Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes

Mecanismes de reconnaissance dans les processus metastatiques : caracterisation d'une lectine membranaire des cellules du carcinome pulmonaire de Lewis

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Acute Rhinosinusitis: A Pharmacoeconomic Review of Antibacterial Use

Acute rhinosinusitis is a common disease, in both children and adult patients, and happens most often in the setting of a viral infection with or without bacterial superinfection. Although spontaneous resolution is common, antibacterials are often prescribed and have a tremendous impact on costs, either directly or through the emergence of resistance in causative or colonising micro-organisms. The purpose of this work was to review published literature from 1989 to 2002 on antibacterial treatment in acute rhinosinusitis from a clinical and economical perspective. A relatively small number of studies have compared antibacterials with placebo and few have suggested that antibacterials are superior to placebo, except when a bacterial cause is established or in the presence of specific CT-scan findings. On the other hand, 58 randomised controlled trials were published between 1989 and 2002, that compared the relative efficacy of various antibacterials. Most of these studies had serious methodological flaws, and no single antibacterial proved superior to its comparators. Economic data are scarce and indicate cost of disease is high. Of the different treatment strategies assessed symptomatic treatment (patients being treated with antibacterials only if they failed to improve after 7 days) was the most cost-effective approach, compared with treating patients on the basis of specific clinical criteria, empirical treatment (all patients initially treated with antibacterials), or radiology-guided treatment. Cost effectiveness varied with disease prevalence. In conclusion, this pharmacoeconomic review of antibacterial use in acute rhinosinusitis shows the need for improvement in the quality of the studies feeding economic analyses, but suggests that huge financial interests are at stake. Savings achievable, by better targeting patients needing antibacterial treatment, could be substantial, and more practical and precise diagnostic procedures are clearly needed. Acute rhinosinusitis is a typical example of a clinical dilemma in which good clinical practice must be balanced against imperfect information and patientsAntibacterials, Cost-effectiveness, Cost-of-illness, Sinusitis

A key enzyme of the leloir pathway is involved in pathogenicity of Leptosphaeria maculans toward oilseed rape

International audienceAgrobacterium tumefaciens-mediated random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m186, is analyzed in detail here. Microscopic analyses of infected plant tissues revealed that m186 is specifically blocked at the invasive growth phase after an unaffected initial penetration stage and is unable to switch to the necrotrophic lifestyle. In addition, m186 exhibits an altered cell wall and seems to be affected in its ability to produce cell-wall-degrading enzymes. The T-DNA insertion occurs in the intergenic region between two head-to-tail genes, leading to a constitutive upregulation of their expression. Complementation experiments showed that only one of these two genes, Lmepi, fully accounts for the mutant phenotype. Bioinformatics and expression analyses along with functional studies suggested that the Lmepi gene encodes for the highly conserved UDP-glucose-4-epimerase, a key enzyme of the Leloir pathway involved in galactose metabolism. For the third time, this study highlights the intimate connection between primary metabolism and pathogenicity in L. maculans. This finding, along with similar data obtained from the related species Stagonospora nodorum, indicates the importance of in planta nutrition for the success of infection of plants by fungi belonging to class Dothideomycete

Investigating the involvement of the chromatin state in the control of effector gene expression in Leptospharia maculans

Leptosphaeria maculans, a hemibiotrophic fungus responsible of stem canker, colonises oilseed rape in two stages: an early stage of cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically without visible symptom the plant before stem canker appears. L. maculans presents a bipartite genome structure alternating gene-rich and transposable element (TE)-rich regions. TE-rich regions, which encompass one third of the genome, are enriched in putative effector-encoding genes that present the same expression pattern (no or a low expression level during in vitro growth and a strong over-expression during early infection in cotyledons and leaves; ‘early’ effectors). In contrast, gene-rich regions were recently reported to contain putative effector-encoding genes specifically expressed during the late stages of stem infection (‘late’ effectors). We have previously investigated the involvement of the chromatin structure of repeat-rich regions on the expression of ‘early’ effector genes: RNAi silencing of two genes encoding key players in heterochromatin assembly through histone modification H3K9me3, HP1 and KMT1, induced an over-expression of genes located in AT-isochores, particularly ‘early’ effector genes but no modification of ‘late’ effector genes expression. Here, we performed analysis of nucleosome positioning, chromatin structure and gene expression at the genome scale combining MAINE-seq, ChIP-seq and RNA-seq data during in vitro growth of L. maculans. We analysed in vitro ChIP-seq data targeting heterochromatin modifications, H3K9me3 and H3K27me3, and a euchromatin mark, H3K4me2 and found that gene-rich regions are associated with H3K4me2 and H3K27me3 while TE-rich regions are associated with H3K9me3. Effector genes are also associated with distinct heterochromatin marks according to their genomic location and expression kinetics: while ‘early’ effector genes located in TE-rich regions are associated with H3K9me3, ‘late’ effector genes located in gene-rich region are associated with H3K27me3. Genome-wide nuclesome positioning was analysed using MAINE-seq data, showing distinct nucleosome organization for genes located in TE-rich or gene-rich regions, and according to gene expression level. Finally, we recently investigated the role of another key player in heterochromatin assembly, KMT6, involved in the heterochromatic-associated histone modification H3K27me3, on the control of effector gene expression. RNAi silencing of KMT6 leads to a deregulation of genes not only associated with H3K27me3 in the wild type strain, suggesting a relocation of different histone modification

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus

Leptosphaeria maculans is a hemibiotophic ascomycete which causes stem canker of oilseed rape. That phytopathogenic fungus interacts with its host (Brassica napus) according to the gene-for-gene concept. The most economically and environment friendly method of control of stem canker is the genetic control by using host resistance. Single gene resistance is extremely efficient, but races of the pathogen virulent towards a resistance gene can appear in a few years and necessitates continuously new breeding programs. Moreover, specific resistances are rare in oilseed rape, and a lot of efforts are made to find other resistance genes in other Brassica species. To date, 11 interactions were genetically characterized between L. maculans avirulence genes and corresponding resistance genes in Brassica, and 5 of those avirulence genes were cloned. Recently, the avirulence gene AvrLm10 which is recognized by the resistance gene Rlm10 of the black mustard (Brassica nigra) has been cloned. AvrLm10 corresponds in fact to two avirulence genes AvrLm10_1 and AvrLm10_2 which are located in the same AT-rich genomic region. They encore for small secreted proteins (SSP), are co-regulated and over-expressed 7 days post-infection. Each of them is necessary but not sufficient to induce resistance towards Rlm10. Silencing of one of those genes is sufficient to abolish recognition by Rlm10. Silencing by RNA interference of AvrLm10-1 induces an increase of lesion size on oilseed rape leaves while silencing of AvrLm10-2 has no major effect on aggressiveness of the fungus. That interaction of two avirulence genes against one resistance gene is therefore different from the classical gene-for-gene concept. It suggests that AvrLm10_1 and AvrLm10_2 could directly interact and / or that they could target the same plant protein. A Y2H screen suggested a direct interaction between AvrLm10-1 and AvrLm10-2. This interaction was confirmed with Bimolecular Fluorescence Complementation (BiFC) experiments. Coimmunoprecipitation experiments are also in progress to confirm this interaction

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus. 7th Effectome meetin