Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide RING1B binding in Fbxl10 overexpressing 3T3-L1 cells at day 2 of differentiation

ABSTRACT: We analyzed RING1B binding regions in 3T3-L1 cell lines transduced with the retroviral vector for V5-tagged Fbxl10 or its dF-box mutant. RING1B ChIP-seq in empty, Fbxl10-1, and dF-box mutant vector transduced 3T3-L1 preadipocytes, in duplicate, and V5-Fbxl10 ChIP-seq in Fbxl10 overexpressing 3T3-L1 cells

ORGANISM(S): Mus musculus

SUBMITTER: Takeshi Inagaki

PROVIDER: E-GEOD-64312 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The FBXL10/KDM2B scaffolding protein associates with novel polycomb repressive complex-1 to regulate adipogenesis.

Inagaki Takeshi T Iwasaki Satoshi S Matsumura Yoshihiro Y Kawamura Takeshi T Tanaka Toshiya T Abe Yohei Y Yamasaki Ayumu A Tsurutani Yuya Y Yoshida Ayano A Chikaoka Yoko Y Nakamura Kanako K Magoori Kenta K Nakaki Ryo R Osborne Timothy F TF Fukami Kiyoko K Aburatani Hiroyuki H Kodama Tatsuhiko T Sakai Juro J

The Journal of biological chemistry 20141222 7

Polycomb repressive complex 1 (PRC1) plays an essential role in the epigenetic repression of gene expression during development and cellular differentiation via multiple effector mechanisms, including ubiquitination of H2A and chromatin compaction. However, whether it regulates the stepwise progression of adipogenesis is unknown. Here, we show that FBXL10/KDM2B is an anti-adipogenic factor that is up-regulated during the early phase of 3T3-L1 preadipocyte differentiation and in adipose tissue in ...[more]

PMID: 25533466

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Project description: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.

Dataset Information

Genome-wide RING1B binding in Fbxl10 overexpressing 3T3-L1 cells at day 2 of differentiation

Publications

The FBXL10/KDM2B scaffolding protein associates with novel polycomb repressive complex-1 to regulate adipogenesis.

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