Neural stem cell quiescence and stemness are molecularly distinct outputs of the notch3 signalling cascade in the vertebrate adult brain

International audienceNeural stem cells (NSCs) in the adult vertebrate brain are found in a quiescent state and can preserve long-lasting progenitor potential (stemness). Whether and how these two properties are linked, and to what extent they can be independently controlled by NSC maintenance pathways, is unresolved. We have previously identified Notch3 signalling as a major quiescence-promoting pathway in adult NSCs of the zebrafish pallium. We now show that Notch3 also controls NSC stemness. Using parallel transcriptomic characterizations of notch3 mutant NSCs and adult NSC physiological states, we demonstrate that a set of potentially direct Notch3 target genes distinguishes quiescence and stemness control. As a proof of principle, we focus on one 'stemness' target, encoding the bHLH transcription factor Hey1, that has not yet been analysed in adult NSCs. We show that abrogation of Hey1 function in adult pallial NSCs in vivo, including quiescent NSCs, leads to their differentiation without affecting their proliferation state. These results demonstrate that quiescence and stemness are molecularly distinct outputs of Notch3 signalling, and identify Hey1 as a major Notch3 effector controlling NSC stemness in the vertebrate adult brain

Ancestrally duplicated conserved noncoding element suggests dual regulatory roles of HOTAIR in <i>cis </i>and <i>trans</i>

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.publishedVersio

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

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

Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis

Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.publishedVersio

MIR21 drives resistance to Heat Shock Protein 90 inhibition in cholangiocarcinoma

Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors

Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis

Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronicRNA(sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates

Regulatory mechanisms of non-coding RNAs during zebrafish embryogenesis

For many years, RNAs were thought to be intermediate products between DNA and protein. The discovery of RNA interference (RNAi), a regulatory process that uses small non-coding RNAs to regulate gene expression at the post-transcriptional level, changed our view about RNAs. However, the discovery of microRNAs was the realization of RNAs as the regulatory elements. In recent years, many highthroughput sequencing studies have identified hundreds to thousands of various kinds of non-coding RNAs. The existence and biological relevance of these noncoding RNAs detected in large-scale analysis of human tissues have not yet been characterized in a vertebrate animal in vivo. To gain insight into the existence and biological relevance of these non-coding RNAs in vertebrate animal in vivo, we have set out to generate the first global description of TSS usage during key stages of vertebrate embryonic development at single nucleotide resolution. We have coupled CAGE maps to protein-coding and non-coding transcripts by RNA sequencing (providing a quantitative description of TSS usage on a genome scale) and anchored to posttranslational histone modifications (H3K4me3) by ChIP sequencing. We reveal an extraordinary dynamics of promoter usage that takes place during development of the vertebrate embryo. We showed that the onset of transcription and subsequent differentiation of the embryo is characterized by the developmentally regulated appearance of 5’-ends of intragenic RNAs on many genes, and of an entire hitherto unknown layer of RNA species overlapping known genes and having specific signatures occurring in exons, introns and 3’-UTRs of developmentally active genes. We characterize the pervasive production of intragenic processed RNAs including exonic and intron-5’ end specific RNAs and provide the first indication for the biological processes in which they may function. Notably, intron 5’ end associated non-coding RNAs are active zygotically and restricted to genes that encode RNA processing and the splicing proteins in both fish and human. We demonstrated evidence that exonic RNAs are produced by a non-canonical posttranscriptional mechanism independent of the gene 5’ end. We show the initiation landscape and developmental dynamics of lincRNAs; we show the evolutionary conserved process of developmentally regulated posttranscriptional processing of lincRNAs into intragenic RNAs, which demonstrate the utility of zebrafish in studying mammalian lincRNA processing. The main aim of this work was to provide a (currently non-existent) annotation of miRNA promoters and characterize their common characteristics features at transcription, post transcription and chromatin level. We describe the first genomewide identification of miRNA promoters in zebrafish active during the early embryonic developmental stages. We identified a small number of maternally transcribed miRNAs, one MBT specific miRNA and the majority that are zygotically transcribed. We report the first evidence of moRNAs in zebrafish and pufferfish that were previously reported in human and Ciona intestinalis. We show evidence for unexpected enrichment of pre-miRNA sites with promoter-associated histone modification marks (H3K4me3 and H2A.Z) suggesting chromatin regulation and potential involvement of transcription machinery in pre-miRNA processing, suggesting co-transcriptional splicing of pre-miRNAs and pri-miRNA. We have provided a catalogue of intermediate-sized non-coding RNAs in zebrafish, by making RNA library enriched for intermediate-sized (50-500 nt) non-coding RNAs, collected from zebrafish larvae (5-7 days post fertilization). In particular, we validated most annotated snoRNAs and identified few hundreds of novel snoRNAs making the most comprehensive annotations of zebrafish snoRNAs. Host genes for most snoRNAs showed no evidence for independent transcription of snoRNAs, suggesting they are co-transcribed by host genes. Interestingly, host (coding and non-coding) genes require non-canonical transcription initiation machinery, as indicated by TCT initiation signals, that is associated with translation machinery. 5’-end of many snoRNAs overlaps with CAGE 5’-ends, suggesting either they are capped or undergo post-transcriptional modification, which is also evolutionary conserved in human snoRNAs. Small RNAs derived from snoRNAs are generated from most snoRNAs and provide first evidence of sd-snoRNAs produced in oocytes, suggesting their potential importance during early embryogenesis