A proximal activator of transcription in epithelial-mesenchymal transition
Christo D. Venkov, Andrew J. Link, Jennifer L. Jennings, David Plieth, Tsutomu Inoue, Kojiro Nagai, Carol Xu, Yoana N. Dimitrova, Frank J. Rauscher III, Eric G. Neilson
Christo D. Venkov, Andrew J. Link, Jennifer L. Jennings, David Plieth, Tsutomu Inoue, Kojiro Nagai, Carol Xu, Yoana N. Dimitrova, Frank J. Rauscher III, Eric G. Neilson
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Research Article

A proximal activator of transcription in epithelial-mesenchymal transition

Abstract

Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site–1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box–binding factor–A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, β-catenin, ZO 1, vimentin, α1(I) collagen, and α–smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

Authors

Christo D. Venkov, Andrew J. Link, Jennifer L. Jennings, David Plieth, Tsutomu Inoue, Kojiro Nagai, Carol Xu, Yoana N. Dimitrova, Frank J. Rauscher III, Eric G. Neilson

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Figure 3

Overexpression of CBF-A in the epithelial cells leads to formation of the FTS-1 complex and expression of fibroblast markers.

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Overexpression of CBF-A in the epithelial cells leads to formation of th...
(A) Transcripts for rCBF-A and FSP1 increase in PCR cycles after rCBF-A induction. (B) ChIP assay shows formation of the FTS-1 complex in epithelial cells expressing rCBF-A (+Dox; doxycycline) compared to 3T3 cells. IgG was a loading control. Immunoprecipitated DNA was used in PCR with primers for FTS-1. (C) Nuclear protein from MCT/rCBF-A+ or 3T3 fibroblasts form FTS-1 complexes in EMSA with similar affinity, challenged by 10× or 20× molar excess of nonlabeled FTS-1 probe (Cold probe). MCT cells were the negative control. (D) Differential expression of epithelial and mesenchymal protein markers in MCT/rCBF-A+ cells at different hours after induction of rCBF-A compared with 3T3 fibroblasts. Coo, Coomassie staining. (E) Increased expression of CBF-A and FSP1 in UUO undergoing EMT (28% and 26%, respectively) as measured by qRT-PCR and represented graphically in arbitrary units. Included for comparison are E-cadherin, α-SMA, and KAP-1. (F) CBF-A (yellow/orange) accumulates in the nuclei of tubular and interstitial cells from the UUO kidney in FSP1.GFP mice. FSP1+ fibroblasts expressing GFP under the control of the FSP1 promoter were recolored dark blue, and nuclei were counterstained red. Normal contralateral kidney tissue demonstrates CBF-A in the tubular cytoplasm (speckled green). In the cytoplasm, CBF-A and FSP1 staining merge to become light blue. Original magnification, ×630.