Comparative analyses of bidirectional promoters in vertebrates

<p>Abstract</p> <p>Background</p> <p>Orthologous genes with deep phylogenetic histories are likely to retain similar regulatory features. In this report we utilize orthology assignments for pairs of genes co-regulated by bidirectional promoters to map the ancestral history of the promoter regions.</p> <p>Results</p> <p>Our mapping of bidirectional promoters from humans to fish shows that many such promoters emerged after the divergence of chickens and fish. Furthermore, annotations of promoters in deep phylogenies enable detection of missing data or assembly problems present in higher vertebrates. The functional importance of bidirectional promoters is indicated by selective pressure to maintain the arrangement of genes regulated by the promoter over long evolutionary time spans. Characteristics unique to bidirectional promoters are further elucidated using a technique for unsupervised classification, known as ESPERR.</p> <p>Conclusion</p> <p>Results of these analyses will aid in our understanding of the evolution of bidirectional promoters, including whether the regulation of two genes evolved as a consequence of their proximity or if function dictated their co-regulation.</p

Amphioxus functional genomics and the origins of vertebrate gene regulation.

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

CYNTENATOR: Progressive Gene Order Alignment of 17 Vertebrate Genomes

Whole genome gene order evolution in higher eukaryotes was initially considered as a random process. Gene order conservation or conserved synteny was seen as a feature of common descent and did not imply the existence of functional constraints. This view had to be revised in the light of results from sequencing dozens of vertebrate genomes

Transcriptional regulation of bidirectional gene pairs by 17-&#946;-estradiol in MCF-7 breast cancer cells

Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5&#8217;flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40% of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17% of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17&#946;-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17&#946;-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs

Co-regulated expression of HAND2 and DEIN by a bidirectional promoter with asymmetrical activity in neuroblastoma

<p>Abstract</p> <p>Background</p> <p><it>HAND2</it>, a key regulator for the development of the sympathetic nervous system, is located on chromosome 4q33 in a head-to-head orientation with <it>DEIN</it>, a recently identified novel gene with stage specific expression in primary neuroblastoma (NB). Both genes are expressed in primary NB as well as most NB cell lines and are separated by a genomic sequence of 228 bp. The similar expression profile of both genes suggests a common transcriptional regulation mediated by a bidirectional promoter.</p> <p>Results</p> <p>Northern Blot analysis of <it>DEIN </it>and <it>HAND2 </it>in 20 primary NBs indicated concurrent expression levels of the two genes, which was confirmed by microarray analysis of 236 primary NBs (Pearson's correlation coefficient r = 0.65). While <it>DEIN </it>expression in the latter cohort was associated with stage 4S (p = 0.02), <it>HAND2 </it>expression was not associated with tumor stage. In contrast, both <it>HAND2 </it>and <it>DEIN </it>transcript levels were highly associated with age at diagnosis <12 months (p = 0.001). The intergenic region shows substantial homology in different species (89%, 72% and 53% identity between human and mouse, chicken and zebrafish, respectively) and contains many highly conserved putative transcription factor binding sites. Using luciferase reporter gene constructs, asymmetrical bidirectional promoter activity was found in four NB cell lines: In <it>DEIN </it>orientation, an average 3.4 fold increase in activity was observed as compared to the promoterless vector, whereas an average 15.4 fold activation was detected in <it>HAND2 </it>orientation. The presence of two highly conserved putative regulatory elements, one of which was shown to enhance <it>HAND2 </it>expression in branchial arches previously, displayed weak repressor activity for both genes.</p> <p>Conclusion</p> <p><it>HAND2 </it>and <it>DEIN </it>represent a gene pair that is tightly linked by a bidirectional promoter in an evolutionary highly conserved manner. Expression of both genes in NB is co-regulated by asymmetrical activity of this promoter and modulated by the activity of two cis-regulatory elements acting as weak repressors. The concurrent quantitative and tissue specific expression of <it>HAND2 </it>and <it>DEIN </it>suggests a functional link between both genes.</p

Sorting out inherent features of head-to-head gene pairs by evolutionary conservation

BACKGROUND: A ‘head-to-head’ (h2h) gene pair is defined as a genomic locus in which two adjacent genes are divergently transcribed from opposite strands of DNA. In our previous work, this gene organization was found to be ancient and conserved, which subjects functionally related genes to transcriptional co-regulation. However, some of the biological features of h2h pairs still need further clarification. RESULTS: In this work, we assorted human h2h pairs into four sequentially inclusive sets of gradually incremental conservation, and examined whether those previously asserted features were conserved or sharpened in the more conserved h2h pair sets in order to identify the inherent features of the h2h gene organization. The features of TSS distance, expression correlation within h2h pairs and among h2h genes, transcription factor association and functional similarities of h2h genes were examined. Our conservation-based analyses found that the bi-directional promoters of h2h gene pairs are most likely shorter than 100 bp; h2h gene pairs generally have only significant positive expression correlation but not negative correlation, and remarkably high positive expression correlations exist among h2h genes, as well as between h2h pairs observed in our previous study; h2h paired genes tend to share transcription factors. In addition, expression correlation of h2h pairs is positively related with the TF-sharing and functional coordination, while not related with TSS distance. CONCLUSIONS: Our findings remove the uncertainties of h2h genes about TSS distance, expression correlation and functional coordination, which provide insights into the study on the molecular mechanisms and functional consequences of the transcriptional regulation based on this special gene organization

Conservation of different mechanisms of Hox cluster regulation within chordates

[eng] In this thesis we have covered the importance of finding underlying conservation events to better understand the regulatory mechanisms of important development orchestrators like the Hox cluster. As an example of these non-evident conservation, we have shown two cases, as described below. The first case studied, after developing a software able to detect homologous long noncoding RNAs by means of microsynteny analyses, is the conservation of Hotairm1 in Chordata. For assessing the homology of this lncRNA, first we had to identify the lncRNA fraction within the B. lanceolatum transcriptome. With a reliable lincRNA dataset, we used our pipeline, LincOFinder, to identify orthologs between human and amphioxus through microsynteny. After the identification of Hotairm1 as one of the lincRNAs with conserved microsynteny, we used Xenopus as a proxy to analyse the homologies in the expression and the function. We had to proceed this way due to the difficulties associated with the inhibition of genes in B. lanceolatum, and the unavailability of expression patterns for Hotairm1 in the bibliography. After we successfully characterised Hotairm1 expression in amphioxus and Xenopus, we injected morpholino oligonucleotides to target and inhibit the splicing of Hotairm1 to promote an isoform imbalance. Through the phenotype obtained and the performing of qPCRs, we were able to deduct the mechanism of Hotairm1 and successfully relate this mechanism with the one described in human cells. With all the data obtained we were able to strongly suggest that the amphioxus Hotairm1 is homologous to the Xenopus and human Hotairm1, thus being conserved in most of the lineages within chordates. The second case studied was the conservation of the regulation of the Hox cluster mediated by Cdx. When analysing the B. floridae knockouts of Cdx and Pdx obtained using the TALEN technique, we found a severe phenotype of the developing larvae in Cdx-/- and a mild phenotype in Pdx-/-. The Cdx-/- phenotype consisted in the disruption of posterior gut development, as well as an underdevelopment of the postanal tail, coupled with a non-opening anus. When looking at changes in the expression of the Hox cluster in this Cdx-/- embryos, we found collinear misregulation of the expressed Hox genes, with the most anterior Hox cluster genes upregulated, and the most posterior ones downregulated. This is very similar to findings seen in triple morpholino knockdowns of the Cdx genes in Xenopus, indicating that in both, Xenopus and amphioxus, Cdx is regulating the Hox cluster through a homologous mechanism