Publications by authors named "Boris Lenhard"

138 Publications

Neuronal genes deregulated in Cornelia de Lange Syndrome respond to removal and re-expression of cohesin.

Nat Commun 2021 05 18;12(1):2919. Epub 2021 May 18.

Lymphocyte Development Group, Epigenetics Section, MRC London Institute of Medical Sciences, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS.
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http://dx.doi.org/10.1038/s41467-021-23141-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131595PMC
May 2021

Computational Resources for Molecular Biology 2021.

J Mol Biol 2021 05 24;433(11):166962. Epub 2021 Mar 24.

Structural Bioinformatics Group, Centre for Integrative Systems Biology and Bioinformatics, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address:

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http://dx.doi.org/10.1016/j.jmb.2021.166962DOI Listing
May 2021

Germ cell differentiation requires Tdrd7-dependent chromatin and transcriptome reprogramming marked by germ plasm relocalization.

Dev Cell 2021 03 1;56(5):641-656.e5. Epub 2021 Mar 1.

Institute of Cancer and Genomics Sciences, Birmingham Centre for Genome Biology, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK. Electronic address:

In many animal models, primordial germ cell (PGC) development depends on maternally deposited germ plasm, which prevents somatic cell fate. Here, we show that PGCs respond to regulatory information from the germ plasm in two distinct phases using two distinct mechanisms in zebrafish. We demonstrate that PGCs commence zygotic genome activation together with the somatic blastocysts with no demonstrable differences in transcriptional and chromatin opening. Unexpectedly, both PGC and somatic blastocysts activate germ-cell-specific genes, which are only stabilized in PGCs by cytoplasmic germ plasm determinants. Disaggregated perinuclear relocalization of germ plasm during PGC migration is regulated by the germ plasm determinant Tdrd7 and is coupled to dramatic divergence between PGC and somatic transcriptomes. This transcriptional divergence relies on PGC-specific cis-regulatory elements characterized by promoter-proximal distribution. We show that Tdrd7-dependent reconfiguration of chromatin accessibility is required for elaboration of PGC fate but not for PGC migration.
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http://dx.doi.org/10.1016/j.devcel.2021.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957325PMC
March 2021

Functional reconstruction of human AML reveals stem cell origin and vulnerability of treatment-resistant MLL-rearranged leukemia.

Sci Transl Med 2021 02;13(582)

Leukaemia and Stem Cell Biology Group, School of Cancer and Pharmaceutical Sciences, King's College, London SE5 9NU, UK.

Chemoresistance remains the major challenge for successful treatment of acute myeloid leukemia (AML). Although recent mouse studies suggest that treatment response of genetically and immunophenotypically indistinguishable AML can be influenced by their different cells of origin, corresponding evidence in human disease is still largely lacking. By combining prospective disease modeling using highly purified human hematopoietic stem or progenitor cells with retrospective deconvolution study of leukemia stem cells (LSCs) from primary patient samples, we identified human hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) as two distinctive origins of human AML driven by Mixed Lineage Leukemia (MLL) gene fusions (MLL-AML). Despite LSCs from either MLL-rearranged HSCs or MLL-rearranged CMPs having a mature CD34/CD38 immunophenotype in both a humanized mouse model and primary patient samples, the resulting AML cells exhibited contrasting responses to chemotherapy. HSC-derived MLL-AML was highly resistant to chemotherapy and expressed elevated amounts of the multispecific anion transporter ABCC3. Inhibition of ABCC3 by shRNA-mediated knockdown or with small-molecule inhibitor fidaxomicin, currently used for diarrhea associated with infection, effectively resensitized HSC-derived MLL-AML toward standard chemotherapeutic drugs. This study not only functionally established two distinctive origins of human LSCs for MLL-AML and their role in mediating chemoresistance but also identified a potential therapeutic avenue for stem cell-associated treatment resistance by repurposing a well-tolerated antidiarrhea drug already used in the clinic.
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http://dx.doi.org/10.1126/scitranslmed.abc4822DOI Listing
February 2021

The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus.

Nat Commun 2021 01 4;12(1):99. Epub 2021 Jan 4.

MRC London Institute of Medical Sciences, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.

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, Cd4Cd8a selection intermediates appear before Cd4Cd8a 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.
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http://dx.doi.org/10.1038/s41467-020-20306-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782583PMC
January 2021

TBPL2/TFIIA complex establishes the maternal transcriptome through oocyte-specific promoter usage.

Nat Commun 2020 12 22;11(1):6439. Epub 2020 Dec 22.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 67404, Illkirch, France.

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.
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http://dx.doi.org/10.1038/s41467-020-20239-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7755920PMC
December 2020

Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors.

EMBO J 2021 Mar 19;40(5):e105564. Epub 2020 Dec 19.

MRC London Institute of Medical Sciences, London, UK.

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.
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http://dx.doi.org/10.15252/embj.2020105564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917550PMC
March 2021

Conservative route to genome compaction in a miniature annelid.

Nat Ecol Evol 2021 02 16;5(2):231-242. Epub 2020 Nov 16.

Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway.

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-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus 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 D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus 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 Takifugu rubripes.
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http://dx.doi.org/10.1038/s41559-020-01327-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854359PMC
February 2021

Functional annotation of human long noncoding RNAs via molecular phenotyping.

Genome Res 2020 07 27;30(7):1060-1072. Epub 2020 Jul 27.

Department of Computational Systems Biology, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow 119991, Russia.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for and .
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http://dx.doi.org/10.1101/gr.254219.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397864PMC
July 2020

Embryonic tissue differentiation is characterized by transitions in cell cycle dynamic-associated core promoter regulation.

Nucleic Acids Res 2020 09;48(15):8374-8392

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK.

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.
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http://dx.doi.org/10.1093/nar/gkaa563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470974PMC
September 2020

------Widespread conservation and lineage-specific diversification of genome-wide DNA methylation patterns across arthropods.

PLoS Genet 2020 06 25;16(6):e1008864. Epub 2020 Jun 25.

MRC London Institute of Medical Sciences, London, United Kingdom.

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. A similar set of genes is 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.
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http://dx.doi.org/10.1371/journal.pgen.1008864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343188PMC
June 2020

Computational Resources for Molecular Biology: Special Issue 2020.

J Mol Biol 2020 05 14;432(11):3361-3363. Epub 2020 Apr 14.

Structural Bioinformatics Group, Centre for Integrative Systems Biology and Bioinformatics, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address:

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http://dx.doi.org/10.1016/j.jmb.2020.04.010DOI Listing
May 2020

Ancestrally Duplicated Conserved Noncoding Element Suggests Dual Regulatory Roles of HOTAIR in cis and trans.

iScience 2020 Apr 25;23(4):101008. Epub 2020 Mar 25.

Biotech Research and Innovation Centre, Department of Health and Medical Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark. Electronic address:

HOTAIR was proposed to regulate either HoxD cluster genes in trans or HoxC cluster genes in cis, a mechanism that remains unclear. We have identified a 32-nucleotide conserved noncoding element (CNE) as HOTAIR ancient sequence that likely originated at the root of vertebrate. The second round of whole-genome duplication resulted in one copy of the CNE within HOTAIR and another copy embedded in noncoding transcript of HOXD11. Paralogous CNEs underwent compensatory mutations, exhibit sequence complementarity with respect to transcripts directionality, and have high affinity in vitro. The HOTAIR CNE resembled a poised enhancer in stem cells and an active enhancer in HOTAIR-expressing cells. HOTAIR expression is positively correlated with HOXC11 in cis and negatively correlated with HOXD11 in trans. We propose a dual modality of HOTAIR regulation where transcription of HOTAIR and its embedded enhancer regulates HOXC11 in cis and sequence complementarity between paralogous CNEs suggests HOXD11 regulation in trans.
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http://dx.doi.org/10.1016/j.isci.2020.101008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139118PMC
April 2020

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions.

Nat Commun 2020 02 24;11(1):1018. Epub 2020 Feb 24.

RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, 230-0045, Japan.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.
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http://dx.doi.org/10.1038/s41467-020-14337-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039879PMC
February 2020

Dual-initiation promoters with intertwined canonical and TCT/TOP transcription start sites diversify transcript processing.

Nat Commun 2020 01 10;11(1):168. Epub 2020 Jan 10.

Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

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.
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http://dx.doi.org/10.1038/s41467-019-13687-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954239PMC
January 2020

JASPAR 2020: update of the open-access database of transcription factor binding profiles.

Nucleic Acids Res 2020 01;48(D1):D87-D92

Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, 0318 Oslo, Norway.

JASPAR (http://jaspar.genereg.net) is an open-access database of curated, non-redundant transcription factor (TF)-binding profiles stored as position frequency matrices (PFMs) for TFs across multiple species in six taxonomic groups. In this 8th release of JASPAR, the CORE collection has been expanded with 245 new PFMs (169 for vertebrates, 42 for plants, 17 for nematodes, 10 for insects, and 7 for fungi), and 156 PFMs were updated (125 for vertebrates, 28 for plants and 3 for insects). These new profiles represent an 18% expansion compared to the previous release. JASPAR 2020 comes with a novel collection of unvalidated TF-binding profiles for which our curators did not find orthogonal supporting evidence in the literature. This collection has a dedicated web form to engage the community in the curation of unvalidated TF-binding profiles. Moreover, we created a Q&A forum to ease the communication between the user community and JASPAR curators. Finally, we updated the genomic tracks, inference tool, and TF-binding profile similarity clusters. All the data is available through the JASPAR website, its associated RESTful API, and through the JASPAR2020 R/Bioconductor package.
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http://dx.doi.org/10.1093/nar/gkz1001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145627PMC
January 2020

Understanding the genetics of neuropsychiatric disorders: the potential role of genomic regulatory blocks.

Mol Psychiatry 2020 01 15;25(1):6-18. Epub 2019 Oct 15.

MRC London Institute of Medical Sciences, London, W12 0NN, UK.

Recent genome-wide association studies have identified numerous loci associated with neuropsychiatric disorders. The majority of these are in non-coding regions, and are commonly assigned to the nearest gene along the genome. However, this approach neglects the three-dimensional organisation of the genome, and the fact that the genome contains arrays of extremely conserved non-coding elements termed genomic regulatory blocks (GRBs), which can be utilized to detect genes under long-range developmental regulation. Here we review a GRB-based approach to assign loci in non-coding regions to potential target genes, and apply it to reanalyse the results of one of the largest schizophrenia GWAS (SWG PGC, 2014). We further apply this approach to GWAS data from two related neuropsychiatric disorders-autism spectrum disorder and bipolar disorder-to show that it is applicable to developmental disorders in general. We find that disease-associated SNPs are overrepresented in GRBs and that the GRB model is a powerful tool for linking these SNPs to their correct target genes under long-range regulation. Our analysis identifies novel genes not previously implicated in schizophrenia and corroborates a number of predicted targets from the original study. The results are available as an online resource in which the genomic context and the strength of enhancer-promoter associations can be browsed for each schizophrenia-associated SNP.
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http://dx.doi.org/10.1038/s41380-019-0518-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906185PMC
January 2020

CNEr: A toolkit for exploring extreme noncoding conservation.

PLoS Comput Biol 2019 08 26;15(8):e1006940. Epub 2019 Aug 26.

Computational Regulatory Genomics Group, MRC London Institute of Medical Sciences, United Kingdom.

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 CNEr, a R/Bioconductor toolkit for large-scale identification of CNEs and for studying their genomic properties. We apply CNEr 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. CNEr is available at Bioconductor (https://bioconductor.org/packages/CNEr/) and maintained at github (https://github.com/ge11232002/CNEr).
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http://dx.doi.org/10.1371/journal.pcbi.1006940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730951PMC
August 2019

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE.

J Vis Exp 2019 06 26(148). Epub 2019 Jun 26.

Institute of Clinical Sciences, Faculty of Medicine, Imperial College London; MRC London Institute of Medical Sciences; Sars International Centre for Marine Molecular Biology, University of Bergen;

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 °C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5'-centric expression profiling.
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http://dx.doi.org/10.3791/59805DOI Listing
June 2019

Computation Resources for Molecular Biology: Special Issue 2019.

J Mol Biol 2019 06 30;431(13):2395-2397. Epub 2019 May 30.

Structural Bioinformatics Group, Centre for Integrative systems Biology and Bioinformatics, Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Electronic address:

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http://dx.doi.org/10.1016/j.jmb.2019.05.034DOI Listing
June 2019

Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation.

PLoS Biol 2019 04 12;17(4):e2006506. Epub 2019 Apr 12.

Unit of Computational Medicine, Department of Medicine, Solna, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.

The differentiation of self-renewing progenitor cells requires not only the regulation of lineage- and developmental stage-specific genes but also the coordinated adaptation of housekeeping functions from a metabolically active, proliferative state toward quiescence. How metabolic and cell-cycle states are coordinated with the regulation of cell type-specific genes is an important question, because 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 expression requires a feedforward circuit whereby Ikaros down-regulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping states can be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.
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http://dx.doi.org/10.1371/journal.pbio.2006506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481923PMC
April 2019

DNA stretching induces Cas9 off-target activity.

Nat Struct Mol Biol 2019 03 25;26(3):185-192. Epub 2019 Feb 25.

Molecular Virology, Department of Medicine, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1038/s41594-019-0188-zDOI Listing
March 2019

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions.

FEMS Yeast Res 2019 03;19(2)

Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden.

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 TSSs 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 spatial distribution of transcription initiation events, described by the shape index, has a surprisingly strong negative correlation with measured gene expression levels, meaning that genes with higher expression levels tend to have a broader distribution of TSSs. 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.
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http://dx.doi.org/10.1093/femsyr/foy128DOI Listing
March 2019

A novel measure of non-coding genome conservation identifies genomic regulatory blocks within primates.

Bioinformatics 2019 07;35(14):2354-2361

Computational Regulatory Genomics Group, MRC London Institute of Medical Sciences.

Motivation: Clusters of extremely conserved non-coding elements (CNEs) mark genomic regions devoted to cis-regulation of key developmental genes in Metazoa. We have recently shown that their span coincides with that of topologically associating domains (TADs), making them useful for estimating conserved TAD boundaries in the absence of Hi-C data. The standard approach-detecting CNEs in genome alignments and then establishing the boundaries of their clusters-requires tuning of several parameters and breaks down when comparing closely related genomes.

Results: We present a novel, kurtosis-based measure of pairwise non-coding conservation that requires no pre-set thresholds for conservation level and length of CNEs. We show that it performs robustly across a large span of evolutionary distances, including across the closely related genomes of primates for which standard approaches fail. The method is straightforward to implement and enables detection and comparison of clusters of CNEs and estimation of underlying TADs across a vastly increased range of Metazoan genomes.

Availability And Implementation: The data generated for this study, and the scripts used to generate the data, can be found at https://github.com/alexander-nash/kurtosis_conservation.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty1014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612856PMC
July 2019

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Nature 2018 12 21;564(7734):64-70. Epub 2018 Nov 21.

Genoscope, Institut de biologie François-Jacob, Commissariat à l'Energie Atomique (CEA), Université Paris-Saclay, Evry, France.

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.
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http://dx.doi.org/10.1038/s41586-018-0734-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292497PMC
December 2018

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA.

Genome Res 2018 12 7;28(12):1943-1956. Epub 2018 Nov 7.

Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom.

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 the 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 µg), 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. This 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 11.5 primordial germ cells.
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http://dx.doi.org/10.1101/gr.235937.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280763PMC
December 2018

Control of inducible gene expression links cohesin to hematopoietic progenitor self-renewal and differentiation.

Nat Immunol 2018 09 20;19(9):932-941. Epub 2018 Aug 20.

Lymphocyte Development Group, Epigenetics Section, MRC London Institute of Medical Sciences, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.

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.
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http://dx.doi.org/10.1038/s41590-018-0184-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195188PMC
September 2018

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

Brain 2018 08;141(8):2299-2311

Département de Génétique, Hôpital La Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.
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http://dx.doi.org/10.1093/brain/awy173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061686PMC
August 2018
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