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Background: The TGx-DDI biomarker identifies transcripts specifically induced by primary DNA damage. Profiling similarity of TGx-DDI signatures can allow clustering compounds by genotoxic mechanism. This transcriptomics-based approach complements conventional toxicology testing by enhancing mechanistic resolution., Methods: Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding (tSNE) were utilized to assess similarity of publicly-available per- and polyfluoroalkyl substances (PFAS) and ToxCast chemicals based on TGx-DDI modulation. TempO-seq transcriptomic data after highest chemical concentrations were analyzed., Results: Clustering discriminated between genotoxic and non-genotoxic compounds while drawing similarity among chemicals with shared mechanisms. PFAS largely clustered distinctly from classical mutagens. However, dynamic range across PFAS types and durations indicated variable potential for DNA damage. tSNE visualization reinforced phenotypic groupings, with genotoxins clustering separately from non-DNA damaging agents., Discussion: Unsupervised learning approaches applied to TGx-DDI profiles effectively categorizes chemical genotoxicity potential, aiding elucidation of biological response pathways. This transcriptomics-based strategy gives further insight into the role and effect of individual TGx-DDI biomarker genes and complements existing assays by enhancing mechanistic resolution. Overall, TGx-DDI biomarker profiling holds promise for predictive safety screening.

To identify pathway perturbations and examine biological modes of action (MOAs) for various perfluoroalkyl substances, we re-analyzed published in vitro gene expression studies from human primary liver spheroids. With treatment times ranging from 10 to 14 days, shorter-chain PFAS (those with 6 or fewer fluorinated carbon atoms in the alkyl chain) showed enrichment for pathways of fatty acid metabolism and fatty acid beta-oxidation with upregulated genes. Longer-chain PFAS compounds, specifically PFOS (perfluorooctane sulfonate), PFDS (perfluorodecane sulfonate), and higher doses of PFOA (perfluorooctanoic acid), had enrichment for pathways involved in steroid metabolism, fatty acid metabolism, and biological oxidation for downregulated genes. Although PFNA (perfluorononanoic acid), PFDA (perfluorodecanoic acid), and PFUnDA (perfluoroundecanoic acid) were more toxic and could only be examined after a 1-day treatment, all three had enrichment patterns similar to those observed with PFOS. With PFOA there were dose-dependent changes in pathway enrichment, shifting from upregulation of fatty acid metabolism and downregulation of steroid metabolism to downregulation of both at higher doses. The response to PFHpS (perfluoroheptanesulfonic acid) was similar to the PFOA pattern at the lower treatment dose. Based on results of transcription factor binding sites analyses, we propose that downregulation of pathways of lipid metabolism by longer chain PFAS may be due to inhibitory interactions of PPARD on genes controlled by PPARA and PPARG. In conclusion, our transcriptomic analysis indicates that the biological MOAs of PFAS compounds differ according to chain length and dose, and that risk assessments for PFAS should consider these differences in biological MOAs when evaluating mixtures of these compounds., Competing Interests: Declaration of competing interest The authors are employed by ScitoVation LLC., (Copyright © 2023. Published by Elsevier Inc.)

Single, high doses of TCDD in rats are known to cause wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD - 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, ≤ 22 ng/kg/day in the CL and ≤ 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and high-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs which showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shift circadian processes away from the resting state, leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth, and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes., Competing Interests: Declaration of competing interest During the course of this study, A.R.B, M.B.B. and M.E.A. were employed by ScitoVation LLC. J.A.H. is employed by the EPA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Toxicological risk assessment increasingly utilizes transcriptomics to derive point of departure (POD) and modes of action (MOA) for chemicals. One essential biological process that allows a single gene to generate several different RNA isoforms is called alternative splicing. To comprehensively assess the role of splicing dysregulation in toxicological evaluation and elucidate its potential as a complementary endpoint, we performed RNA-seq on A549 cells treated with five oxidative stress modulators across a wide dose range. Differential gene expression (DGE) showed limited pathway enrichment except at high concentrations. However, alternative splicing analysis revealed variable intron retention events affecting diverse pathways for all chemicals in the absence of significant expression changes. For instance, diazinon elicited negligible gene expression changes but progressive increase in the number of intron retention events, suggesting splicing alterations precede expression responses. Benchmark dose modeling of intron retention data highlighted relevant pathways overlooked by expression analysis. Systematic integration of splicing datasets should be a useful addition to the toxicogenomic toolkit. Combining both modalities paint a more complete picture of transcriptomic dose-responses. Overall, evaluating intron retention dynamics afforded by toxicogenomics may provide biomarkers that can enhance chemical risk assessment and regulatory decision making. This work highlights splicing-aware toxicogenomics as a possible additional tool for examining cellular responses., Competing Interests: Authors ARB, MB, RS, SS, PM, and AN were employed by the ScitoVation LLC., (Copyright © 2024 Barutcu, Black, Samuel, Slattery, McMullen and Nong.)

Introduction: While targeted investigation of key toxicity pathways has been instrumental for biomarker discovery, unbiased and holistic analysis of transcriptomic data provides a complementary systems-level perspective. However, in a systematic context, this approach has yet to receive comprehensive and methodical implementation. Methods: Here, we took an integrated bioinformatic approach by re-analyzing publicly available MCF7 cell TempO-seq data for 44 ToxCast chemicals using an alternative pipeline to demonstrate the power of this approach. The original study has focused on analyzing the gene signature approach and comparing them to in vitro biological pathway altering concentrations determined from ToxCast HTS assays. Our workflow, in comparison, involves sequential differential expression, gene set enrichment, benchmark dose modeling, and identification of commonly perturbed pathways by network visualization. Results: Using this approach, we identified dose-responsive molecular changes, biological pathways, and points of departure in an untargeted manner. Critically, benchmark dose modeling based on pathways recapitulated points of departure for apical endpoints, while also revealing additional perturbed mechanisms missed by single endpoint analyses. Discussion: This systems-toxicology approach provides multifaceted insights into the complex effects of chemical exposures. Our work highlights the importance of unbiased data-driven techniques, alongside targeted methods, for comprehensively evaluating molecular initiating events, dose-response relationships, and toxicity pathways. Overall, integrating omics assays with robust bioinformatics holds promise for improving chemical risk assessment and advancing new approach methodologies (NAMs)., Competing Interests: Authors AB, MB, and AN were employed by ScitoVation LLC., (Copyright © 2023 Barutcu, Black and Nong.)

New Approach Methodologies (NAMs) are being widely used to reduce, refine, and replace, animal use in studying toxicology. For respiratory toxicology, this includes both in silico and in vitro alternatives to replace traditional in vivo inhalation studies. 1,3-Dichloropropene (1,3-DCP) is a volatile organic compound that is widely used in agriculture as a pre-planting fumigant. Short-term exposure of humans to 1,3-DCP can result in mucous membrane irritation, chest pain, headache, and dizziness. In our previous work, we exposed differentiated cells representing different parts of the respiratory epithelium to 1,3-DCP vapor, measured cytotoxicity, and did In Vitro to In Vivo Extrapolation (IVIVE). We have extended our previous study with 1,3-DCP vapors by conducting transcriptomics on acutely exposed nasal cultures and have implemented a separate 5-day repeated exposure with multiple endpoints to gain further molecular insight into our model. MucilAir™ Nasal cell culture models, representing the nasal epithelium, were exposed to six sub-cytotoxic concentrations of 1,3-DCP vapor at the air-liquid interface, and the nasal cultures were analyzed by different methodologies, including histology, transcriptomics, and glutathione (GSH) -depletion assays. We observed the dose-dependent effect of 1,3-DCP in terms of differential gene expression, change in cellular morphology from pseudostratified columnar epithelium to squamous epithelium, and depletion of GSH in MucilAir™ nasal cultures. The MucilAir™ nasal cultures were also exposed to 3 concentrations of 1,3-DCP using repeated exposure 4 h per day for 5 days and the histological analyses indicated changes in cellular morphology and a decrease in ciliated bodies and an increase in apoptotic bodies, with increasing concentrations of 1,3-DCP. Altogether, our results suggest that sub-cytotoxic exposures to 1,3-DCP lead to several molecular and cellular perturbations, providing significant insight into the mode-of-action (MoA) of 1,3-DCP using an innovative NAM model., Competing Interests: Declaration of Competing Interest ScitoVation reports financial support was provided by American Chemistry Council., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: The AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells (i.e., transiently expressed) remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation., Methods: We established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivo. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions., Results: Persistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes., Conclusions: Our results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process., (© 2022. The Author(s).)

The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression., Competing Interests: Declaration of interests B.J.B. is a member of the Molecular Cell advisory board. The authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nuclear RNA and the act of transcription have been implicated in nuclear organization. However, their global contribution to shaping fundamental features of higher-order chromatin organization such as topologically associated domains (TADs) and genomic compartments remains unclear. To investigate these questions, we perform genome-wide chromatin conformation capture (Hi-C) analysis in the presence and absence of RNase before and after crosslinking, or a transcriptional inhibitor. TAD boundaries are largely unaffected by RNase treatment, although a subtle disruption of compartmental interactions is observed. In contrast, transcriptional inhibition leads to weaker TAD boundary scores. Collectively, our findings demonstrate differences in the relative contribution of RNA and transcription to the formation of TAD boundaries detected by the widely used Hi-C methodology., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Nuclei require a precise three- and four-dimensional organization of DNA to establish cell-specific gene-expression programs. Underscoring the importance of DNA topology, alterations to the nuclear architecture can perturb gene expression and result in disease states. More recently, it has become clear that not only intrachromosomal interactions, but also interchromosomal interactions, a less studied feature of chromosomes, are required for proper physiological gene-expression programs. Here, we review recent studies with emerging insights into where and why cross-chromosomal communication is relevant. Specifically, we discuss how long noncoding RNAs (lncRNAs) and three-dimensional gene positioning are involved in genome organization and how low-throughput (live-cell imaging) and high-throughput (Hi-C and SPRITE) techniques contribute to understand the fundamental properties of interchromosomal interactions., (© 2018 Maass et al.)

Background: Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors., Results: For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body., Conclusions: A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.

Chromosomes occupy distinct interphase territories in the three-dimensional nucleus. However, how these chromosome territories are arranged relative to one another is poorly understood. Here, we investigated the inter- chromosomal interactions between chromosomes 2q, 12, and 17 in human mesenchymal stem cells (MSCs) and MSC-derived cell types by DNA-FISH We compared our findings in normal karyotypes with a three-generation family harboring a 2q37-deletion syndrome, featuring a heterozygous partial deletion of histone deacetylase 4 ( HDAC4 ) on chr2q37. In normal karyotypes, we detected stable, recurring arrangements and interactions between the three chromosomal territories with a tissue-specific interaction bias at certain loci. These inter- chromosomal interactions were confirmed by Hi-C. Interestingly, the disease-related HDAC4 deletion resulted in displaced inter- chromosomal arrangements and altered interactions between the deletion-affected chromosome 2 and chromosome 12 and/or 17 in 2q37-deletion syndrome patients. Our findings provide evidence for a direct link between a structural chromosomal aberration and altered interphase architecture that results in a nuclear configuration, supporting a possible molecular pathogenesis., (© 2018 The Authors.)

The binding of the transcriptional regulator CTCF to the genome has been implicated in the formation of topologically associated domains (TADs). However, the general mechanisms of folding the genome into TADs are not fully understood. Here we test the effects of deleting a CTCF-rich locus on TAD boundary formation. Using genome-wide chromosome conformation capture (Hi-C), we focus on one TAD boundary on chromosome X harboring ~ 15 CTCF binding sites and located at the long non-coding RNA (lncRNA) locus Firre. Specifically, this TAD boundary is invariant across evolution, tissues, and temporal dynamics of X-chromosome inactivation. We demonstrate that neither the deletion of this locus nor the ectopic insertion of Firre cDNA or its ectopic expression are sufficient to alter TADs in a sex-specific or allele-specific manner. In contrast, Firre's deletion disrupts the chromatin super-loop formation of the inactive X-chromosome. Collectively, our findings suggest that apart from CTCF binding, additional mechanisms may play roles in establishing TAD boundary formation.

Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Imaging and chromatin capture techniques have provided important insights into our understanding of nuclear organization. A limitation of these techniques is the inability to resolve allele-specific spatiotemporal properties of genomic loci in living cells. Here, we describe an allele-specific CRISPR live-cell DNA imaging technique (SNP-CLING) to provide the first comprehensive insights into allelic positioning across space and time in mouse embryonic stem cells and fibroblasts. With 3D imaging, we studied alleles on different chromosomes in relation to one another and relative to nuclear substructures such as the nucleolus. We find that alleles maintain similar positions relative to each other and the nucleolus; however, loci occupy unique positions. To monitor spatiotemporal dynamics by SNP-CLING, we performed 4D imaging and determined that alleles are either stably positioned or fluctuating during cell state transitions, such as apoptosis. SNP-CLING is a universally applicable technique that enables the dissection of allele-specific spatiotemporal genome organization in live cells.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression., (© 2017 Wiley Periodicals, Inc.)

Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix-attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix-associated genome is highly cell-context dependent. J. Cell. Physiol. 232: 1295-1305, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The eukaryotic genome is partitioned into topologically associating domains (TADs). Despite recent advances characterizing TADs and TAD boundaries, the organization of these structures is an important dimension of genome architecture and function that is not well understood. Recently, we demonstrated that knockdown of BRG1, an ATPase driving the chromatin remodeling activity of mammalian SWI/SNF enzymes, globally alters long-range genomic interactions and results in a reduction of TAD boundary strength. We provided evidence suggesting that this effect may be due to BRG1 affecting nucleosome occupancy around CTCF sites present at TAD boundaries. In this review, we elaborate on our findings and speculate that BRG1 may contribute to the regulation of the structural and functional properties of chromatin at TAD boundaries by affecting the function or the recruitment of CTCF and DNA topoisomerase complexes.

RUNX1 is a transcription factor functioning both as an oncogene and a tumor suppressor in breast cancer. RUNX1 alters chromatin structure in cooperation with chromatin modifier and remodeling enzymes. In this study, we examined the relationship between RUNX1-mediated transcription and genome organization. We characterized genome-wide RUNX1 localization and performed RNA-seq and Hi-C in RUNX1-depleted and control MCF-7 breast cancer cells. RNA-seq analysis showed that RUNX1 depletion led to up-regulation of genes associated with chromatin structure and down-regulation of genes related to extracellular matrix biology, as well as NEAT1 and MALAT1 lncRNAs. Our ChIP-Seq analysis supports a prominent role for RUNX1 in transcriptional activation. About 30% of all RUNX1 binding sites were intergenic, indicating diverse roles in promoter and enhancer regulation and suggesting additional functions for RUNX1. Hi-C analysis of RUNX1-depleted cells demonstrated that overall three-dimensional genome organization is largely intact, but indicated enhanced association of RUNX1 near Topologically Associating Domain (TAD) boundaries and alterations in long-range interactions. These results suggest an architectural role for RUNX1 in fine-tuning local interactions rather than in global organization. Our results provide novel insight into RUNX1-mediated perturbations of higher-order genome organization that are functionally linked with RUNX1-dependent compromised gene expression in breast cancer cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization., (© 2016 Barutcu et al.; Published by Cold Spring Harbor Laboratory Press.)

The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signaling, cell-cycle regulation, and tumor suppression. However, despite extensive study, confusion exists regarding its mechanism of action: is activity driven by the transcript acting in trans, in cis, or by an underlying functional enhancer? Here, using a knockout mouse model and a massively parallel enhancer assay, we delineate the functional elements at this locus. We observe that, even in tissues with no detectable Linc-p21 transcript, deletion of the locus significantly affects local gene expression, including of the cell-cycle regulator Cdkn1a. To characterize this RNA-independent regulatory effect, we systematically interrogated the underlying DNA sequence for enhancer activity at nucleotide resolution and confirmed the existence of multiple enhancer elements. Together, these data suggest that, in vivo, the cis-regulatory effects mediated by Linc-p21, in the presence or absence of transcription, are due to DNA enhancer elements., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear-structure building blocks in directly supporting nuclear function in a physiologically responsive manner., (© 2015 Wiley Periodicals, Inc.)

Three-dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher-order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or "3C" technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C-based methods and how they can be used to answer distinct biological questions., (© 2015 Wiley Periodicals, Inc.)

Background: Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines., Results: Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells., Conclusions: We show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer.

Runx2 is a developmentally regulated gene in vertebrates and is essential for bone formation and skeletal homeostasis. The induction of runx2-P1 isoform transcripts is a hallmark of early osteoblastogenesis. Although previous in vitro studies have defined a minimal Runx2-P1 promoter sequence with well-characterized functional elements, several lines of evidence suggest that transcription of the Runx2-P1 isoform relies on elements that extend beyond the previously defined P1 promoter boundaries. In this study, we examined Runx2-P1 transcriptional regulation in a cellular in vivo context during early osteoblastogenesis of MC3T3-E1 cultures and BMSCs induced towards the bone lineage by multi-layered analysis of the Runx2-P1 gene promoter using the following methodologies: 1) sequence homology among several mammalian species, 2) DNaseI hypersensitivity coupled with massively parallel sequencing (DNase-seq), and 3) chromatin immunoprecipitation of activating histone modifications coupled with massively parallel sequencing (ChIP-seq). These epigenetic features have allowed the demarcation of boundaries that redefine the minimal Runx2-P1 promoter to include a 336-bp sequence that mediates responsiveness to osteoblast differentiation. We also find that an additional level of control is contributed by a regulatory region in the 5'-UTR of Runx2-P1., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Differentiation signaling results in reprogramming of cellular gene expression that leads to morphological changes and functional specialization of a precursor cell. This global change in gene expression involves temporal regulation of differentiation-specific genes that are located throughout the genome, raising the idea that genome structure may also be re-organized during cell differentiation to facilitate regulated gene expression. Using in vitro adipocyte differentiation as a model, we explored whether gene organization within the nucleus is altered upon exposure of precursor cells to signaling molecules that induce adipogenesis. The peroxisome proliferator-activated receptor gamma (PPARγ) nuclear hormone receptor is a master determinant of adipogenesis and is required for adipose differentiation. We utilized the chromosome conformation capture (3C) assay to determine whether the position of the PPARγ locus relative to other adipogenic genes is changed during differentiation. We report that the PPARγ2 promoter is transiently positioned in proximity to the promoters of genes encoding adipokines and lipid droplet associated proteins at 6 hours post-differentiation, a time that precedes expression of any of these genes. In contrast, the PPARγ2 promoter was not in proximity to the EF1α promoter, which drives expression of a constitutively active, housekeeping gene that encodes a translation elongation factor, nor was the PPARγ2 promoter in proximity to the promoter driving the expression of the C/EBPα regulatory protein. The formation of the long-range, intergenic interactions involving the PPARγ2 promoter required the regulatory factor C/EBPβ, elevated cyclic AMP (cAMP) levels, and protein kinase A (PKA) signaling. We conclude that genome organization is dynamically remodeled in response to adipogenic signaling, and we speculate that these transient inter-genic interactions may be formed for the purposes of selecting some of the transcriptionally silent tissue-specific loci for subsequent transcriptional activation.

Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2. These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Tumor-specific, coordinate expression of cancer-testis (CT) genes, mapping to the X chromosome, is observed in more than 60% of non-small cell lung cancer (NSCLC) patients. Although CT gene expression has been unequivocally related to DNA demethylation of promoter regions, the underlying mechanism leading to loss of promoter methylation remains elusive. Polymorphisms of enzymes within the 1-carbon pathway have been shown to affect S-adenosyl methionine (SAM) production, which is the sole methyl donor in the cell. Allelic variants of several enzymes within this pathway have been associated with altered SAM levels either directly, or indirectly as reflected by altered levels of SAH and Homocysteine levels, and altered levels of DNA methylation. We, therefore, asked whether the five most commonly occurring polymorphisms in four of the enzymes in the 1-carbon pathway associated with CT gene expression status in patients with NSCLC., Methods: Fifty patients among a cohort of 763 with NSCLC were selected based on CT gene expression status and typed for five polymorphisms in four genes known to affect SAM generation by allele specific q-PCR and RFLP., Results: We identified a significant association between CT gene expression and the MTHFR 677 CC genotype, as well as the C allele of the SNP, in this cohort of patients. Multivariate analysis revealed that the genotype and allele strongly associate with CT gene expression, independent of potential confounders., Conclusions: Although CT gene expression is associated with DNA demethylation, in NSCLC, our data suggests this is unlikely to be the result of decreased MTHFR function.

Self-renewal of human pluripotent embryonic stem cells proceeds via an abbreviated cell cycle with a shortened G(1) phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G(1)/S-phase transition in pluripotent cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure--hypersensitivity and histone protein modifications--between human embryonic stem (hES) and normal diploid cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5' and 3' regions of the gene. Thus, progression through the abbreviated G(1) phase involves cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem cell renewal.

Gateway-compatible yeast one-hybrid (Y1H) assays provide a convenient gene-centered (DNA to protein) approach to identify transcription factors that can bind a DNA sequence of interest. We present Y1H resources, including clones for 988 of 1,434 (69%) predicted human transcription factors, that can be used to detect both known and new interactions between human DNA regions and transcription factors.