, ethnicity, gravida, parity, primary infertility, secondary infertility, spontaneous conception, single or multiple gestation, and surgical complications. Fetal chromosome detection was significantly higher without maternal contamination in group 2 (88.5%) compared with group 1 (64.8%) (p < .001). There was no significant between-group difference in postoperative retained products of conception. CONCLUSION: Obtaining fetal genetics can be useful when planning for a future successful pregnancy. The addition of operative hysteroscopy to biopsy the gestational sac, chorionic villi, and/or fetus significantly decreases the risk of maternal contamination and increases the ability to detect fetal chromosomes for genetic analysis without an increased risk of surgical complications. Despite the low risk of surgical complications, immediate second-look hysteroscopy after the completion of suction evacuation does not reduce the risk of postoperative retained products of conception.

Kettin, the large actin-binding protein with multiple immunoglobulin domains, is essential for sarcomeric actin assembly and larval development in Caenorhabditis elegans.

Among many essential genes in the nematode Caenorhabditis elegans, let-330 is located on the left arm of chromosome V and was identified as the largest target of a mutagen in this region. However, let-330 gene has not been characterized at the molecular level. Here, we report that two sequenced let-330 alleles are nonsense mutations of ketn-1, a previously characterized gene encoding kettin. Kettin is a large actin-binding protein of 472 kDa with 31 immunoglobulin domains and is expressed in muscle cells in C. elegans. let-330/ketn-1 mutants are homozygous lethal at the first larval stage with mild defects in body elongation. These mutants have severe defects in sarcomeric actin and myosin assembly in striated muscle. However, α-actinin and vinculin, which are components of the dense bodies anchoring actin to the membranes, were not significantly disorganized by let-330/ketn-1 mutation. Kettin localizes to embryonic myofibrils before α-actinin is expressed, and α-actinin deficiency does not affect kettin localization in larval muscle. Depletion of vinculin minimally affects kettin localization but significantly reduces colocalization of actin with kettin in embryonic muscle cells. These results indicate that kettin is an essential protein for sarcomeric assembly of actin filaments in muscle cells.

Organization of Chromosomal DNA by SMC Complexes.

Structural maintenance of chromosomes (SMC) complexes are key organizers of chromosome architecture in all kingdoms of life. Despite seemingly divergent functions, such as chromosome segregation, chromosome maintenance, sister chromatid cohesion, and mitotic chromosome compaction, it appears that these complexes function via highly conserved mechanisms and that they represent a novel class of DNA translocases.

Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF.

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk-, with the complexes failing to associate with chromosomes.

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.

InTAD: chromosome conformation guided analysis of enhancer target genes.

BACKGROUND: High-throughput technologies for analyzing chromosome conformation at a genome scale have revealed that chromatin is organized in topologically associated domains (TADs). While TADs are relatively stable across cell types, intra-TAD activities are cell type specific. Epigenetic profiling of different tissues and cell-types has identified a large number of non-coding epigenetic regulatory elements ('enhancers') that can be located far away from coding genes. Linear proximity is a commonly chosen criterion for associating enhancers with their potential target genes. While enhancers frequently regulate the closest gene, unambiguous identification of enhancer regulated genes remains to be a challenge in the absence of sample matched chromosome conformation data. RESULTS: To associate enhancers with their target genes, we have previously developed and applied a method that tests for significant correlations between enhancer and gene expressions across a cohort of samples. To limit the number of tests, we constrain this analysis to gene-enhancer pairs embedded in the same TAD, where information on TAD boundaries is borrowed from publicly available chromosome conformation capturing ('Hi-C') data. We have now implemented this method as an R Bioconductor package 'InTAD' and verified the software package by reanalyzing available enhancer and gene expression data derived from ependymoma brain tumors. CONCLUSION: The open-source package InTAD is an easy-to-use software tool for identifying proximal and distal enhancer target genes by leveraging information on correlated expression of enhancers and genes that are located in the same TAD. InTAD can be applied to any heterogeneous cohort of samples analyzed by a combination of gene expression and epigenetic profiling techniques and integrates either public or custom information of TAD boundaries.