Epigenetic Conservation of Evolutionarily Conserved Cancer/Testis Antigens

DNA cytosine methylation is an important epigenetic modification that plays a key role in gene expression. DNA methylation has been shown to be involved in numerous processes, including X-chromosome inactivation in mammals, retrotransposon silencing, genomic imprinting, carcinogenesis and the regulation of tissue specific gene expression during development. Gene expression is tightly regulated via DNA methylation (5mC) and the aberrant expression of meiotic genes in mitotic cells via CpG promoter hypomethylation has been proposed to cause cancer. Cancer/Testis Antigens (CTAs) are a group of genes that encode tumour specific antigens and are expressed in the testis, certain cancers but not in normal post-natal somatic tissues. CpG island methylation and histone modifications appear to play a role in the epigenetic regulation of CTA expression, however, very little is known about their functions in vivo. A widely studied but poorly understood question to date is the mechanisms behind aberrant CTA reactivation in cancer. Given that 5mC mediated gene repression has been found to exist in vertebrate genomes and CTAs have also been identified to be a subset of highly evolutionarily conserved genes, it is critical to understand the role of 5mC mediated CTA silencing in vertebrates. By gaining a deeper understanding into the mechanisms behind this highly conserved pattern of gene repression on a specific subset of genes, we would be able to identify methods to prevent aberrant gene expression. In this study, I analysed publicly available whole genome bisulfite sequencing (WGBS), RNA-seq and chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq) data of developing embryonic and adult somatic tissue of 3 vertebrate species to elucidate the evolutionary epigenetic regulation of CTAs in vertebrate genomes. Integrative WGBS, RNA-seq and ChIP-seq analysis revealed that CTAs are evolutionarily conserved in zebrafish, mice and humans and mechanisms of their epigenetic regulation are also conserved. I observed that histone modifications could potentially serve as an indicator of the methylation status of CTA gene promoters and that the expression of CTAs was inversely related to gene promoter 5mC levels. I demonstrate that CTAs when over-expressed cause embryonic lethality in zebrafish and the same genes are aberrantly hypomethylated at their CpG islands in a subset of human cancers. Overall, my work shows that CTAs are epigenetically regulated in an evolutionarily conserved manner and possibly via a conserved transcription factor, ETS1, that is expressed both in embryonic and cancerous tissue

Long noncoding RNA genes: conservation of sequence and brain expression among diverse amniotes

BACKGROUND: Long considered to be the building block of life, it is now apparent that protein is only one of many functional products generated by the eukaryotic genome. Indeed, more of the human genome is transcribed into noncoding sequence than into protein-coding sequence. Nevertheless, whilst we have developed a deep understanding of the relationships between evolutionary constraint and function for protein-coding sequence, little is known about these relationships for non-coding transcribed sequence. This dearth of information is partially attributable to a lack of established non-protein-coding RNA (ncRNA) orthologs among birds and mammals within sequence and expression databases. RESULTS: Here, we performed a multi-disciplinary study of four highly conserved and brain-expressed transcripts selected from a list of mouse long intergenic noncoding RNA (lncRNA) loci that generally show pronounced evolutionary constraint within their putative promoter regions and across exon-intron boundaries. We identify some of the first lncRNA orthologs present in birds (chicken), marsupial (opossum), and eutherian mammals (mouse), and investigate whether they exhibit conservation of brain expression. In contrast to conventional protein-coding genes, the sequences, transcriptional start sites, exon structures, and lengths for these non-coding genes are all highly variable. CONCLUSIONS: The biological relevance of lncRNAs would be highly questionable if they were limited to closely related phyla. Instead, their preservation across diverse amniotes, their apparent conservation in exon structure, and similarities in their pattern of brain expression during embryonic and early postnatal stages together indicate that these are functional RNA molecules, of which some have roles in vertebrate brain development

Computational modeling of microRNA Biogenesis

Over the past few years it has been observed, thanks in no small part to high-throughput methods, that a large proportion of the human genome is transcribed in a tissue- and time-specific manner. Most of the detected transcripts are non-coding RNAs and their functional consequences are not yet fully understood. Among the different classes of non-coding transcripts, microRNAs (miRNAs) are small RNAs that post-transcriptionally regulate gene expression. Despite great progress in understanding the biological role of miRNAs, our understanding of how miRNAs are regulated and processed is still developing. High-throughput sequencing data have provided a robust platform for transcriptome-level, as well as gene-promoter analyses. In silico predictive models help shed light on the transcriptional and post-transcriptional regulation of miRNAs, including their role in gene regulatory networks. Here we discuss the advances in computational methods that model different aspects of miRNA biogeneis, from transcriptional regulation to post-transcriptional processing. In particular, we show how the predicted miRNA promoters from PROmiRNA, a miRNA promoter prediction tool, can be used to identify the most probable regulatory factors for a miRNA in a specific tissue. As differential miRNA post-transcriptional processing also affects gene-regulatory networks, especially in diseases like cancer, we also describe a statistical model proposed in the literature to predict efficient miRNA processing from sequence features

PuTmiR: A database for extracting neighboring transcription factors of human microRNAs

<p>Abstract</p> <p>Background</p> <p>Some of the recent investigations in systems biology have revealed the existence of a complex regulatory network between genes, microRNAs (miRNAs) and transcription factors (TFs). In this paper, we focus on TF to miRNA regulation and provide a novel interface for extracting the list of putative TFs for human miRNAs. A putative TF of an miRNA is considered here as those binding within the close genomic locality of that miRNA with respect to its starting or ending base pair on the chromosome. Recent studies suggest that these putative TFs are possible regulators of those miRNAs.</p> <p>Description</p> <p>The interface is built around two datasets that consist of the exhaustive lists of putative TFs binding respectively in the 10 kb upstream region (USR) and downstream region (DSR) of human miRNAs. A web server, named as PuTmiR, is designed. It provides an option for extracting the putative TFs for human miRNAs, as per the requirement of a user, based on genomic locality, i.e., any upstream or downstream region of interest less than 10 kb. The degree distributions of the number of putative TFs and miRNAs against each other for the 10 kb USR and DSR are analyzed from the data and they explore some interesting results. We also report about the finding of a significant regulatory activity of the YY1 protein over a set of oncomiRNAs related to the colon cancer.</p> <p>Conclusion</p> <p>The interface provided by the PuTmiR web server provides an important resource for analyzing the direct and indirect regulation of human miRNAs. While it is already an established fact that miRNAs are regulated by TFs binding to their USR, this database might possibly help to study whether an miRNA can also be regulated by the TFs binding to their DSR.</p

Identification and molecular characterization of bone-related micrornas: functional implications

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014MicroRNAs (miRNAs) are a conserved class of small RNAs providing a post-transcriptional mechanism for fine-tuning of intricate physiological and pathological cellular processes, such as those affecting development. Skeletogenesis however, was so far poorly investigated and mainly focused on mammalian models, with a general lack of knowledge concerning other vertebrates. We aimed at the identification of bone-related miRNAs and their characterization from an evolutionary perspective, using fish (mostly zebrafish) as model, in comparison to mammalian systems. First, we focused on miR-223, a miRNA that was associated with bone remodelling. We demonstrated that miR-223 genomic organization/context and primary/secondary structures are largely maintained between human and zebrafish. As in mammals, miR-223 expression in zebrafish was highly correlated with hematopoietic events and osteoclastogenesis. Finally, miR-223 targets identified in mammals were also predicted in zebrafish, supporting a functional conservation of this miRNA. In a second set of experiments, we studied the biological role of miR-29a, a bone-related miRNA that was fairly investigated in mammals, but with no mineralogenic effects yet demonstrated. We took advantage of our fish bone-derived systems to explore miR-29a mineralogenic effects through gain-of-function experiments. We demonstrated a strong stimulation of this process through a mechanism probably involving the canonical Wnt signalling. Once more, through bioinformatics analysis, patterns of expression and target prediction/validation, we provided evidences for miR-29 conservation throughout evolution. Finally, we explored miR-214 putative roles on skeleton formation in vertebrates. Although our initial hypothesis of miR-214 involvement in osteogenesis was recently demonstrated by Wang et al. (2013), we proceeded with our investigation and finally showed that miR-214 is also associated with chondrogenesis. Overexpression of miR-214 in ATDC5 cells mitigated differentiation and down-regulated Mgp and Osteocalcin, probably by targeting Atf4. This work provides novel evidence that some miRNAs have conserved functions across vertebrates and, probably, conserved regulatory mechanisms of action.Nos últimos anos, assistiu-se a uma marcante expansão na área da biologia molecular, devendo-se isto principalmente à descoberta de pequenas moléculas de RNA não codante e ao seu modo peculiar de intervir na regulação genética. Dentro deste grupo de moléculas, os microRNAs (miRNAs) são, definitivamente, a classe melhor compreendida, o que se comprova pelo crescimento exponencial do número de trabalhos publicados desde a sua descoberta. Os miRNAs, na sua forma matura, são RNAs com aproximadamente 22 nucleótidos (nt), altamente conservados em vertebrados e que asseguram um controlo apertado de vários processos celulares através de uma regulação pós-transcricional. Esta regulação ocorre através da ligação específica do miRNA à 3’UTR do RNA mensageiro (mRNA). Neste mecanismo, destaca-se o envolvimento do complexo RISC (RNA-induced silencing complex; associado ao miRNA), a complementaridade da denominada região “seed” (extremidade 5’ do miRNA) ao mRNA, e o consequente bloqueio da tradução ou degradação do mRNA. Desta forma, cada miRNA pode regular centenas de genes transcritos, e de facto, hoje em dia pensa-se que a maioria dos genes humanos são controlados por miRNAs. Assim, os miRNAs são considerados não só importantes reguladores de múltiplos processos biológicos, incluindo desenvolvimento, diferenciação e apoptose celular, mas também responsáveis por vários processos patológicos, como o cancro, onde se observou que inúmeros miRNAs têm a sua expressão desregulada. Assim, a caracterização dos miRNAs (a vários níveis) é fundamental para a compreensão das suas funções, permitindo alargar também o conhecimento dos processos biológicos e patológicos onde estão envolvidos. Apesar do conhecimento sobre miRNAs ter aumentado francamente nos últimos anos, o papel dos miRNAs na formação e homeostasia do osso ainda está pouco caracterizado, e a maioria dos estudos tem abordado principalmente esta forma de regulação em mamíferos, havendo assim uma lacuna de conhecimento na regulação destes processos noutros vertebrados. Neste sentido, este trabalho focou-se na identificação de miRNAs potencialmente envolvidos na regulação do osso e na sua caracterização numa perspectiva evolutiva, usando o peixe (essencialmente o peixe-zebra) como modelo, e em comparação com mamíferos. Numa primeira abordagem, focámos a nossa investigação no estudo do miR-223, um miRNA anteriormente associado à diferenciação celular da linhagem hematopoiética e ao processo de remodelação óssea. Neste estudo, demonstramos que a organização e contexto genómicos do miR-223 estão preservados em vertebrados, verificando-se uma conservação das estruturas primária e secundária do pre-miR-223 em 46 espécies. Este estudo mostra ainda que a expressão deste miRNA se correlaciona com determinadas fases do desenvolvimento do peixe-zebra onde a hematopoiese e a osteoclastogénese são eventos predominantes. Além disso, este estudo mostra que o miR-223 apresenta uma expressão elevada no principal órgão hematopoético de peixes e ratinhos adultos (rim anterior e medula óssea, respectivamente), sugerindo que a função hematopoiética também se encontra conservada. Por último, através de análise bioinformática demonstrámos que a regulação de genes alvo do miR-223 em mamíferos também deverá estar mantida em peixe-zebra. Na secção seguinte estudámos o papel biológico do miR-29a, cujo efeito osteogénico em mamíferos se encontra bem caracterizado, mas sem nenhum fenótipo mineralogénico ainda associado. Neste estudo utilizámos uma linha celular derivada do osso de peixe previamente desenvolvida no nosso laboratório e com capacidade de mineralização in vitro. A fim de explorar os efeitos mineralogénicos do miR-29a foram realizadas experiências de ganho de função. O aumento dos níveis endógenos deste miRNA resultaram num incremento da mineralização da matriz extra-celular, o que provavelmente terá sido devido a uma aceleração da diferenciação celular pelo potenciamento da via de sinalização Wnt, tal como evidenciado pela acumulação de um dos seus principais componentes, a -catenina. Além disso, foi demonstrada a conservação da função deste miRNA através de estudos baseados em homologia de sequências, análise de sintenia, padrão de expressão tecidular e na manutenção da regulação do SPARC, um alvo previamente descrito em mamíferos. Reforçou-se assim a ideia de que o miR-29a é um regulador crucial na diferenciação de osteoblastos, induzindo um aumento da mineralização em sistemas in vitro. Finalmente, explorámos a hipótese do miR-214 ser regulador da formação do esqueleto/osso, em vertebrados. Apesar da nossa primeira hipótese, que consistia no envolvimento do miR-214 na osteogénese, ter sido entretanto demonstrada através do trabalho realizado por Wang et al. (2013), continuámos com este estudo, tentando demonstrar um potencial envolvimento deste miRNA na condrogénese, um processo essencial na formação do esqueleto de vertebrados. Através do padrão de expressão espacial e temporal do miR-214 durante o desenvolvimento do peixe-zebra, verificou-se uma clara associação com estruturas cartilagíneas. Adicionalmente, demonstrámos que a região reguladora (promotor) do transcrito primário deste miRNA se encontra conservada em oito vertebrados, assim como os locais de ligação de factores de transcrição (associados à condrogénese e/ou osteogénese) identificados. De acordo com a análise funcional deste promotor, concluiu-se que esta região reguladora (quer de peixe-zebra quer de humano) é activada e regulada de forma semelhante em condrócitos e osteoblastos. Por último, verificou-se que a sobreexpressão do miR-214 nas células ATDC5, um modelo in vitro para a condrogénese, atenua a diferenciação condrocítica, possivelmente através da regulação do gene Atf4. O decréscimo simultâneo de dois marcadores ósseos, a Mgp e a osteocalcina, aquando da sobreexpressão deste miRNA sugere que a mineralização dos condrócitos poderá estar comprometida nesta condição. Assim, propomos que o miR-214 desempenha um papel fundamental na formação do esqueleto de vertebrados, não apenas pela regulação da osteogénese, mas também pelo controlo da condrogénese, promovendo assim a normal e equilibrada formação de estruturas ósseas e cartilagíneas. No seu conjunto, estes estudos evidenciam uma conservação na função e mecanismos de regulação de muitos dos miRNAs identificados em vertebrados. Este conhecimento é bastante importante, por exemplo para a investigação de tratamento de patologias, uma vez que permite a utilização de modelos alternativos no rastreio de potenciais alvos terapêuticos, com particular destaque para as vias reguladas por miRNAs. Nesta perspectiva, em doenças como por exemplo a osteoporose, onde se verifica uma perda de massa óssea, terapias que estimulem a acção de miRNAs que promovam a osteoblastogénese ou que inibam a osteoclástogénese, são atractivas e com grande potencial na estimulação da formação óssea ou na redução da reabsorção óssea excessiva, respectivamente.Fundação para a Ciência e Tecnologia (SFRH/BD/38607/2007),pelo financiamento da bolsa de doutoramento, Fundação Calouste Gulbenkian, através do programa ‘‘Na Fronteira das Ciências da Vida’’ pelo co-financiamento deste trabalho

Transcriptional Regulation Of MicroRNA Genes And The Regulatory Networks In Which They Participate

MicroRNA genes are short, non-coding RNAs that function as post-transcriptional gene regulators. Although they have been implicated in organismal development as well as a variety of human diseases, there is still surprisingly little known about their transcriptional regulation. The understanding of microRNA transcription is very important for determining their regulators as well as the specific role they may play in signaling cascades. This dissertation focused on the comparison of mammalian microRNA promoters and upstream sequences to those of known protein coding genes. This dissertation is also focused on determining potential regulatory networks that microRNA genes may participate in, particularly those networks involved in the TGFβ / SMAD signaling pathway. The comparison of intergenic microRNA upstream sequences to those of protein coding genes revealed that the former are up to twice as conserved as the latter, except in the first 500 base pairs where the conservation is similar. Further investigation of the upstream sequences by RNA Polymerase II ChIP-chip revealed the transcription start site for 35 primary-microRNA transcripts. The identification of features capable of distinguishing core promoter regions from background sequences using a support vector machine approach revealed that the transcription start site of primary-microRNA genes share the same sequence features as protein coding genes. These results suggest that in fact microRNA genes are transcribed by the same mechanism by which protein coding genes are transcribed. This information allowed us to then identify the regulatory elements of microRNA genes in the same manner in which we use for protein coding genes. Identification of a SMAD family transcription factor binding site upstream of the human let-7d microRNA revealed a feed-forward regulatory circuit involved in epithelial mesenchymal transition. This provided the first evidence of a direct link between a growth factor and the expression of a microRNA gene. The understanding of microRNA transcriptional regulation has great public health significance. The ability to understand how these post-transcriptional gene regulators function in cellular networks may provide new molecular targets for cures or therapies to a variety of human diseases

Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data

Background: MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology: We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion: miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex. © 2009 Corcoran et al

Evolution of MicroRNAs and the Diversification of Species

MicroRNAs (miRNAs) are ancient, short noncoding RNA molecules that regulate the transcriptome through post-transcriptional mechanisms. miRNA riboregulation is involved in a diverse range of biological processes, and misregulation is implicated in disease. It is generally thought that miRNAs function to canalize cellular outputs, for instance as “fail-safe” repressors of gene misexpression. Genomic surveys in humans have revealed reduced genetic polymorphism and the signature of negative selection for both miRNAs themselves and the target sequences to which they are predicted to bind. We investigated the evolution of miRNAs and their binding sites across cichlid fishes from Lake Malawi (East Africa), where hundreds of diverse species have evolved in the last million years. Using low-coverage genome sequence data, we identified 100 cichlid miRNA genes with mature regions that are highly conserved in other animal species. We computationally predicted target sites on the 3′-untranslated regions (3′-UTRs) of cichlid genes to which miRNAs may bind and found that these sites possessed elevated single nucleotide polymorphism (SNP) densities. Furthermore, polymorphic sites in predicted miRNA targets showed higher minor allele frequencies on average and greater genetic differentiation between Malawi lineages when compared with a neutral expectation and nontarget 3′-UTR SNPs. Our data suggest that divergent selection on miRNA riboregulation may have contributed to the diversification of cichlid species and may similarly play a role in rapid phenotypic evolution of other natural systems