Dataset Information

Proteomic characterization of the role of Snail1 in the differentiation of 3T3-L1 fibroblasts

ABSTRACT: Snail1 is a transcriptional repressor required for a correct embryonic development. In cancer, Snail1 promotes the epithelial to mesenchymal transition in tumorigenic epithelial cells. In this work, we have analyzed the control of Snail1 in the differentiation of the 3T3-L1 cell line derived from murine embryo cells. The activation by snail of 3T3-L1 induced typical markers of cancer-activated fibroblasts as S100A4 or CD44. We generated 3T3-L1 cells stably over expressing Snail1 (3T3L1/Snail1) and control (3T3-L1/control) cells. We used SILAC quantitative approach to identify and characterize protein alterations induced by Snail1. Cells were fractionated in 5 subcellular fractions. The nuclear fraction of the cells was separated by 10% SDS-PAGE. Gels with forward and reverse experiments were stained with Coomassie Blue and cut into 18 slices prior to reduction, alkylation and digestion with trypsin. Tryptic peptides were scanned and fragmented with a linear ion trap-Orbitrap Velos (ThermoScientific). We identified a total of 3108 proteins, with 2572 quantified proteins, and 565 proteins modulated >1.5-fold by Snail1 overexpression. Among them, we found interesting up-regulated proteins associated to early differentiation of adipogenesis (C/EBPβ) and down-regulated proteins implicated in the final stages of differentiation to adipocytes (Fatty acid-binding protein or Fatty acid synthase). We also observed as down-regulated proteins important mediators of PPARγ pathway. We also observed downregulation of proteins implicated in mTOR, SRC and JAK/STAT pathway. We validated these proteomics data by western blot and qPCR in 3T3-L1 cells and other types of fibroblasts with capable to differentiate to terminal mesenchymal phenotypes, as well as in mesenchymal stem cells (MSC). This work provided insight into novel proteins with potential roles in the regulation of differentiation of the 3T3-L1 and MSCs as Nr2F6, ASC-1, Prrx1 or Cbx6. These candidates are down regulated due to the overexpression of Snail1 in 3T3-L1 cells. We next investigated the potential binding of Snail1 to promoter of these candidates. In silico analysis with MatInspector program revealed various putative E-box consensus motifs for Snail1. We performed ChIP and Luciferase assay to validate Snail1 binds to different E-box motifs of our candidates. Additionally, we analyzed the ability to prevent the differentiation to adipocytes of the 3T3-L1 cells using siRNAs. This work provided insight into novel proteins with potential roles in the regulation of differentiation to adipocytes of the 3T3-L1 and mMSC cells as Nr2F6, ASC-1, Prrx1 or Cbx6 controlled by Snail1.

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryo, Fibroblast

SUBMITTER: Alberto Pelaez García

LAB HEAD: Ignacio Casal

PROVIDER: PXD001529 | Pride | 2015-01-06

REPOSITORIES: Pride

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A proteomic analysis reveals that Snail regulates the expression of the nuclear orphan receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and interleukin 17 (IL-17) to inhibit adipocyte differentiation.

Peláez-García Alberto A Barderas Rodrigo R Batlle Raquel R Viñas-Castells Rosa R Bartolomé Rubén A RA Torres Sofía S Mendes Marta M Lopez-Lucendo María M Mazzolini Rocco R Bonilla Félix F García de Herreros Antonio A Casal J Ignacio JI

Molecular & cellular proteomics : MCP 20141210 2

Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 57 ...[more]

PMID: 25505127

Publication: 1/2

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Project description:Enhancers are developmentally-controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. Here, we show by genome-wide mapping that the newly discovered DNA modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells as well as during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates like Meis1 in P19 cells and PPARgamma in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5mC hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes Genome-wide 5hmC distribution was determined using hMeDIP-seq. Cells used in this study are P19.6 mouse embryonal carnicoma cells and P19.6 cells treated for 48 hours with 1M-BM-5M all-trans retinoic acid (RA), as well as 3T3-L1 cells and 3T3-L1 derived adipocytes differentiated with dexamethasone, insulin and IBMX (differentiation cocktail - DC). Individual hMeDIP samples from P19.6 ord 3T3-L1 cells were pooled for library preparation. Libraries were prepared and sequenced at the IBL sequencing facility (Lille, France) with an Illumina Genome Analyser II.

			Action	DRS
	3T3L1_SNAIL1_DIRECT.msf	Msf
	3T3L1_SNAIL1_DIRECT.zip	Other
	3T3L1_SNAIL1_REVERSE.msf	Msf
	3T3L1_SNAIL1_REVERSE.zip	Other