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In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked—that is, phased—is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as ‘linked-reads’). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, andPKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).

Metastasis, the development of secondary malignant growths at a distance from a primary tumor, is the cause of death for 90% of cancer patients, but little is known about how metastatic cancer cells adapt to and colonize new tissue environments. Here, using clinical samples, patient-derived xenograft (PDX) samples, PDX cells, and primary/metastatic cell lines, we discovered that liver metastatic colorectal cancer (CRC) cells lose their colon-specific gene transcription program yet gain a liver-specific gene transcription program. We showed that this transcription reprogramming is driven by a reshaped epigenetic landscape of both typical enhancers and super-enhancers. Further, we identified that the liver-specific transcription factors FOXA2 and HNF1A can bind to the gained enhancers and activate the liver-specific gene transcription, thereby driving CRC liver metastasis. Importantly, similar transcription reprogramming can be observed in multiple cancer types. Our data suggest that reprogrammed tissue-specific transcription promotes metastasis and should be targeted therapeutically.

Human induced pluripotent and embryonic stem cell cultures (hiPSC/hESC) are phenotypically heterogeneous and prone to clonal deviations during subculturing and differentiation. Clonal deviations often emerge unnoticed, but they can change the biology of the cell culture with a negative impact on experimental reproducibility. Here, we describe a computational workflow to profile the bulk clonal composition in a hiPSC/hESC culture that can also be used to infer clonal deviations. This workflow processes data obtained with two versions of the same method. The two versions-epigenetic and transcriptomic-rely on a mechanism of stochastic H3K4me3 deposition during hiPSC/hESC derivation. This mechanism generates a signature of ten or more H3K4me3-enriched clustered protocadherin (PCDH) promoters distinct in every single cell. The aggregate of single-cell signatures provides an identificatory feature in every hiPSC/hESC line. This feature is stably transmitted to the cell progeny of the culture even after differentiation unless there is a clonal deviation event that changes the internal balance of single-cell signatures. H3K4me3 signatures can be profiled by chromatin immunoprecipitation and next-generation sequencing (ChIP-seq). Alternatively, an equivalent PCDH-expression version can be profiled by RNA-seq in PCDH-expressing hiPSC/hESC-derived cells (such as neurons, astrocytes, and cardiomyocytes; and, in long-term cultures, such as cerebral organoids). Notably, our workflow can also distinguish genetically identical hiPSC/hESC lines derived from the same patient or generated in the same editing process. Together, we propose a method to improve data sharing and reproducibility in the hiPSC and hESC fields.

MEF2C has been shown to be a critical transcription factor for neurodevelopment, whose loss-of-function mutation in humans results in MEF2C haploinsufficiency syndrome (MHS), a severe form of autism spectrum disorder (ASD)/intellectual disability (ID). Here, we use patient hiPSC-derived cerebrocortical neurons and cerebral organoids to characterize MHS deficits. Unexpectedly, we found an aberrant micro-RNA-mediated gliogenesis pathway that contributes to decreased neurogenesis. We also demonstrate network-level hyperexcitability in neurons, as evidenced by excessive synaptic and extrasynaptic activity contributing to excitatory/inhibitory (E/I) imbalance. Notably, the extrasynaptic NMDA receptor antagonist, NitroSynapsin, corrects this aberrant electrical activity associated with abnormal phenotypes. During neurodevelopment, MEF2C regulates many ASD-associated gene networks suggesting that our approach may lead to personalized therapy for multiple forms of ASD.One sentence summaryAutism-like MEF2C+/- patient hiPSC models show miRNA-mediated overproduction of astrocytes and hyperactivity of neurons.

Summary: Cisplatin is an antineoplastic drug administered at suboptimal and intermittent doses to avoid life-threatening effects. Although this regimen shortly improves symptoms in the short term, it also leads to more malignant disease in the long term. We describe a multilayered analysis ranging from chromatin to translation—integrating chromatin immunoprecipitation sequencing (ChIP-seq), global run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and ribosome profiling—to understand how cisplatin confers (pre)malignant features by using a well-established ovarian cancer model of cisplatin exposure. This approach allows us to segregate the human transcriptome into gene modules representing distinct regulatory principles and to characterize that the most cisplatin-disrupted modules are associated with underlying events of super-enhancer plasticity. These events arise when cancer cells initiate without ultimately ending the program of drug-stimulated death. Using a PageRank-based algorithm, we predict super-enhancer regulator ISL1 as a driver of this plasticity and validate this prediction by using CRISPR/dCas9-KRAB inhibition (CRISPRi) and CRISPR/dCas9-VP64 activation (CRISPRa) tools. Together, we propose that cisplatin reprograms cancer cells when inducing them to undergo near-to-death experiences. : Ovarian tumors acquire new phenotypic features when exposed to anticancer therapy, which helps them endure the struggle for survival and increase malignancy. Using a six-layered multiomic approach, Ma et al. find that selective super-enhancer redistribution is a major driver of the acquisition of new phenotypic features. Keywords: multi-omic approach, gene dysregulation, super-enhancer, cisplatin, drug resistance, ovarian cancer, GRO-seq, ribosome profiling, Seurat, PageRank

In the mammalian genome, the clustered protocadherin (cPcdh) locus is a paradigm of stochastic gene expression with the potential to generate a unique cPcdh combination in every neuron. Here, we report a chromatin-based mechanism emerging during the transition from the naive to the primed states of cell pluripotency that reduces by orders of magnitude the combinatorial potential in the human cPcdh locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPcdh genes after neuronal differentiation in monolayers, months-old organoids, and engrafted cells in the rat spinal cord. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, unless there is a condition of delayed maturation such as Down Syndrome. We therefore propose that a pattern of limited cPcdh diversity is maintained while human neurons still retain fetal-like levels of maturation., Editorial SUMMARY: Short and long-term cultures of human stem cell-derived neurons reveal that a pattern of restricted selection of clustered protocadherin isoforms, pre-established in pluripotent cells, distinguishes immature from mature neurons.

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.

Anticancer drugs with narrow therapeutic window, such as cisplatin, are administrated at suboptimal and intermittent doses to limit their adverse effects in patients. This regimen may allow survival of cancer cells that problematically also gain malignancy as result of the treatment. Here, we describe a multilayered analysis ranging from chromatin to translation (based on ChIP-seq, GRO-seq, RNA-seq, and ribosome profiling) to comprehensively understand how cisplatin confers new cellular properties in a well-established ovarian cancer model of cisplatin exposure. This approach allowed us to segregate the human transcriptome into seventeen modules underlying distinct regulatory principles. The most robustly disrupted modules are primarily associated with super-enhancer reprogramming, induced after cells repeatedly initiate but fail to complete the process of drug-stimulated death (corroborated by CRISPRi/a technology after PageRank-based prediction of candidate mediators). We therefore propose that cisplatin provides survivor cells with new properties, paradoxically, when they undergo a drug-stimulated near to death experience.

Summary Genome-wide association studies (GWASs) are instrumental in identifying loci harboring common single-nucleotide variants (SNVs) that affect human traits and diseases. GWAS hits emerge in clusters, but the focus is often on the most significant hit in each trait- or disease-associated locus. The remaining hits represent SNVs in linkage disequilibrium (LD) and are considered redundant and thus frequently marginally reported or exploited. Here, we interrogate the value of integrating the full set of GWAS hits in a locus repeatedly associated with cardiac conduction traits and arrhythmia, SCN5A-SCN10A. Our analysis reveals 5 common 7-SNV haplotypes (Hap1–5) with 2 combinations associated with life-threatening arrhythmia—Brugada syndrome (the risk Hap1/1 and protective Hap2/3 genotypes). Hap1 and Hap2 share 3 SNVs; thus, this analysis suggests that assuming redundancy among clustered GWAS hits can lead to confounding disease-risk associations and supports the need to deconstruct GWAS data in the context of haplotype composition., Graphical abstract, Highlights Haplotype block analysis separates cis-regulatory regions around SCN5A and SCN10A Haplotype diversity limits to 5 common 7-SNV combinations in the enhancer region Hap1/1 and Hap2/3 are Brugada syndrome risk and protective genotypes, respectively Hap1/1 and Hap2/3 genotypes are anti-correlated with SCN10A expression, The GWAS field tends to focus on the most significant hit in each disease-associated locus, despite that its distinct association is not a proxy of functionality and it is accompanied by other significant clustered signals. Focusing on Brugada syndrome, Pinsach-Abuin et al. show that deconstructing GWAS hit clusters enhances disease-relevant annotations.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription

Mammalian genomes contain thousands of cis-regulatory elements for each transcription factor (TF), but TFs only occupy a relatively small subset referred to as cistrome. Recent studies demonstrate that a TF cistrome might differ among different organisms, tissue types and individuals. In a cell, a TF cistrome might differ among different physiological states, pathological stages and between physiological and pathological conditions. It is, therefore, remarkable how highly plastic these binding profiles are, and how massively they can be reprogrammed in rapid response to intra/extracellular variations and during cell identity transitions and evolution. Biologically, cistrome reprogramming events tend to be followed by changes in transcriptional outputs, thus serving as transformative mechanisms to synchronically alter the biology of the cell. In this review, we discuss the molecular basis of cistrome plasticity and attempt to integrate the different mechanisms and biological conditions associated with cistrome reprogramming. Emerging data suggest that, when altered, these reprogramming events might be linked to tumor development and/or progression, which is a radical conceptual change in our mechanistic understanding of cancer and, potentially, other diseases.

OBJECTIVE: Although the role of reputation in determining the relative standings in the U.S. News & World Report (USNWR) annual rankings of the top 50 hospitals has received analytical attention, the role of reputation in the best children's hospitals pediatric specialty rankings has not been quantified. Our goal was to quantify the role of reputation in determining the relative standings of the top-ranked pediatric specialties and their associated hospitals in the 2008–2010 editions of the USNWR best children's hospital rankings. METHODS: A cross-sectional study of USNWR data collected from the top 30 hospitals in each of 6 (and later 10) specialties was performed. The main outcome measures were rankings based on total USNWR scores and subjective reputation scores. RESULTS: On average, rankings based on reputation scores alone correlated with USNWR overall rankings; correlation coefficients ranged from 0.80 to 0.98 (Spearman Correlation; mean P < .001). This relationship was consistent over all 3 survey years. CONCLUSIONS: The relative standings of the top 30 pediatric hospitals in each of 10 specialties are largely explained by the compelling correlation between subjective reputation scores and ranking scores.

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer-promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

Purpose – The development and proliferation of cellular/wireless technology has changed the competitive environment of traditional cooper based telephony. The complexity in the competitive environment coupled with advances in technology and innovation is requiring management to rethink strategy formulation and implementation. Convergence is discussed in the context of discontinuous competitive environment and possible management responses to changes.Design/methodology/approach – The findings of this paper are based on the analysis of the communications industry, a comprehensive review of trends in innovation and technology, strategic diagnosis and implication for management.Findings – The rate of change in innovation is leading to the creation of new industries and the disintegration of the industry classifications due to convergence of multiple needs previously served by different industry groupings. As such, firms have to upgrade their environmental scanning systems to detect competitive forces beyond t...

Purpose – It is widely acknowledged that the pace of change due to complexity in the competitive environment coupled with advances in technology and innovation is forcing management to rethink strategy formulation and implementation. The purpose of this paper is to discuss convergence in the context of discontinuous competitive environment and possible management responses to changes.Design/methodology/approach – The findings of this paper are based on the analysis of developments in the biotechnology environment. The disruption to pharmaceutical industry is examined from the context of need served.Findings – The rate of change in innovation is leading to the creation of new industries and the disintegration of the industry classifications due to convergence of multiple needs previously served by different industry groupings. As such, firms have to upgrade their environmental scanning systems to detect competitive forces beyond the traditional industrial competitive boundaries.Practical implications – The...

Purpose – The purpose of this paper is to examine the history and development of the automobile industry Furthermore the paper discusses convergence in the context of discontinuous competitive environment and possible management responses to changes.Design/methodology/approach – The findings of this paper are based on the analysis of the auto industry and the changes triggered by advances in related industries and socio‐economic forces.Findings – The rate of change in innovation is leading to the creation of new industries and the disintegration of the industry classifications due to convergence of multiple needs previously served by different industry groupings. As such, firms have to upgrade their environmental scanning systems to detect competitive forces beyond the industrial competitive boundaries.Practical implications – The paper provides a comprehensive review of convergence and disruptive technologies and the resulting implications for the automobile industry.Originality/value – The paper highlig...

While the transcriptional machinery has been extensively dissected at the molecular level, little is known about regulation of chromosomal organization in the three-dimensional space of the nucleus to achieve integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories requiring nuclear actin/myosin-I transport machinery, dynein light chain 1 (DLC1), and a specific subset of transcriptional coactivators and chromatin remodeling complexes. We establish a requirement for the histone lysine demethylase, LSD1, in directing specific interchromosomal interaction loci to distinct interchromatin granules, long thought to be "storage" sites for splicing machinery, and demonstrate that these three-dimensional motor-dependent interactions are required to achieve enhanced transcription of specific estrogen-receptor target genes. These findings reveal roles for the modulation of nuclear architecture in orchestrating regulated gene-expression programs in the mammalian nucleus.

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

ChIP coupled with microarray provides a powerful tool to determine in vivo binding profiling of transcription factors to deduce regulatory circuitries in mammalian cells. Aiming at improving the specificity and sensitivity of such analysis, we developed a new technology called ChIP-DSL using the DNA selection and ligation (DSL) strategy, permitting robust analysis with much reduced materials compared with standard procedures. We profiled general and sequence-specific DNA binding transcription factors using a full human genome promoter array based on the ChIP-DSL technology, revealing an unprecedented number of the estrogen receptor (ERα) target genes in MCF-7 cells. Coupled with gene expression profiling, we found that only a fraction of these direct ERα target genes were highly responsive to estrogen and that the expression of those ERα-bound, estrogen-inducible genes was associated with breast cancer progression in humans. This study demonstrates the power of the ChIP-DSL technology in revealing regulatory gene expression programs that have been previously invisible in the human genome.

SummaryNuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

The proto-oncogene c-Myc plays a central role in cell growth and the development of human tumors. c-Myc interacts with Max and Myc-Max complexes bind to E-box and related sequences to activate transcription. Max also interacts with Mnt but Mnt-Max complexes repress transcription when bound to these sequences. MNT maps to human chromosome 17p13.3, a region frequently deleted in various human tumors, including mammary gland tumors. Consistent with the possibility that Mnt functions as a Myc antagonist, Mnt-deficient fibroblasts exhibit many of the hallmark characteristics of cells that overexpress Myc, and conditional (Cre/Lox) inactivation of Mnt in mammary gland epithelium leads to adenocarcinomas. Here, we further characterize mammary gland tissue following conditional deletion of Mnt in the mammary gland. We show that loss of Mnt severely disrupts mammary gland involution and leads to hyperplastic ducts associated with reduced numbers of apoptotic cells. These findings suggest that loss of Mnt in mammary tissue has similarities to Myc overexpression. We tested this directly by using promoter array analysis and mRNA expression analysis by oligonucleotide arrays. We found that Mnt and c-Myc bound to similar promoters in tumors from MMTV-c-Myc transgenic mice, and mRNA expression patterns were similar between mammary tumors from MMTV-Cre/MntKO/CKO and MMTV-c-Myc transgenic mice. These results reveal an important role for Mnt in pregnancy-associated mammary gland development and suggest that mammary gland tumorigenesis in the absence of Mnt is analogous to that caused by Myc deregulation. (Cancer Res 2006; 66(11): 5565-73)

SummaryDefining the precise molecular strategies that coordinate patterns of transcriptional responses to specific signals is central for understanding normal development and homeostasis as well as the pathogenesis of hormone-dependent cancers. Here we report specific prostate cancer cell/macrophage interactions that mediate a switch in function of selective androgen receptor antagonists/modulators (SARMs) from repression to activation in vivo. This is based on an evolutionarily conserved receptor N-terminal L/HX7LL motif, selectively present in sex steroid receptors, that causes recruitment of TAB2 as a component of an N-CoR corepressor complex. TAB2 acts as a sensor for inflammatory signals by serving as a molecular beacon for recruitment of MEKK1, which in turn mediates dismissal of the N-CoR/HDAC complex and permits derepression of androgen and estrogen receptor target genes. Surprisingly, this conserved sensor strategy may have arisen to mediate reversal of sex steroid-dependent repression of a limited cohort of target genes in response to inflammatory signals, linking inflammatory and nuclear receptor ligand responses to essential reproductive functions.

© 2015 Macmillan Publishers Limited. All rights reserved. dDsk2 is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. Here we report that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1, a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications., This work was supported by grants from MICINN (CSD2006-49, BFU2009-07111 and BFU2012-30724) and the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204). This work was carried out within the framework of the ‘Centre de Referència en Biotecnologia’ of the ‘Generalitat de Catalunya’. R.K. acknowledges receipt of a ‘La Caixa’ PhD fellowship

An important purpose of sequencing the human genome was to determine the number of human genes and to catalog them for future study. The final gene count matched the most conservative predictions, with gene sequences representing only 1.5% of the entire human genome. However, more recent transcriptomic analyses have revealed that the cumulative coverage of transcribed regions is at least 75-85% of the human genome. Moreover, since these analyses did not investigate the process of transcription itself, but only the accumulation of RNAs, the range of cumulative transcribed genome is likely close to the full human genome. The question is how much of the human transcriptome (i.e. the transcribed genome) is functional and how much of the human transcriptome is transcriptional noise. Thus, we return to the question of how many genes (i.e. sequences coding for functional transcripts) are there in the human genome, and to the challenge of cataloging them. In this study, we catalog the full repertoire of long nonc...

In the presence of specific metal-ions (namely zinc but also cadmium, cobalt and manganese), d(GA x TC)n DNA sequences can form non-B-DNA conformations. At low metal-ion concentration they form [GA(GA x TC)] intramolecular triplexes but, upon increasing the metal concentration, the formation of (GA x GA) intramolecular hairpins is detected. In this paper we address the question of the specific effects of zinc on the structure of the d(GA x TC)n sequences. In the presence of zinc, the DMS-reactivity of the (GA x GA) hairpins is strongly reduced suggesting a direct interaction of the metal-ion with the N7-group of the guanines. This effect is specific for antiparallel-stranded d(GA)n homoduplexes. No such strong decrease in DMS-reactivity is observed in B-DNA duplexes or in d(GGA)n and d(GGGA)n homoduplexes. In addition, the thermal stability of antiparallel-stranded d(GA)n homoduplexes increases in the presence of zinc. On the contrary, the melting temperature of similar B-DNA molecules decreases upon increasing the zinc concentration. Altogether, these result indicate that zinc plays an specific role on the stabilization of the (GA x GA) intramolecular hairpins.

The epigenetic control of neuronal gene expression patterns has emerged as an underlying regulatory mechanism for neuronal function, identity, and plasticity, in which short- to long-lasting adaptation is required to dynamically respond and process external stimuli. To achieve a comprehensive understanding of the physiology and pathology of the brain, it becomes essential to understand the mechanisms that regulate the epigenome and transcriptome in neurons. Here, we review recent advances in the study of regulated neuronal gene expression, which are dramatically expanding as a result of the development of new and powerful contemporary methodologies, based on next-generation sequencing. This flood of new information has already transformed our understanding of many biological processes and is now driving discoveries elucidating the molecular mechanisms of brain function in cognition, behavior, and disease and may also inform the study of neuronal identity, diversity, and neuronal reprogramming.

While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1-S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.

Although the role of liganded nuclear receptors in mediating coactivator/corepressor exchange is well-established, little is known about the potential regulation of chromosomal organization in the 3-dimensional space of the nucleus in achieving integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among specific subsets of estrogen receptor α-bound transcription units, with a dramatic reorganization of nuclear territories, which depends on the actions of nuclear actin/myosin-I machinery and dynein light chain 1. The histone lysine demethylase, LSD1, is required for these ligand-induced interactive loci to associate with distinct interchromatin granules, long thought to serve as “storage” sites for the splicing machinery, some critical transcription elongation factors, and various chromatin remodeling complexes. We demonstrate that this 2-step nuclear rearrangement is essential for achieving enhanced, coordinated transcription of nuclear receptor target genes.

While the transcriptional machinery has been extensively dissected at the molecular level, little is known about regulation of chromosomal organization in the three-dimensional space of the nucleus to achieve integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories requiring nuclear actin/myosin-I transport machinery, dynein light chain 1 (DLC1), and a specific subset of transcriptional coactivators and chromatin remodeling complexes. We establish a requirement for the histone lysine demethylase, LSD1, in directing specific interchromosomal interaction loci to distinct interchromatin granules, long thought to be "storage" sites for splicing machinery, and demonstrate that these three-dimensional motor-dependent interactions are required to achieve enhanced transcription of specific estrogen-receptor target genes. These findings reveal roles for the modulation of nuclear architecture in orchestrating regulated gene-expression programs in the mammalian nucleus.

SAP18 is a highly conserved protein that was proposed to be involved in multiple cellular processes from autophagy to gene regulation and mRNA processing. In this paper we show that, in Drosophila, dSAP18 is a predominantly nuclear protein that associates to both chromosomes and the nuclear matrix. dSAP18 becomes nuclear early during development, at the onset of cellularization, and remains so all through embryo development. dSAP18 is also nuclear in salivary glands, ovaries and cultured S2 cells. Here we also show that dSAP18 forms a complex with the Drosophila homolog of pinin (dPnn), a protein factor involved in mRNA splicing. dSAP18-dPnn interaction was confirmed in vivo, through co-immunoprecipitation experiments, as well as in vitro, through GST pull-down assays. These results are discussed in the context of the possible functions played by SAP18. © Springer 2006., This work was financed by grants from the MCyT (BMC2002-00905, BMC2003-243), the CIRIT (2001SGR00344) and the EU (LSHB-CT- 2004-511965). EC acknowledges receipt of a doctoral fellowship from PCB. SP acknowledges receipt of a doctoral FU fellowship from MEC. This work was carried out within the framework of the FCeRBa_ of the Generalitat de Catalunya

Multiple enzymatic activities are required for transcriptional initiation. The enzyme DNA topoisomerase II associates with gene promoter regions and can generate breaks in double-stranded DNA (dsDNA). Therefore, it is of interest to know whether this enzyme is critical for regulated gene activation. We report that the signal-dependent activation of gene transcription by nuclear receptors and other classes of DNA binding transcription factors, including activating protein 1, requires DNA topoisomerase IIβ-dependent, transient, site-specific dsDNA break formation. Subsequent to the break, poly(adenosine diphosphate–ribose) polymerase–1 enzymatic activity is induced, which is required for a nucleosome-specific histone H1–high-mobility group B exchange event and for local changes of chromatin architecture. Our data mechanistically link DNA topoisomerase IIβ–dependent dsDNA breaks and the components of the DNA damage and repair machinery in regulated gene transcription.

Platinum-based antineoplastic drugs, such as cisplatin, are DNA damaging agents typically utilized in cancer treatment. In ovarian cancer, however, these drugs have a relatively low therapeutic index (which is a measure of clinical benefit over toxicity of these drugs), thus even a small change (reduction) in the cellular sensitivity to these chemotherapeutic agents leads to clinical treatment failure, also known as ‘drug resistance’. To increase the clinical efficacy of platinum-based antineoplastic drugs, we need to develop therapies that are less likely to result in drug resistance, or we need to modify the current therapies to prevent or reduce drug resistance. To gain mechanistic insights into the process of drug resistance that may help us to develop new or modified therapies that are less prone to clinical drug resistance, we have focused our studies on the comprehensive analysis of two still poorly studied cisplatin-associated processes at genome-wide scale: the processes of RNA nascent transcription and RNA translation. In particular, we investigate by global run-on combined with next-generation sequencing (GRO-seq) how cisplatin-sensitive A2780 ovarian cancer cells and its cisplatin-resistance derivative, A2780cis cells, respond to acute cisplatin treatment at the level of the nascent transcriptome. We also investigate, in this case by ribosome profiling, how the same cells respond to acute cisplatin treatment at the level of the ribosome-associated transcriptome (also known as translatome). These two massive analyses, complemented with conventional profiling of steady-state RNA (profiled by RNA-seq) provide the first multidimensional analysis of the actions induced by any chemotherapeutic agent at the level of the three basic states of the transcriptome (nascent RNA, steady-state RNA, and translated RNA). Interestingly, this analysis reveals an unexpected large fraction of uncoupled events between these three transcriptomic states, which challenges the conventional one-dimensional approach based exclusively on RNA-seq for the study of drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Carlos Mackintosh, Sergiy Konovalov, Ivan Garcia-Bassets. Dissecting the cellular response to cisplatin from RNA transcription to translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2358. doi:10.1158/1538-7445.AM2014-2358

Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences.

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses., Author Summary The innate immune response is a complex biological program that is configured to allow host cells to rapidly respond to infection and tissue injury. An essential feature of this response is the sequential activation of large numbers of genes that play roles in amplification of the initial inflammatory response, exert anti-microbial activities, and initiate acquired immunity. Here, we use a combination of genome-wide approaches to characterize the basal and activated states of promoters that drive the expression of genes that are turned on at immediate/early or late times in macrophages following their stimulation with a mimetic of bacterial infection. These studies identify genetically encoded features that establish basal levels of expression and distinct temporal profiles of signal-dependent gene activation required for effective immune responses. The general features of immediate/early and late genes defined by these studies are likely to be instructive for understanding how other high-magnitude, temporally orchestrated programs of gene expression are established.

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell type-specific gene expression programs; however, the potential that there are pre-established enhancers that permit alternative signal-dependent transcriptional responses has remained unexplored. Here we present evidence that cell lineage-specific factors, such as FoxA1, which contribute to the activation of cell-type specific enhancers by selectively favoring the recruitment of key regulated transcription factors such as the androgen receptor (AR) in prostate cancer cells, can simultaneously restrict these same factors from activating additional (alternative) pre-established but yet inactive enhancers, thus licensing specific signal-activated responses and restricting others. Importantly, these alternative hormonal responses are linked to distinct structural and functional classes of enhancers. Consequently, FoxA1 down-regulation, an unfavorable prognostic sign in some sets of advanced prostate tumors, causes a massive switch in AR binding from one functional enhancer class to another, which massively reprograms the hormonal response. The molecular basis for this switch lies in the release of FoxA1-mediated “restriction” of AR binding to the new enhancer class that requires no apparent nucleosome remodeling for their activation. Multiple global analyses of enhancer formation, structure, looping, and activation were combined to support these conclusions, including: H3K4me1/me3, H3K27ac, and H4K4ac mapping (ChIP-seq); nucleosomal H3K4me2 mapping (nucleosomal ChIP-seq); FoxA1, AR, p300, and MED12 mapping (ChIP-seq), also supported by the 3C assay; enhancer ncRNA or eRNAs generation analysis (GRO-seq); and gene expression analysis (microarray and GRO-seq). Together, these findings reveal a large repository of pre-determined enhancers in the human genome that can be dynamically tuned to induce alternative gene expression programs, which -we speculate- may underlie many sequential gene expression events in development or during cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 930. doi:10.1158/1538-7445.AM2011-930