Functional diversification of sonic hedgehog paralog enhancers identified by phylogenomic reconstruction

Investigation of the ar-C midline enhancer of sonic hedgehog orthologs and paralogs from distantly related vertebrate lineages identified lineage-specific motif changes; exchanging motifs between paralog enhancers resulted in the reversal of enhancer specificity

Hedgehog signaling patterns the outgrowth of unpaired skeletal appendages in zebrafish

Background: Little is known about the control of the development of vertebrate unpaired appendages such as the caudal fin, one of the key morphological specializations of fishes. Recent analysis of lamprey and dogshark median fins suggests the co-option of some molecular mechanisms between paired and median in Chondrichthyes. However, the extent to which the molecular mechanisms patterning paired and median fins are shared remains unknown.Results: Here we provide molecular description of the initial ontogeny of the median fins in zebrafish and present several independent lines of evidence that Sonic hedgehog signaling emanating from the embryonic midline is essential for establishment and outgrowth of the caudal fin primordium. However, gene expression analysis shows that the primordium of the adult caudal fin does not harbor a Sonic hedgehog-expressing domain equivalent to the Shh secreting zone of polarizing activity ( ZPA) of paired appendages.Conclusion: Our results suggest that Hedgehog proteins can regulate skeletal appendage outgrowth independent of a ZPA and demonstrates an unexpected mechanism for mediating Shh signals in a median fin primordium. The median fins evolved before paired fins in early craniates, thus the patterning of the median fins may be an ancestral mechanism that controls the outgrowth of skeletogenic appendages in vertebrates

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

Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position

<p>Abstract</p> <p>Background</p> <p>The modern coelacanth (<it>Latimeria</it>) is the extant taxon of a basal sarcopterygian lineage and sister group to tetrapods. Apart from certain apomorphic traits, its morphology is characterized by a high degree of retention of ancestral vertebrate structures and little morphological change. An insight into the molecular evolution that may explain the unchanged character of <it>Latimeria </it>morphology requires the analysis of the expression patterns of developmental regulator genes and their <it>cis</it>-regulatory modules (CRMs).</p> <p>Results</p> <p>We describe the comparative and functional analysis of the <it>sonic hedgehog </it>(<it>shh</it>) genomic region of <it>Latimeria menadoensis</it>. Several putative enhancers in the <it>Latimeria shh </it>locus have been identified by comparisons to sarcopterygian and actinopterygian extant species. Specific sequence conservation with all known actinopterygian enhancer elements has been detected. However, these elements are selectively missing in more recently diverged actinopterygian and sarcopterygian species. The functionality of the putative <it>Latimeria </it>enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos.</p> <p>Conclusions</p> <p><it>Latimeria shh </it>CRMs represent the ancestral set of enhancers that have emerged before the split of lobe-finned and ray-finned fishes. In contrast to lineage-specific losses and differentiations in more derived lineages, <it>Latimeria shh </it>enhancers reveal low levels of sequence diversification. High overall sequence conservation of <it>shh </it>conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving <it>Latimeria </it>genome.</p

CMTr mediated 2`-O-ribose methylation status of cap adjacent-nucleotides across animals

ACKNOWLEDGMENTS We thank Mark Carrington and Nancy Standard for T. brucei total RNA, Jane Nimmo for honeybees, Caroline Chadwick for mouse tissue, Pawel Grzechnik for HEK293T cells, Roland Arnold for HCT116 cells, Rupert Fray for plasmids, and the National BioResource Project, Tokyo, Japan for the C. elegans CMTr2 strain. M.S. acknowledges funding from the Leverhulme Trust and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/R002932/1), J.P. and B.M. from BBSRC (BB/T002859/1), and F.M. from the Wellcome Trust (106955/Z/15/Z).Peer reviewedPublisher PD

Intra-promoter switch of transcription initiation sites in proliferation signaling-dependent RNA metabolism

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.</p

The miR-430 locus with extreme promoter density forms a transcription body during the minor wave of zygotic genome activation

In anamniote embryos, the major wave of zygotic genome activation starts during the mid-blastula transition. However, some genes escape global genome repression, are activated substantially earlier, and contribute to the minor wave of genome activation. The mechanisms underlying the minor wave of genome activation are little understood. We explored the genomic organization and cis -regulatory mechanisms of a transcrip-tion body, in which the minor wave of genome activation is first detected in zebrafish. We identified the miR-430 cluster as having excessive copy number and the highest density of Pol-II-transcribed promoters in the genome, and this is required for forming the transcription body. However, this transcription body is not essential for, nor does it encompasse, minor wave transcription globally. Instead, distinct minor-wave -specific promoter architecture suggests that promoter-autonomous mechanisms regulate the minor wave of genome activation. The minor-wave-specific features also suggest distinct transcription initiation mecha-nisms between the minor and major waves of genome activation

Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish. RESULTS: flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development. CONCLUSIONS: In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic brain development.Funding for this project was provided by the Myrovlytis Trust and a KWF travel grant to Monique Luijten.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s12861-016-0119-

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