Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

<p>Abstract</p> <p>Background</p> <p>The <it>mce </it>operons play an important role in the entry of <it>M. tuberculosis </it>into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of <it>mce </it>mutants. Recently, <it>mce1 </it>operon was found to extend over 13 genes, <it>fadD5 </it>(Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to <it>mce1 </it>among the four <it>mce </it>operons of <it>M.tuberculosis</it>. We have examined the function of this region.</p> <p>Results</p> <p>We predicted putative promoter activity of the 200 base pairs of non-coding, intergenic region between Rv0166 and Rv0167 <it>in silico </it>using MEME software and designate it as intergenic promoter, IGPr. We demonstrate both promoter activity and a putative negative regulatory function of this fragment by reporter assays carried out in the surrogate host <it>M.smegmatis</it>. We find that the repressive elements not only control the native promoter but also repress a heterologous promoter of <it>M.smegmatis</it>. The higher activity of the intergenic promoter in a clinical isolate in comparison with the wild type sequence from <it>M.tuberculosis </it>H37Rv could be correlated with a point mutation within the negative element. We have mapped two transcription start sites for <it>mce1 </it>operon both of which are utilized in <it>M.tuberculosis </it>H37Rv as well as the clinical isolate VPCI591. Our studies show that the promoter activity in the non-coding region is relevant not only in reporter gene expression but also in the expression of <it>mce1 </it>operon in <it>M. tuberculosis </it>cells grown in synthetic medium.</p> <p>Conclusion</p> <p>The <it>mce </it>operon of <it>M.tuberculosis </it>H37Rv potentially can be transcribed from two promoters P1 and P2, former mapping upstream of Rv0166 and the latter in the non-coding intergenic region between Rv0166 and Rv0167. The transcription initiation from P1 results in a transcript with Rv0166 while that from P2 will be without it. The sequences between the translation start site of Rv0167 and the promoter P2 have a negative regulatory role, as point mutation within the sequence leads to enhanced activity of P2 as well as a heterologous promoter from <it>M.smegmatis</it>. The mutation detected in the clinical isolate VPCI591 therefore behaves like a gain-of-function mutation.</p

Quantitative Spatial and Temporal Assessment of Regulatory element activity in Zebrafish

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation

Discovery and assessment of conserved Pax6 target genes and enhancers

The characterization of transcriptional networks (TNs) is essential for understanding complex biological phenomena such as development, disease, and evolution. In this study, we have designed and implemented a procedure that combines in silico target screens with zebrafish and mouse validation, in order to identify cis-elements and genes directly regulated by Pax6. We chose Pax6 as the paradigm because of its crucial roles in organogenesis and human disease. We identified over 600 putative Pax6 binding sites and more than 200 predicted direct target genes, conserved in evolution from zebrafish to human and to mouse. This was accomplished using hidden Markov models (HMMs) generated from experimentally validated Pax6 binding sites. A small sample of genes, expressed in the neural lineage, was chosen from the predictions for RNA in situ validation using zebrafish and mouse models. Validation of DNA binding to some predicted cis-elements was also carried out using chromatin immunoprecipitation (ChIP) and zebrafish reporter transgenic studies. The results show that this combined procedure is a highly efficient tool to investigate the architecture of TNs and constitutes a useful complementary resource to ChIP and expression data sets because of its inherent spatiotemporal independence. We have identified several novel direct targets, including some putative disease genes, among them Foxp2; these will allow further dissection of Pax6 function in development and disease

SBE6, a novel long-range enhancer involved in driving Sonic Hedgehog expression in neural progenitor cells

The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (<i>Shh</i>) that plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both <i>in vivo</i> and <i>in vitro. Shh</i> expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh-Shh-brain-enhancer-6 (SBE6) that is located 100 kb upstream of <i>Shh</i> and that is required for the proper induction of <i>Shh</i> expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to <i>in vivo</i> testing, can be used to identify new cell-type specific <i>cis</i>-regulatory elements and points to yet further complexity in the control of <i>Shh</i> expression during embryonic brain development

Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability.

Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use

Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA Hottip

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis

Functional Assessment of Disease-Associated Regulatory Variants <i>In Vivo</i> Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function