169 results found
Karimi MM, Guo Y, Cui X, et al., 2021, The order and logic of CD4 CD8 lineage choice and differentiation in mouse thymus, Nature Communications, Vol: 12, ISSN: 2041-1723
CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.
Yu C, Cvetesic N, Hisler V, et al., 2020, TBPL2/TFIIA complex establishes the maternal transcriptome through oocyte-specific promoter usage, Nature Communications, Vol: 11, ISSN: 2041-1723
During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.
Beltran T, Pahita E, Ghosh S, et al., 2020, Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors, The EMBO Journal, Pages: 1-17, ISSN: 0261-4189
Piwi‐interacting RNAs (piRNAs) play key roles in germline development and genome defence in metazoans. In C. elegans, piRNAs are transcribed from > 15,000 discrete genomic loci by RNA polymerase II (Pol II), resulting in 28 nt short‐capped piRNA precursors. Here, we investigate transcription termination at piRNA loci. We show that the Integrator complex, which terminates snRNA transcription, is recruited to piRNA loci. Moreover, we demonstrate that the catalytic activity of Integrator cleaves nascent capped piRNA precursors associated with promoter‐proximal Pol II, resulting in termination of transcription. Loss of Integrator activity, however, does not result in transcriptional readthrough at the majority of piRNA loci. Taken together, our results draw new parallels between snRNA and piRNA biogenesis in nematodes and provide evidence of a role for the Integrator complex as a terminator of promoter‐proximal RNA polymerase II during piRNA biogenesis.
Cvetesic N, Borkowska M, Hatanaka Y, et al., 2020, Global regulatory transitions at core promoters demarcate the mammalian germline cycle
<jats:title>Abstract</jats:title><jats:p>Core promoters integrate regulatory inputs of genes<jats:sup>1–3</jats:sup>. Global dynamics of promoter usage can reveal systemic changes in how genomic sequence is interpreted by the cell<jats:sup>4</jats:sup> Here we report the first analysis of promoter dynamics and code switching in the mammalian germ line, characterising the full cycle of transitions from embryonic stem cells through germline, oogenesis, and zygotic genome activation. Using Super Low Input Carrier-CAGE<jats:sup>5,6</jats:sup> (SLIC-CAGE) we show that mouse germline development starts with the somatic promoter code, followed by a prominent switch to the maternal code during follicular oogenesis. The sequence features underlying the shift from somatic to maternal code are conserved across vertebrates, despite large differences in promoter nucleotide compositions. In addition, we show that, prior to this major shift, the promoters of gonadal germ cells diverge from the canonical somatic transcription initiation. This divergence is distinct from the promoter code used later by developing oocytes and reveals genome-wide promoter remodelling associated with alternative nucleosome positioning during early female and male germline development. Collectively, our findings establish promoter-level regulatory transitions as a central, conserved feature of the vertebrate life cycle.</jats:p>
Wragg JW, Roos L, Vucenovic D, et al., 2020, Embryonic tissue differentiation is characterized by transitions in cell cycle dynamic-associated core promoter regulation, NUCLEIC ACIDS RESEARCH, Vol: 48, Pages: 8374-8392, ISSN: 0305-1048
Yu C, Cvetesic N, Hisler V, et al., 2020, TBPL2/TFIIA complex establishes the maternal transcriptome by an oocyte-specific promoter usage
<jats:title>Abstract</jats:title><jats:p>During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.</jats:p>
Lewis SH, Ross L, Bain SA, et al., 2020, ------Widespread conservation and lineage-specific diversification of genome-wide DNA methylation patterns across arthropods, PLOS GENETICS, Vol: 16, ISSN: 1553-7404
Lenhard B, Sternberg MJE, 2020, Computational Resources for Molecular Biology: Special Issue 2020, JOURNAL OF MOLECULAR BIOLOGY, Vol: 432, Pages: 3361-3363, ISSN: 0022-2836
Martín-Durán JM, Vellutini BC, Marlétaz F, et al., 2020, Conservative route to genome compaction in a miniature annelid
<jats:title>Summary</jats:title><jats:p>The causes and consequences of genome reduction in animals are unclear, because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8 Mb genome of <jats:italic>Dimorphilus gyrociliatus</jats:italic>, a meiobenthic segmented worm. The <jats:italic>D. gyrociliatus</jats:italic> genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit, and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the <jats:italic>D. gyrociliatus</jats:italic> epigenome revealed canonical features of genome regulation, excluding the presence of operons and <jats:italic>trans</jats:italic>-splicing. Instead, the gene dense <jats:italic>D. gyrociliatus</jats:italic> genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation, and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate <jats:italic>Takifugu rubripes</jats:italic>.</jats:p>
Nepal C, Taranta A, Hadzhiev Y, et al., 2020, Ancestrally Duplicated Conserved Noncoding Element Suggests Dual Regulatory Roles of HOTAIR in cis and trans, ISCIENCE, Vol: 23
Bonetti A, Agostini F, Suzuki AM, et al., 2020, RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723
Lewis S, Ross L, Bain SA, et al., 2020, Widespread conservation and lineage-specific diversification of genome-wide DNA methylation patterns across arthropods, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Cytosine methylation is an ancient epigenetic modification yet its function and extent within genomes is highly variable across eukaryotes. In mammals, methylation controls transposable elements and regulates the promoters of genes. In insects, DNA methylation is generally restricted to a small subset of transcribed genes, with both intergenic regions and transposable elements (TEs) depleted of methylation. The evolutionary origin and the function of these methylation patterns are poorly understood. Here we characterise the evolution of DNA methylation across the arthropod phylum. While the common ancestor of the arthropods had low levels of TE methylation and did not methylate promoters, both of these functions have evolved independently in centipedes and mealybugs. In contrast, methylation of the exons of a subset of transcribed genes is ancestral and widely conserved across the phylum, but has been lost in specific lineages. Remarkably the same set of genes are likely to be methylated in all species that retained exon-enriched methylation. We show that these genes have characteristic patterns of expression correlating to broad transcription initiation sites and well-positioned nucleosomes, providing new insights into potential mechanisms driving methylation patterns over hundreds of millions of years.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p>Animals develop from a single cell to form a complex organism with many specialised cells. Almost all of the fantastic variety of cells must have the same sequence of DNA, and yet they have distinct identities that are preserved even when they divide. This remarkable process is achieved by turning different sets of genes on or off in different types of cell using molecular mechanisms known as “epigenetic gene regulation”.</jats:p><jats:p>Surprisingly, though all animals need epigenetic gene
Nepal C, Hadzhiev Y, Balwierz P, et al., 2020, Dual-initiation promoters with intertwined canonical and TCT/TOP transcription start sites diversify transcript processing, Nature Communications, Vol: 11, ISSN: 2041-1723
Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5'-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5'-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs.
Fornes O, Castro-Mondragon JA, Khan A, et al., 2020, JASPAR 2020: update of the open-access database of transcription factor binding profiles, NUCLEIC ACIDS RESEARCH, Vol: 48, Pages: D87-D92, ISSN: 0305-1048
Baresic A, Nash AJ, Dahoun T, et al., 2020, Understanding the genetics of neuropsychiatric disorders: the potential role of genomic regulatory blocks, MOLECULAR PSYCHIATRY, Vol: 25, Pages: 6-18, ISSN: 1359-4184
Tan G, Polychronopoulos D, Lenhard B, 2019, CNEr: A toolkit for exploring extreme noncoding conservation, PLOS COMPUTATIONAL BIOLOGY, Vol: 15, ISSN: 1553-734X
Nash AJ, Lenhard B, 2019, A novel measure of non-coding genome conservation identifies genomic regulatory blocks within primates, BIOINFORMATICS, Vol: 35, Pages: 2354-2361, ISSN: 1367-4803
Cvetesic N, Pahita E, Lenhard B, 2019, Transcription Start Site Mapping Using Super-low Input Carrier-CAGE, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
Lenhard B, Sternberg MJE, 2019, Computation resources for molecular biology: Special issue 2019, Journal of Molecular Biology, Vol: 431, Pages: 2395-2397, ISSN: 0022-2836
Ferreirós-Vidal I, Carroll T, Zhang T, et al., 2019, Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation, PLoS Biology, Vol: 17, ISSN: 1544-9173
The differentiation of self-renewingprogenitor cells requires not only the regulation of lineage-and developmental stage-specific genes, but also the coordinated adaptation of housekeeping functionsfrom a metabolically active, proliferative state towards quiescence. How metabolic and cell cycle states are coordinated with the regulation of cell type-specific genes is an important question, as dissociation between differentiation, cell cycle, and metabolic states is a hallmark of cancer. Here we use a model system to systematically identify key transcriptional regulators of Ikaros-dependent B cell progenitor differentiation. We find that the coordinated regulation of housekeeping functions and tissue-specific gene expressionrequires afeedforward circuit whereby Ikarosdownregulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping statescan be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.
Tan G, Polychronopoulos D, Lenhard B, 2019, CNEr: a toolkit for exploring extreme noncoding conservation, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Conserved Noncoding Elements (CNEs) are elements exhibiting extreme noncoding conservation in Metazoan genomes. They cluster around developmental genes and act as long-range enhancers, yet nothing that we know about their function explains the observed conservation levels. Clusters of CNEs coincide with topologically associating domains (TADs), indicating ancient origins and stability of TAD locations. This has suggested further hypotheses about the still elusive origin of CNEs, and has provided a comparative genomics-based method of estimating the position of TADs around developmentally regulated genes in genomes where chromatin conformation capture data is missing. To enable researchers in gene regulation and chromatin biology to start deciphering this phenomenon, we developed<jats:italic>CNEr</jats:italic>, a R/Bioconductor toolkit for large-scale identification of CNEs and for studying their genomic properties. We apply<jats:italic>CNEr</jats:italic>to two novel genome comparisons - fruit fly vs tsetse fly, and two sea urchin genomes - and report novel insights gained from their analysis. We also show how to reveal interesting characteristics of CNEs by coupling CNEr with existing Bioconductor packages.<jats:italic>CNEr</jats:italic>is available at Bioconductor (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://bioconductor.org/packages/CNEr/">https://bioconductor.org/packages/CNEr/</jats:ext-link>) and maintained at github (<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/ge11232002/CNEr">https://github.com/ge11232002/CNEr</jats:ext-link>).</jats:p>
Borlin CS, Cvetesic N, Holland P, et al., 2019, Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions, FEMS YEAST RESEARCH, Vol: 19, ISSN: 1567-1356
CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
Newton M, Taylor B, Driessen R, et al., 2018, DNA stretching induces Cas9 off-target binding and cleavage, Nature Structural and Molecular Biology, ISSN: 1545-9985
CRISPR/Cas9 is a powerful genome editing tool, but spurious off-target edits present a barrier towards therapeutic applications. To understand how CRISPR/Cas9 discrimi-nates between on- and off-targets, we have developed a single-molecule assay com-bining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule FRET and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with 10 mis-matches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (e.g., transcription, replication, etc) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
Marletaz F, Firbas PN, Maeso I, et al., 2018, Amphioxus functional genomics and the origins of vertebrate gene regulation, Nature, Vol: 564, Pages: 64-70, ISSN: 0028-0836
Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.
Cvetesic N, Leitch HG, Borkowska M, et al., 2018, SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA, Genome Research, Vol: 28, Pages: 1943-1956, ISSN: 1088-9051
Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5′ ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of rules of transcription initiation, led to discovery of new core promoter sequence features and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 micrograms), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5′ ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods by generating a complex, high quality library from mouse embryonic day (E) 11.5 primordial germ cells.
Merkenschlager M, Cuartero S, Weiss F, et al., 2018, Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation, Nature Immunology, Vol: 19, Pages: 932-941, ISSN: 1529-2908
Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.
Börlin CS, Cvetesic N, Holland P, et al., 2018, Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions, Publisher: Cold Spring Harbor Laboratory
<jats:title>ABSTRACT</jats:title><jats:p>One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of transcription start sites at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the shape index, a characteristic feature of each TSS describing the spatial distribution of transcription initiation events, has a surprisingly strong negative correlation with the measured expression levels. Our analysis supplies a set of high quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.</jats:p>
Lessel D, Gehbauer C, Bramswig NC, et al., 2018, BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells, BRAIN, Vol: 141, Pages: 2299-2311, ISSN: 0006-8950
Li C, Lenhard B, Luscombe NM, 2018, Integrated analysis sheds light on evolutionary trajectories of young transcription start sites in the human genome, Genome Research, Vol: 28, Pages: 676-688, ISSN: 1088-9051
Understanding the molecular mechanisms and evolution of the gene regulatory system remains a major challenge in biology. Transcription start sites (TSSs) are especially interesting because they are central to initiating gene expression. Previous studies revealed widespread transcription initiation and fast turnover of TSSs in mammalian genomes. Yet, how new TSSs originate and how they evolve over time remain poorly understood. To address these questions, we analyzed ∼200,000 human TSSs by integrating evolutionary (inter- and intra-species) and functional genomic data, particularly focusing on evolutionarily young TSSs that emerged in the primate lineage. TSSs were grouped according to their evolutionary age using sequence alignment information as a proxy. Comparisons of young and old TSSs revealed that (1) new TSSs emerge through a combination of intrinsic factors, like the sequence properties of transposable elements and tandem repeats, and extrinsic factors such as their proximity to existing regulatory modules; (2) new TSSs undergo rapid evolution that reduces the inherent instability of repeat sequences associated with a high propensity of TSS emergence; and (3) once established, the transcriptional competence of surviving TSSs is gradually enhanced, with evolutionary changes subject to temporal (fewer regulatory changes in younger TSSs) and spatial constraints (fewer regulatory changes in more isolated TSSs). These findings advance our understanding of how regulatory innovations arise in the genome throughout evolution and highlight the genomic robustness and evolvability in these processes.
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