De-regulated expression of the BRG1 chromatin remodeling factor in bone marrow mesenchymal stromal cells induces senescence associated with the silencing of NANOG and changes in the levels of chromatin proteins

<p>Stem cells have a peculiar chromatin architecture that contributes to their unique properties, including uncommitted status, multi/pluripotency and self-renewal. We analyzed the effect of the de-regulation of the SWI/SNF chromatin remodeling complex in mesenchymal stromal cells (MSC) through the silencing and up-regulation of BRG1, which is the ATPase subunit of the complex. The altered expression of BRG1 promoted the senescence of MSC with suppression of the NANOG transcription, which is part of the transcriptional circuitry governing stem cell functions. To gain insight on the way NANOG was silenced, we evaluated how the de-regulated BRG1 expression affect the binding of activators and repressors on the NANOG promoter. We found 4 E2F binding motifs on NANOG promoter, which can be occupied by RB1 and RB2/P130. These are members of the retinoblastoma gene family. In MSC with a silenced BRG1, the relative binding of the 2 retinoblastoma proteins increased, and this was associated with the recruitment of DNMT1. This induced the methylation of CpG on the NANOG promoter. Opposingly, when a high level of BRG1 was present, the same E2F binding motifs were docking sites for BRG1, which induced chromatin compaction without CpG methylation but with increased histone deacetylation, associated with the presence of HDAC1 on E2F binding sites. Besides the sharp regulation of the NANOG expression, we evidenced, through proteomic analysis, that the de-regulation of the SWI/SNF function affected the expression of histones and other nuclear proteins involved in “nuclear architecture,” suggesting that BRG1 may act as global regulator of gene expression.</p

Bar chart of the enriched biological functions of differentially expressed proteins in pFF+ versus pFF- samples.

<p>The assessment of significantly enriched biological functions for differentially expressed proteins was performed by IPA software. p-values and the number (#) of molecules mapped on the enriched categories are reported. </p

Mapping of a subset of differentially expressed proteins in pFF+ versus pFF- samples on the complement system canonical pathway.

<p>According to IPA categorization, up-regulated proteins are coloured in red. The assessment of significantly enriched processes networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test. The pathway components identified by the algorithm or with no significant differences in their expression levels are reported in white and blue, respectively. Molecules are named according to IPA software.</p

Mapping of a subset of differentially expressed proteins in pFF+ versus pFF- samples on the acute phase response signaling canonical pathway.

<p>According to IPA categorization, up-regulated proteins are coloured in red. The assessment of significantly enriched processes networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test. The pathway components identified by the algorithm or with no significant differences in their expression levels are reported in white and blue, respectively. Molecules are named according to IPA software.</p

Expression levels of chemokines in hFF samples.

<p>hFF samples were simultaneously screened for the concentration of the chemokines Eotaxin (A), IP-10 (B), MIP-1α (C), MIP-1β (D), MCP-1 (E) and RANTES (F) by using the Bio-Plex 27-plex human cytokine kit from BioRad according to the manufacturer’s protocol. Measurements were performed on individual hFF samples diluted (1:2) using the standard curves optimization and the calculation of analyte concentrations of the Bio-Plex Manager software. Data are reported as scatter plots and average concentrations. ** p-value≤ 0.05 FF+ <i>versus</i> FF- dataset.</p

Mapping of a subset of differentially expressed proteins in pFF+ versus pFF- samples on the coagulation system pathway.

<p>According to IPA categorization, up-regulated proteins are coloured in red. The assessment of significantly enriched processes networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test. The pathway components identified by the algorithm or with no significant differences in their expression levels are reported in white and blue, respectively. Molecules are named according to IPA software.</p

Multiple sequence alignments showing similarity and conserved domains identified by the Interpro resource available at http://www.ebi.ac.uk/interpro/in A) Pep-13 transglutaminase peptide elicitors (according to Brunner et al., 2002 [52]), B) berberin-like proteins (Pfam PF08031), C) carbonic anhydrases (PROSITE PS51144; Pfam PF00194) and D) pectinesterase/pectate lyases (Pfam PF01095/SUPERFAMILY SSF51126).

<p>Signal sequences at N-terminus (SignP) and the consensus sequences are also reported.</p

<i>Fagus sylvatica</i> root exudate characterization.

<p>A) Representative photograph shoving the ability of <i>Fagus sylvatica</i> root exudate to attract <i>P. plurivora</i> zoospores. B) ¹H NMR spectrum of <i>F. sylvatica</i> root exudate acquired at 300.03 MHz in methanol-d4-buffer phosphate 1∶1. Protons responsible for NMR signals of molecules are highlighted in red in the structures. Signals of anomeric protons are marked with asterisks. C) Free amino acid profile of <i>F. sylvatica</i> root exudate (lower panel) compared to standards (upper panel). D) Bar chart showing the amount (nmol/mg of root exudate) of free amino acids detected in the <i>F. sylvatica</i> root exudate.</p

List of effectors identified in the <i>P. plurivora</i> secretome by high resolution LC MS/MS.

<p>*Sequences containing the RXLR motif.</p><p>**Nucleotide sequence submitted to the EMBL/GenBank/DDBJ databases.</p><p>The secretion prediction according to signal peptide probability of Signal P 4.1 server is reported. Y and N indicate the presence or absence of the signal peptide for secretion, respectively.</p><p>List of effectors identified in the <i>P. plurivora</i> secretome by high resolution LC MS/MS.</p

Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides

Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane