PloS one

Collagen 6A3 (Col6a3), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that Col6a3 is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated Col6a3 expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing Taf4(-/-) MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in Taf4(-/-) MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, Wnt9a is activated and the Sfrp2 antagonist of Wnt signalling is repressed. Surprisingly, treatment of Taf4(-/-) MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses Col6a3 expression independently of TAF4 expression and Col6a3 silencing is sufficient to restore contact inhibition in Taf4(-/-) MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing Sfrp2 expression to antagonize Wnt signalling. All together, these results reveal a critical role for Col6a3 in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of Col6a3. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth

Genome Sequence of Halomonas sp. Strain A3H3, Isolated from Arsenic-Rich Marine Sediments

We report the genome sequence of Halomonas sp. strain A3H3, a bacterium with a high tolerance to arsenite, isolated from multicontaminated sediments of the l'Estaque harbor in Marseille, France. The genome is composed of a 5,489,893-bp chromosome and a 157,085-bp plasmid

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.Peer reviewe

An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells

International audienceGenome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion, the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells

TAF4 Inactivation Reveals the 3 Dimensional Growth Promoting Activities of Collagen 6A3

<div><p>Collagen 6A3 (<i>Col6a3</i>), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that <i>Col6a3</i> is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated <i>Col6a3</i> expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing <i>Taf4^−/−</i> MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in <i>Taf4^−/−</i> MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, <i>Wnt9a</i> is activated and the <i>Sfrp2</i> antagonist of Wnt signalling is repressed. Surprisingly, treatment of <i>Taf4^−/−</i> MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses <i>Col6a3</i> expression independently of TAF4 expression and <i>Col6a3</i> silencing is sufficient to restore contact inhibition in <i>Taf4^−/−</i> MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing <i>Sfrp2</i> expression to antagonize Wnt signalling. All together, these results reveal a critical role for <i>Col6a3</i> in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of <i>Col6a3</i>. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth.</p></div

Wnt signalling is required for 3D growth.

<p><b>A–C</b>. Results of RT-qPCR analysis of the indicated genes in C1 or C3 cells grown for 3 and 9–10 days in monolayer cluture, 10 days as fibrospheres (F1 and F2) or in C3 cells expressing the indicated shRNAs. <b>D</b> Phase contrast microscopy (20× magnification) of C3 cells grown in presence or absence of the indicated Wnt pathway inhibitors or expressing control shRNA and shRNA directed againt <i>Wnt9a</i>. <b>E</b>. Confocal microscopy sections through foci grown in the presence or absence of XAV939 showing that the nuclear β-catenin localisation seen in the control C3 cells is lost in presence of XAV939. <b>F</b>. Immunostaining for YAP1 and SOX2 in foci in presence or absence of XAV939.</p

3D growth of C3 cells.

<p><b>A</b>. Phase contrast microscopy (20× magnification) of C1 and C3 cells grown as dense cultures for 3 days. <b>B</b>. Growth of C1 and C3 cells after 10 days in soft agar. <b>C</b>. Phase contrast microscopy (12× magnification) of C1 cells or C3 cells in grown for 10 days under non-adherent conditions.</p

Changes in gene expression upon 3D growth of C3 cells.

<p><b>A–B</b> Venn diagrammes showing overlapping changes in gene expression upon growth as dense cultures or fibrospheres. <b>C–F</b>. Ontology analysis (<a href="http://david.abcc.ncifcrf.gov/" target="_blank">http://david.abcc.ncifcrf.gov/</a>) of the deregulated genes showing some relevant categories from the CC-FAT and BP FAT classifications. The number of genes and the associated <i>P</i> values for each category are indicated.</p

Differential regulation of Hippo signalling in C1 and C3 MEFs.

<p><b>A</b> Results of RT-qPCR analysis of the indicated genes in C1 or C3 cells grown for 3 and 10 days in monolayer culture, or 10 days as fibrospheres (F1). <b>B</b> Immunostaining of C1 and C3 MEFs for YAP1 at low or high densities. <b>C</b> Immunostaining of C3 MEFs grown at high density for YAP1, SOX2 or β-catenin as indicated.</p

ATRA induced changes in properties of C3 MEFs.

<p><b>A</b>. Phase contrast microscopy (20× magnification) of C3 cells grown as dense cultures for 3 days in presence or absence of ATRA. <b>B</b>. Phase contrast microscopy (12× magnification) of C3 cells in grown for 10 days as fibrospheres in presence or absence of ATRA. <b>C</b>–<b>D</b> Effect of ATRA on <i>Col6a3</i> expression in C3 and C1 cells grown for the indicated number of days in presence or absence of ATRA.</p