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Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5′-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5′ transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination., MCIN/AEI/10.13039/501100011033 [RYC2018-024108-I, PID2019-108577GA-I00 to J.G.B.]. Funding for open access charge: CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Promoters are consensus DNA sequences located near the transcription start sites and they play an important role in transcription initiation. Due to their importance in biological processes, the identification of promoters is significantly important for characterizing the expression of the genes. Numerous computational methods have been proposed to predict promoters. However, it is difficult for these methods to achieve satisfactory performance in multiple species. In this study, we propose a novel weighted average ensemble learning model, termed iPro-WAEL, for identifying promoters in multiple species, including Human, Mouse, E.coli, Arabidopsis, B.amyloliquefaciens, B.subtilis and R.capsulatus. Extensive benchmarking experiments illustrate that iPro-WAEL has optimal performance and is superior to the current methods in promoter prediction. The experimental results also demonstrate a satisfactory prediction ability of iPro-WAEL on cross-cell lines, promoters annotated by other methods and distinguishing between promoters and enhancers. Moreover, we identify the most important transcription factor binding site (TFBS) motif in promoter regions to facilitate the study of identifying important motifs in the promoter regions. The source code of iPro-WAEL is freely available at https://github.com/HaoWuLab-Bioinformatics/iPro-WAEL.

CMTR1 (cap methyltransferase 1) catalyses methylation of the first transcribed nucleotide of RNAPII transcripts (N1 2′-O-Me), creating part of the mammalian RNA cap structure. In addition to marking RNA as self, N1 2′-O-Me has ill-defined roles in RNA expression and translation. Here, we investigated the gene specificity of CMTR1 and its impact on RNA expression in embryonic stem cells. Using chromatin immunoprecipitation, CMTR1 was found to bind to transcription start sites (TSS) correlating with RNAPII levels, predominantly binding at histone genes and ribosomal protein (RP) genes. Repression of CMTR1 expression resulted in repression of RNAPII binding at the TSS and repression of RNA expression, particularly of histone and RP genes. In correlation with regulation of histones and RP genes, CMTR1 repression resulted in repression of translation and induction of DNA replication stress and damage. Indicating a direct role for CMTR1 in transcription, addition of recombinant CMTR1 to purified nuclei increased transcription of the histone and RP genes. CMTR1 was found to be upregulated during neural differentiation and there was an enhanced requirement for CMTR1 for gene expression and proliferation during this process. We highlight the distinct roles of the cap methyltransferases RNMT and CMTR1 in target gene expression and differentiation.

The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses., Graphical Abstract Graphical AbstractRNAPII is recruited to a promoter and forms a pre-initiation complex. Conversion of the PIC to an elongation complex involves loading multiple factors that promote elongation, modify chromatin, and process the emerging transcript. Many of these factors are associated with the heptad repeats of the CTD of Rpb1 (gray shapes bound to green line). The Spt6 core domain has conserved functions in RNAPII elongation, and we propose that the Spt6-tSH2 domain coordinates these elongation functions with the activities of the factors associated with the Rpb1 CTD by orienting the CTD and mediating interactions among the factors, the other domains of Spt6, and the catalytic engine of RNAPII.

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the ‘head-to-head’ orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5′ untranslated region (5′ UTR). In the ‘tail-to-tail’ arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3′ UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

Variable Number Tandem Repeats (VNTRs) are tandem repeat (TR) loci that vary in copy number across a population. Using our program, VNTRseek, we analyzed human whole genome sequencing datasets from 2,770 individuals in order to detect minisatellite VNTRs, i.e., those with pattern sizes ≥7 bp. We detected 35,638 VNTR loci and classified 5,676 as commonly polymorphic (i.e., with non-reference alleles occurring in >5% of the population). Commonly polymorphic VNTR loci were found to be enriched in genomic regions with regulatory function, i.e., transcription start sites and enhancers. Investigation of the commonly polymorphic VNTRs in the context of population ancestry revealed that 1,096 loci contained population-specific alleles and that those could be used to classify individuals into super-populations with near-perfect accuracy. Search for quantitative trait loci (eQTLs), among the VNTRs proximal to genes, indicated that in 187 genes expression differences correlated with VNTR genotype. We validated our predictions in several ways, including experimentally, through the identification of predicted alleles in long reads, and by comparisons showing consistency between sequencing platforms. This study is the most comprehensive analysis of minisatellite VNTRs in the human population to date.

Deregulation of microRNA (miRNA) expression plays a critical role in the transition from a physiological to a pathological state. The accurate miRNA promoter identification in multiple cell types is a fundamental endeavor towards understanding and characterizing the underlying mechanisms of both physiological as well as pathological conditions. DIANA-miRGen v4 (www.microrna.gr/mirgenv4) provides cell type specific miRNA transcription start sites (TSSs) for over 1500 miRNAs retrieved from the analysis of >1000 cap analysis of gene expression (CAGE) samples corresponding to 133 tissues, cell lines and primary cells available in FANTOM repository. MiRNA TSS locations were associated with transcription factor binding site (TFBSs) annotation, for >280 TFs, derived from analyzing the majority of ENCODE ChIP-Seq datasets. For the first time, clusters of cell types having common miRNA TSSs are characterized and provided through a user friendly interface with multiple layers of customization. DIANA-miRGen v4 significantly improves our understanding of miRNA biogenesis regulation at the transcriptional level by providing a unique integration of high-quality annotations for hundreds of cell specific miRNA promoters with experimentally derived TFBSs.

The triple-negative breast cancer (TNBC), a subtype of breast cancer which lacks of targeted therapies, exhibits a poor prognosis. It was shown recently that the PIM1 oncogene is highly related to the proliferation of TNBC cells. A quadruplex–duplex hybrid (QDH) forming sequence was recently found to exist near the transcription start site of PIM1. This structure could be an attractive target for regulation of the PIM1 gene expression and thus the treatment of TNBC. Here, we present the solution structures of two QDHs that could coexist in the human PIM1 gene. Form 1 is a three-G-tetrad-layered (3+1) G-quadruplex containing a propeller loop, a lateral loop and a stem-loop made up of three G•C Watson–Crick base pairs. On the other hand, Form 2 is an anti-parallel G-quadruplex comprising two G-tetrads and a G•C•G•C tetrad; the structure has three lateral loops with the middle stem-loop made up of two Watson-Crick G•C base pairs. These structures provide valuable information for the design of G-quadruplex-specific ligands for PIM1 transcription regulation.

The ChIP-exo assay precisely delineates protein-DNA crosslinking patterns by combining chromatin immunoprecipitation with 5′ to 3′ exonuclease digestion. Within a regulatory complex, the physical distance of a regulatory protein to DNA affects crosslinking efficiencies. Therefore, the spatial organization of a protein-DNA complex could potentially be inferred by analyzing how crosslinking signatures vary between the subunits of a regulatory complex. Here, we present a computational framework that aligns ChIP-exo crosslinking patterns from multiple proteins across a set of coordinately bound regulatory regions, and which detects and quantifies protein-DNA crosslinking events within the aligned profiles. By producing consistent measurements of protein-DNA crosslinking strengths across multiple proteins, our approach enables characterization of relative spatial organization within a regulatory complex. We demonstrate that our approach can recover aspects of regulatory complex spatial organization when applied to collections of ChIP-exo data that profile regulatory machinery at yeast ribosomal protein genes and yeast tRNA genes. We also demonstrate the ability to quantify changes in protein-DNA complex organization across conditions by applying our approach to data profiling Drosophila Pol II transcriptional components. Our results suggest that principled analyses of ChIP-exo crosslinking patterns enable inference of spatial organization within protein-DNA complexes.

The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5′ and 3′ ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.

The histone demethylase LSD1 is a key chromatin regulator that is often deregulated in cancer. Its ortholog, dLsd1 plays a crucial role in Drosophila oogenesis; however, our knowledge of dLsd1 function is insufficient to explain its role in the ovary. Here, we have performed genome-wide analysis of dLsd1 binding in the ovary, and we document that dLsd1 is preferentially associated to the transcription start site of developmental genes. We uncovered an unanticipated interplay between dLsd1 and the GATA transcription factor Serpent and we report an unexpected role for Serpent in oogenesis. Besides, our transcriptomic data show that reducing dLsd1 levels results in ectopic transposable elements (TE) expression correlated with changes in H3K4me2 and H3K9me2 at TE loci. In addition, our results suggest that dLsd1 is required for Piwi dependent TE silencing. Hence, we propose that dLsd1 plays crucial roles in establishing specific gene expression programs and in repressing transposons during oogenesis.

Transcription start sites (TSS) in eukaryotes are characterized by a nucleosome-depleted region (NDR), which appears to be flanked upstream and downstream by strongly positioned nucleosomes incorporating the histone variant H2A.Z. H2A.Z associates with both active and repressed TSS and is important for priming genes for rapid transcriptional activation. However, the determinants of H2A.Z occupancy at specific nucleosomes and its relationship to transcription initiation remain unclear. To further elucidate the specificity of H2A.Z, we determined its genomic localization at single nucleosome resolution, as well as the localization of its chromatin remodelers Swr1 and Ino80. By analyzing H2A.Z occupancy in conjunction with RNA expression data that captures promoter-derived antisense initiation, we find that H2A.Z’s bimodal incorporation on either side of the NDR is not a general feature of TSS, but is specifically a marker for bidirectional transcription, such that the upstream flanking −1 H2A.Z-containing nucleosome is more appropriately considered as a +1 H2A.Z nucleosome for antisense transcription. The localization of H2A.Z almost exclusively at the +1 nucleosome suggests that a transcription-initiation dependent process could contribute to its specific incorporation.

Virus integration into the human genome occurs frequently and represents a key driving event in human disease. Many studies have reported viral integration sites (VISs) proximal to structural or functional regions of the human genome. Here, we systematically collected and manually curated all VISs reported in the literature and publicly available data resources to construct the Viral Integration Site DataBase (VISDB, https://bioinfo.uth.edu/VISDB). Genomic information including target genes, nearby genes, nearest transcription start site, chromosome fragile sites, CpG islands, viral sequences and target sequences were integrated to annotate VISs. We further curated VIS-involved oncogenes and tumor suppressor genes, virus–host interactions involved in non-coding RNA (ncRNA), target gene and microRNA expression in five cancers, among others. Moreover, we developed tools to visualize single integration events, VIS clusters, DNA elements proximal to VISs and virus–host interactions involved in ncRNA. The current version of VISDB contains a total of 77 632 integration sites of five DNA viruses and four RNA retroviruses. VISDB is currently the only active comprehensive VIS database, which provides broad usability for the study of disease, virus related pathophysiology, virus biology, host–pathogen interactions, sequence motif discovery and pattern recognition, molecular evolution and adaption, among others.

The ‘open’ and ‘compact’ regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Oxidation of the guanine (G) heterocycle to 8-oxo-7,8-dihydroguanine (OG) in mammalian gene promoters was demonstrated to induce transcription. Potential G-quadruplex forming sequences (PQSs) in promoters have a high density of G nucleotides rendering them highly susceptible to oxidation and possible gene activation. The VEGF PQS with OG or an abasic site were synthesized at key locations in the SV40 or HSV-TK model promoters to determine the location dependency in the gene expression profile in human cells. The PQS location with respect to the transcription start site (TSS) and strand of occupancy (coding versus non-coding strand) are key parameters that determine the magnitude and direction in which gene expression changes with the chemically modified VEGF PQS. The greatest impact observed for OG or F in the PQS context in these promoters was within ∼200 bp of the TSS. Established PQSs found to occur naturally in a similar location relative to the TSS for possible oxidation-induced gene activation include c-MYC, KRAS, c-KIT, HIF-1α, PDGF-A and hTERT. The studies provide experimental constraints that were used to probe bioinformatic data regarding PQSs in the human genome for those that have the possibility to be redox switches for gene regulation.

The Mouse Genome Database (MGD; http://www.informatics.jax.org) is the community model organism genetic and genome resource for the laboratory mouse. MGD is the authoritative source for biological reference data sets related to mouse genes, gene functions, phenotypes, and mouse models of human disease. MGD is the primary outlet for official gene, allele and mouse strain nomenclature based on the guidelines set by the International Committee on Standardized Nomenclature for Mice. In this report we describe significant enhancements to MGD, including two new graphical user interfaces: (i) the Multi Genome Viewer for exploring the genomes of multiple mouse strains and (ii) the Phenotype-Gene Expression matrix which was developed in collaboration with the Gene Expression Database (GXD) and allows researchers to compare gene expression and phenotype annotations for mouse genes. Other recent improvements include enhanced efficiency of our literature curation processes and the incorporation of Transcriptional Start Site (TSS) annotations from RIKEN’s FANTOM 5 initiative.

R-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere homeostasis, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localisation was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the intergenic regions of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination, most T. brucei R-loops act in a novel role, promoting RNA Pol II movement or mRNA processing. Finally, we show there is little evidence for correlation between R-loop localisation and mapped sites of DNA replication initiation.

Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11–13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.

DYRK1A, dual-specificity tyrosine phosphorylation-regulated kinase 1A, which is linked to mental retardation and microcephaly, is a member of the CMGC group of kinases. It has both cytoplasmic and nuclear functions, however, molecular mechanisms of how DYRK1A regulates gene expression is not well understood. Here, we identify two histone acetyltransferases, p300 and CBP, as interaction partners of DYRK1A through a proteomics study. We show that overexpression of DYKR1A causes hyperphosphorylation of p300 and CBP. Using genome-wide location (ChIP-sequencing) analysis of DYRK1A, we show that most of the DYRK1A peaks co-localize with p300 and CBP, at enhancers or near the transcription start sites (TSS). Modulation of DYRK1A, by shRNA mediated reduction or transfection mediated overexpression, leads to alteration of expression of downstream located genes. We show that the knockdown of DYRK1A results in a significant loss of H3K27acetylation at these enhancers, suggesting that DYRK1A modulates the activity of p300/CBP at these enhancers. We propose that DYRK1A functions in enhancer regulation by interacting with p300/CBP and modulating their activity. Overall, DYRK1A function in the regulation of enhancer activity provides a new mechanistic understanding of DYRK1A mediated regulation of gene expression, which may help in better understanding of the roles of DYRK1A in human pathologies.

Despite recent progress on synthetic transcription factor generation in eukaryotes, there remains a need for high-activity bacterial versions of these systems. In synthetic biology applications, it is useful for transcription factors to have two key features: they should be orthogonal (influencing only their own targets, with minimal off-target effects), and programmable (able to be directed to a wide range of user-specified transcriptional start sites). The RNA polymerase of the bacteriophage T7 has a number of appealing properties for synthetic biological designs: it can produce high transcription rates; it is a compact, single-subunit polymerase that has been functionally expressed in a variety of organisms; and its viral origin reduces the connection between its activity and that of its host's transcriptional machinery. We have created a system where a T7 RNA polymerase is recruited to transcriptional start sites by DNA binding proteins, either directly or bridged through protein–protein interactions, yielding a modular and programmable system for strong transcriptional activation of multiple orthogonal synthetic transcription factor variants in Escherichia coli. To our knowledge this is the first exogenous, programmable activator system in bacteria.

Mammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in D. melanogaster. Surprisingly, we find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The different layers of regulation mediated by gene-distal enhancers and gene promoters are also reflected in chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

Canonical bacterial transcription activators bind to non-transcribed promoter elements to increase transcription of their target genes. Here we report crystal structures of binary complexes comprising domains of Caulobacter crescentus GcrA, a noncanonical bacterial transcription factor, that support a novel mechanism for transcription activation through the preferential binding of methylated cis-regulatory elements and the promotion of open complex formation through an interaction with region 2 of the principal σ factor, σ70. We present crystal structures of the C-terminal, σ factor-interacting domain (GcrA-SID) in complex with domain 2 of σ70 (σ702), and the N-terminal, DNA-binding domain (GcrA-DBD) in complex with methylated double-stranded DNA (dsDNA). The structures reveal interactions essential for transcription activation and DNA recognition by GcrA. These structures, along with mutational analyses, support a mechanism of transcription activation in which GcrA associates with RNA polymerase (RNAP) prior to promoter binding through GcrA-SID, arming RNAP with a flexible GcrA-DBD. The RNAP–GcrA complex then binds and activates target promoters harboring a methylated GcrA binding site either upstream or downstream of the transcription start site.

The cell-specific information of transcriptional regulation on microRNAs (miRNAs) is crucial to the precise understanding of gene regulations in various physiological and pathological processes existed in different tissues and cell types. The database, mirTrans, provides comprehensive information about cell-specific transcription of miRNAs including the transcriptional start sites (TSSs) of miRNAs, transcription factor (TF) to miRNA regulations and miRNA promoter sequences. mirTrans also maps the experimental H3K4me3 and DHS (DNase-I hypersensitive site) marks within miRNA promoters and expressed sequence tags (ESTs) within transcribed regions. The current version of database covers 35 259 TSSs and over 2.3 million TF-miRNA regulations for 1513 miRNAs in a total of 54 human cell lines. These cell lines span most of the biological systems, including circulatory system, digestive system and nervous system. Information for both the intragenic miRNAs and intergenic miRNAs is offered. Particularly, the quality of miRNA TSSs and TF-miRNA regulations is evaluated by literature curation. 23 447 TSS records and 2148 TF-miRNA regulations are supported by special experiments as a result of literature curation. EST coverage is also used to evaluate the accuracy of miRNA TSSs. Interface of mirTrans is friendly designed and convenient to make downloads (http://mcube.nju.edu.cn/jwang/lab/soft/mirtrans/ or http://120.27.239.192/mirtrans/).

H2A.Z histone variant is an important regulator of gene transcription, which is enriched at regulatory regions but is also found within gene bodies. Recent evidence suggests that active recruitment of H2A.Z within gene bodies is required to induce gene repression. In contrast to this view, we show that global inhibition of transcription results in H2A.Z accumulation at gene transcription start sites, as well as within gene bodies. Our results indicate that accumulation of H2A.Z within repressed genes can also be a consequence of the repression of gene transcription rather than an active mechanism required to establish the repression.

Histone variants and their chaperones are key regulators of eukaryotic transcription, and are critical for normal development. The histone variant H3.3 has been shown to play important roles in pluripotency and differentiation, and although its genome-wide patterns have been investigated, little is known about the role of its dynamic turnover in transcriptional regulation. To elucidate the role of H3.3 dynamics in embryonic stem cell (ESC) biology, we generated mouse ESC lines carrying a single copy of a doxycycline (Dox)-inducible HA-tagged version of H3.3 and monitored the rate of H3.3 incorporation by ChIP-seq at varying time points following Dox induction, before and after RA-induced differentiation. Comparing H3.3 turnover profiles in ESCs and RA-treated cells, we identified a hyperdynamic H3.3-containing nucleosome at the −1 position in promoters of genes expressed in ESCs. This dynamic nucleosome is restricted and shifted downstream into the +1 position following differentiation. We suggest that histone turnover dynamics provides an additional mechanism involved in expression regulation, and that a hyperdynamic −1 nucleosome marks promoters in ESCs. Our data provide evidence for regional regulation of H3.3 turnover in ESC promoters, and calls for testing, in high resolution, the dynamic behavior of additional histone variants and other structural chromatin proteins.

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination.

Chd1 (Chromodomain Helicase DNA Binding Protein 1) is a conserved ATP-dependent chromatin remodeler that maintains the nucleosomal structure of chromatin, but the determinants of its specificity and its impact on gene expression are not well defined. To identify the determinants of Chd1 binding specificity in the yeast genome, we investigated Chd1 occupancy in mutants of several candidate factors. We found that several components of the PAF1 transcription elongation complex contribute to Chd1 recruitment to highly transcribed genes and identified Spt4 as a factor that appears to negatively modulate Chd1 binding to chromatin. We discovered that CHD1 loss alters H3K4me3 and H3K36me3 patterns throughout the yeast genome. Interestingly, the aberrant histone H3 methylation patterns were predominantly observed within 1 kb from the transcription start site, where both histone H3 methylation marks co-occur. A reciprocal change between the two marks was obvious in the absence of Chd1, suggesting a role for CHD1 in establishing or maintaining the boundaries of these largely mutually exclusive histone marks. Strikingly, intron-containing genes were most susceptible to CHD1 loss and exhibited a high degree of histone H3 methylation changes. Intron retention was significantly lower in the absence of CHD1, suggesting that CHD1 function as a chromatin remodeler could indirectly affect RNA splicing.

Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression inSaccharomycescerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation ratein vivo. However, we also find that determination of elongation ratein vivoby chromatin IP can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants’ growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.

Borrelia burgdorferi, the bacterial pathogen responsible for Lyme disease, modulates its gene expression profile in response to the environments encountered throughout its tick-mammal infectious cycle. To begin to characterize the B. burgdorferi transcriptome during murine infection, we previously employed an in vivo expression technology-based approach (BbIVET). This identified 233 putative promoters, many of which mapped to un-annotated regions of the complex, segmented genome. Herein, we globally identify the 5′ end transcriptome of B. burgdorferi grown in culture as a means to validate non-ORF associated promoters discovered through BbIVET. We demonstrate that 119 BbIVET promoters are associated with transcription start sites (TSSs) and validate novel RNA transcripts using Northern blots and luciferase promoter fusions. Strikingly, 49% of BbIVET promoters were not found to associate with TSSs. This finding suggests that these sequences may be primarily active in the mammalian host. Furthermore, characterization of the 6042 B. burgdorferi TSSs reveals a variety of RNAs including numerous antisense and intragenic transcripts, leaderless RNAs, long untranslated regions and a unique nucleotide frequency for initiating intragenic transcription. Collectively, this is the first comprehensive map of TSSs in B. burgdorferi and characterization of previously un-annotated RNA transcripts expressed by the spirochete during murine infection.

ENPP2, which encodes for the enzyme autotaxin (ATX), is overexpressed during chronic inflammatory diseases and various cancers. However, the molecular mechanism involved in the ENPP2 transcription remains elusive. Here, in HEK 293T cells, we demonstrated that lipopolysaccharide (LPS) increased the transcription process at ENPP2 locus through a NF-кB pathway and a reduction of H3K27me3 level, a histone repressive mark, by the demethylase UTX. Simultaneously, the H3K27me3 demethylase JMJD3/KDM6B was recruited to the transcription start site (TSS), within the gene body and controlled the expression of ENPP2 in a non-enzymatic manner. Mass spectrometry data revealed a novel interaction for JMJD3 with DDX21, a RNA helicase that unwinds R-loops created by nascent transcript and DNA template. Upon LPS treatment, JMJD3 is necessary for DDX21 recruitment at ENPP2 locus allowing the resolution of aberrant R-loops. CRISPR-Cas9-mediated deletion of a distant-acting enhancer decreased the expression of ENPP2 and lowered the recruitment of JMJD3–DDX21 complex at TSS and its progression through the gene body. Taken together, these findings revealed that enhancer-mediated enrichment of novel JMJD3–DDX21 interaction at ENPP2 locus is necessary for nascent transcript synthesis via the resolution of aberrant R-loops formation in response to inflammatory stimulus.