Nicolás Bellora

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer.

Notch has been linked to β-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/β-catenin (down-regulated when blocking Wnt/β-catenin) that are directly regulated by Notch (repressed by γ-secretase inhibitors and up-regulated by active Notch1 in the absence of β-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through β-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/β-catenin pathway in tumors implanted s.c. in nude mice. Crossing APCMin/+ with Jagged1+/Δ mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear β-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by β-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.

Housekeeping genes tend to show reduced upstream sequence conservation

BackgroundUnderstanding the constraints that operate in mammalian gene promoter sequences is of key importance to understand the evolution of gene regulatory networks. The level of promoter conservation varies greatly across orthologous genes, denoting differences in the strength of the evolutionary constraints. Here we test the hypothesis that the number of tissues in which a gene is expressed is related in a significant manner to the extent of promoter sequence conservation.ResultsWe show that mammalian housekeeping genes, expressed in all or nearly all tissues, show significantly lower promoter sequence conservation, especially upstream of position -500 with respect to the transcription start site, than genes expressed in a subset of tissues. In addition, we evaluate the effect of gene function, CpG island content and protein evolutionary rate on promoter sequence conservation. Finally, we identify a subset of transcription factors that bind to motifs that are specifically over-represented in housekeeping gene promoters.ConclusionThis is the first report that shows that the promoters of housekeeping genes show reduced sequence conservation with respect to genes expressed in a more tissue-restricted manner. This is likely to be related to simpler gene expression, requiring a smaller number of functional cis-regulatory motifs.

Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

Matrin3 is an RNA‐ and DNA‐binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co‐regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3.

The translational landscape of the splicing factor SRSF1 and its role in mitosis.

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer. DOI: http://dx.doi.org/10.7554/eLife.02028.001

Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

BackgroundThe arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes.ResultsWe observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters.ConclusionThe study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

Chromatin-Bound IκBα Regulates a Subset of Polycomb Target Genes in Differentiation and Cancer

IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation.

Evolution of primate orphan proteins

Genomes contain a large number of genes that do not have recognizable homologues in other species. These genes, found in only one or a few closely related species, are known as orphan genes. Their limited distribution implies that many of them are probably involved in lineage-specific adaptive processes. One important question that has remained elusive to date is how orphan genes originate. It has been proposed that they might have arisen by gene duplication followed by a period of very rapid sequence divergence, which would have erased any traces of similarity to other evolutionarily related genes. However, this explanation does not seem plausible for genes lacking homologues in very closely related species. In the present article, we review recent efforts to identify the mechanisms of formation of primate orphan genes. These studies reveal an unexpected important role of transposable elements in the formation of novel protein-coding genes in the genomes of primates.

A chromatin code for alternative splicing involving a putative association between CTCF and HP1α proteins

BackgroundAlternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of the heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated with chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing.ResultsUsing chromatin immunoprecipitation sequencing (ChIP-Seq) data for CTCF, AGO1, HP1α, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, we have analyzed the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two cell lines, MCF7 and MCF10. Using Machine Learning techniques, we identified the changes in chromatin signals that are most significantly associated with splicing regulation between these two cell lines. Moreover, we have built a map of the chromatin signals on the pre-mRNA, that is, a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1α, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, we found a significant association of HP1α and CTCF activities around the regulated exons and a putative DNA binding site for HP1α.ConclusionsOur results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1α and CTCF near regulated exons. Additionally, we find further evidence for the involvement of HP1α and AGO1 in chromatin-related splicing regulation.

In mammalian foetal testes, SOX9 regulates expression of its target genes by binding to genomic regions with conserved signatures

Abstract In mammalian embryonic gonads, SOX9 is required for the determination of Sertoli cells that orchestrate testis morphogenesis. To identify genetic networks directly regulated by SOX9, we combined analysis of SOX9-bound chromatin regions from murine and bovine foetal testes with sequencing of RNA samples from mouse testes lacking Sox9. We found that SOX9 controls a conserved genetic programme that involves most of the sex-determining genes. In foetal testes, SOX9 modulates both transcription and directly or indirectly sex-specific differential splicing of its target genes through binding to genomic regions with sequence motifs that are conserved among mammals and that we called ‘Sertoli Cell Signature’ (SCS). The SCS is characterized by a precise organization of binding motifs for the Sertoli cell reprogramming factors SOX9, GATA4 and DMRT1. As SOX9 biological role in mammalian gonads is to determine Sertoli cells, we correlated this genomic signature with the presence of SOX9 on chromatin in foetal testes, therefore equating this signature to a genomic bar code of the fate of foetal Sertoli cells. Starting from the hypothesis that nuclear factors that bind to genomic regions with SCS could functionally interact with SOX9, we identified TRIM28 as a new SOX9 partner in foetal testes.

The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators

Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery.