Identification of a novel PPARß/δ/miR-21-3p axis in UV-induced skin inflammation.

Although excessive exposure to UV is widely recognized as a major factor leading to skin perturbations and cancer, the complex mechanisms underlying inflammatory skin disorders resulting from UV exposure remain incompletely characterized. The nuclear hormone receptor PPARß/δ is known to control mouse cutaneous repair and UV-induced skin cancer development. Here, we describe a novel PPARß/δ-dependent molecular cascade involving TGFß1 and miR-21-3p, which is activated in the epidermis in response to UV exposure. We establish that the passenger miRNA miR-21-3p, that we identify as a novel UV-induced miRNA in the epidermis, plays a pro-inflammatory function in keratinocytes and that its high level of expression in human skin is associated with psoriasis and squamous cell carcinomas. Finally, we provide evidence that inhibition of miR-21-3p reduces UV-induced cutaneous inflammation in ex vivo human skin biopsies, thereby underlining the clinical relevance of miRNA-based topical therapies for cutaneous disorders.

Birth and expression evolution of mammalian microRNA genes.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression, yet their origins and functional evolution in mammals remain little understood due to the lack of appropriate comparative data. Using RNA sequencing, we have generated extensive and comparable miRNA data for five organs in six species that represent all main mammalian lineages and birds (the evolutionary outgroup) with the aim to unravel the evolution of mammalian miRNAs. Our analyses reveal an overall expansion of miRNA repertoires in mammals, with threefold accelerated birth rates of miRNA families in placentals and marsupials, facilitated by the de novo emergence of miRNAs in host gene introns. Generally, our analyses suggest a high rate of miRNA family turnover in mammals with many newly emerged miRNA families being lost soon after their formation. Selectively preserved mammalian miRNA families gradually evolved higher expression levels, as well as altered mature sequences and target gene repertoires, and were apparently mainly recruited to exert regulatory functions in nervous tissues. However, miRNAs that originated on the X chromosome evolved high expression levels and potentially diverse functions during spermatogenesis, including meiosis, through selectively driven duplication-divergence processes. Overall, our study thus provides detailed insights into the birth and evolution of mammalian miRNA genes and the associated selective forces.

Overexpression of transcription factor Sp1 leads to gene expression perturbations and cell cycle inhibition.

BACKGROUND: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. METHODOLOGY AND PRINCIPAL FINDINGS: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. CONCLUSION: This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

GC content evolution of the human and mouse genomes: insights from the study of processed pseudogenes in regions of different recombination rates.

Processed pseudogenes are generated by reverse transcription of a functional gene. They are generally nonfunctional after their insertion and, as a consequence, are no longer subjected to the selective constraints associated with functional genes. Because of this property they can be used as neutral markers in molecular evolution. In this work, we investigated the relationship between the evolution of GC content in recently inserted processed pseudogenes and the local recombination pattern in two mammalian genomes (human and mouse). We confirmed, using original markers, that recombination drives GC content in the human genome and we demonstrated that this is also true for the mouse genome despite lower recombination rates. Finally, we discussed the consequences on isochores evolution and the contrast between the human and the mouse pattern.

Homology-dependent methylation in primate repetitive DNA.

In mammals, several studies have suggested that levels of methylation are higher in repetitive DNA than in nonrepetitive DNA, possibly reflecting a genome-wide defense mechanism against deleterious effects associated with transposable elements (TEs). To analyze the determinants of methylation patterns in primate repetitive DNA, we took advantage of the fact that the methylation rate in the germ line is reflected by the transition rate at CpG sites. We assessed the variability of CpG substitution rates in nonrepetitive DNA and in various TE and retropseudogene families. We show that, unlike other substitution rates, the rate of transition at CpG sites is significantly (37%) higher in repetitive DNA than in nonrepetitive DNA. Moreover, this rate of CpG transition varies according to the number of repeats, their length, and their level of divergence from the ancestral sequence (up to 2.7 times higher in long, lowly divergent TEs compared with unique sequences). This observation strongly suggests the existence of a homology-dependent methylation (HDM) mechanism in mammalian genomes. We propose that HDM is a direct consequence of interfering RNA-induced transcriptional gene silencing.

Recombination drives the evolution of GC-content in the human genome.

Unraveling the evolutionary forces responsible for variations of neutral substitution patterns among taxa or along genomes is a major issue in the identification of functional sequence features. Mammalian genomes show large-scale regional variations of GC-content (the isochores), but the substitution processes at the origin of this structure are poorly understood. We have analyzed the pattern of neutral substitutions in 14.3 Mb of primate noncoding regions. We show that the GC-content toward which sequences are evolving is strongly correlated (r(2) = 0.61, P

Heterotachy and functional shift in protein evolution.

Study of structure/function relationships constitutes an important field of research, especially for modification of protein function and drug design. However, the fact that rational design (i.e. the modification of amino acid sequences by means of directed mutagenesis, based on knowledge of the three-dimensional structure) appears to be much less efficient than irrational design (i.e. random mutagenesis followed by in vitro selection) clearly indicates that we understand little about the relationships between primary sequence, three-dimensional structure and function. The use of evolutionary approaches and concepts will bring insights to this difficult question. The increasing availability of multigene family sequences that has resulted from genome projects has inspired the creation of novel in silico evolutionary methods to predict details of protein function in duplicated (paralogous) proteins. The underlying principle of all such approaches is to compare the evolutionary properties of homologous sequence positions in paralogs. It has been proposed that the positions that show switches in substitution rate over time--i.e., 'heterotachous sites'--are good indicators of functional divergence. However, it appears that heterotachy is a much more general process, since most variable sites of homologous proteins with no evidence of functional shift are heterotachous. Similarly, it appears that switches in substitution rate are as frequent when paralogous sequences are compared as when orthologous sequences are compared. Heterotachy, instead of being indicative of functional shift, may more generally reflect a less specific process related to the many intra- and inter-molecular interactions compatible with a range of more or less equally viable protein conformations. These interactions will lead to different constraints on the nature of the primary sequences, consistently with theories suggesting the non-independence of substitutions in proteins. However, a specific type of amino acid variation might constitute a good indicator of functional divergence: substitutions occurring at positions that are generally slowly evolving. Such substitutions at constrained sites are indeed much more frequent soon after gene duplication. The identification and analysis of these sites by complementing structural information with evolutionary data may represent a promising direction to future studies dealing with the functional characterization of an ever increasing number of multi-gene families identified by complete genome analysis.