Targeted phasing of 2-200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method.

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2-200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2-200 kb targets using a short-read sequencer.

TREX1 is required for microglial cholesterol homeostasis and oligodendrocyte terminal differentiation in human neural assembloids.

Three Prime Repair Exonuclease 1 (TREX1) gene mutations have been associated with Aicardi-Goutières Syndrome (AGS) - a rare, severe pediatric autoimmune disorder that primarily affects the brain and has a poorly understood etiology. Microglia are brain-resident macrophages indispensable for brain development and implicated in multiple neuroinflammatory diseases. However, the role of TREX1 - a DNase that cleaves cytosolic nucleic acids, preventing viral- and autoimmune-related inflammatory responses - in microglia biology remains to be elucidated. Here, we leverage a model of human embryonic stem cell (hESC)-derived engineered microglia-like cells, bulk, and single-cell transcriptomics, optical and transmission electron microscopy, and three-month-old assembloids composed of microglia and oligodendrocyte-containing organoids to interrogate TREX1 functions in human microglia. Our analyses suggest that TREX1 influences cholesterol metabolism, leading to an active microglial morphology with increased phagocytosis in the absence of TREX1. Notably, regulating cholesterol metabolism with an HMG-CoA reductase inhibitor, FDA-approved atorvastatin, rescues these microglial phenotypes. Functionally, TREX1 in microglia is necessary for the transition from gliogenic intermediate progenitors known as pre-oligodendrocyte precursor cells (pre-OPCs) to precursors of the oligodendrocyte lineage known as OPCs, impairing oligodendrogenesis in favor of astrogliogenesis in human assembloids. Together, these results suggest routes for therapeutic intervention in pathologies such as AGS based on microglia-specific molecular and cellular mechanisms.

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method.

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, and PKI3CA). 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.

Analysis of Clonal Composition in Human iPSC and ESC and Derived 2D and 3D Differentiated Cultures.

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.

Analysis of Brugada syndrome loci reveals that fine-mapping clustered GWAS hits enhances the annotation of disease-relevant variants.

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.

Enhancer release and retargeting activates disease-susceptibility genes.

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).

Super-Enhancer Redistribution as a Mechanism of Broad Gene Dysregulation in Repeatedly Drug-Treated Cancer Cells.

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.

Tissue-specific transcription reprogramming promotes liver metastasis of colorectal cancer.

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.

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells.

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and 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, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation.

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

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.

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes.

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.

Decoding a signature-based model of transcription cofactor recruitment dictated by cardinal cis-regulatory elements in proximal promoter regions.

Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between -150 bp and +50 bp (-150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the -150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed 'cardinal' motifs) prefer acting individually, rather than in fixed combinations, within the -150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription.

Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

Analysis of the levels of lysine-specific demethylase 1 (LSD1) mRNA in human ovarian tumors and the effects of chemical LSD1 inhibitors in ovarian cancer cell lines.

BACKGROUND: Lysine-specific demethylase 1 (LSD1, also known as KDM1A and AOF2) is a chromatin-modifying activity that catalyzes the removal of methyl groups from lysine residues in histone and non-histone proteins, regulating gene transcription. LSD1 is overexpressed in several cancer types, and chemical inhibition of the LSD1 activity has been proposed as a candidate cancer therapy. Here, we examine the levels of LSD1 mRNA in human ovarian tumors and the cytotoxicity of several chemical LSD1 inhibitors in a panel of ovarian cancer cell lines. METHODS: We measured LSD1 mRNA levels in a cohort of n = 177 normal and heterogeneous tumor specimens by quantitative real time-PCR (qRT-PCR). Tumors were classified by FIGO stage, FIGO grade, and histological subtypes. We tested the robustness of our analyses in an independent cohort of n = 573 serous tumor specimens (source: TCGA, based on microarray). Statistical analyses were based on Kruskal-Wallis/Dunn's and Mann Whitney tests. Changes in LSD1 mRNA levels were also correlated with transcriptomic alterations at genome-wide scale. Effects on cell viability (MTS/PMS assay) of six LSD1 inhibitors (pargyline, TCP, RN-1, S2101, CAS 927019-63-4, and CBB1007) were also evaluated in a panel of ovarian cancer cell lines (SKOV3, OVCAR3, A2780 and cisplatin-resistant A2780cis). RESULTS: We found moderate but consistent LSD1 mRNA overexpression in stage IIIC and high-grade ovarian tumors. LSD1 mRNA overexpression correlated with a transcriptomic signature of up-regulated genes involved in cell cycle and down-regulated genes involved in the immune/inflammatory response, a signature previously observed in aggressive tumors. In fact, some ovarian tumors showing high levels of LSD1 mRNA are associated with poor patient survival. Chemical LSD1 inhibition induced cytotoxicity in ovarian cancer lines, which roughly correlated with their reported LSD1 inhibitory potential (RN-1,S2101 >> pargyline,TCP). CONCLUSIONS: Our findings may suggest a role of LSD1 in the biology of some ovarian tumors. It is of special interest to find a correlation of LSD1 mRNA overexpression with a transcriptomic signature relevant to cancer. Our findings, therefore, prompt further investigation of the role of LSD1 in ovarian cancer, as well as the study of its enzymatic inhibition in animal models for potential therapeutic purposes in the context of this disease.

"Seq-ing" insights into the epigenetics of neuronal gene regulation.

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.

Cistrome plasticity and mechanisms of cistrome reprogramming.

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.

Mechanisms establishing TLR4-responsive activation states of inflammatory response genes.

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.

Role of reputation in top pediatric specialties rankings.

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.

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA.

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.

PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression.

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.