TSHZ3 and SOX9 regulate the timing of smooth muscle cell differentiation in the ureter by reducing myocardin activity
Authors: Andreas Kispert
Elise Martin
Laurent Fasano
Xavier Caubit
Rannar Airik
Christine Vola
Ahmed Fatmi
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Smooth muscle cells; visceral organs; urogenital system; mouse ureter; transcriptional regulators
Affiliations: Aix-Marseille Université, CNRS, IBDM UMR7288, Marseille, France.

Abstract:  Smooth muscle cells are of key importance for the proper functioning of different visceral organs including those of the urogenital system. In the mouse ureter, the two transcriptional regulators TSHZ3 and SOX9 are independently required for initiation of smooth muscle differentiation from uncommitted mesenchymal precursor cells. However, it has remained unclear whether TSHZ3 and SOX9 act independently or as part of a larger regulatory network. Here, we set out to characterize the molecular function of TSHZ3 in the differentiation of the ureteric mesenchyme. Using a yeast-two-hybrid screen, we identified SOX9 as an interacting protein. We show that TSHZ3 also binds to the master regulator of the smooth muscle program, MYOCD, and displaces it from the coregulator SRF, thereby disrupting the activation of smooth muscle specific genes. We found that the initiation of the expression of smooth muscle specific genes in MYOCD-positive ureteric mesenchyme coincides with the down regulation of Sox9 expression, identifying SOX9 as a possible negative regulator of smooth muscle cell differentiation. To test this hypothesis, we prolonged the expression of Sox9 in the ureteric mesenchyme in vivo. We found that Sox9 does not affect Myocd expression but significantly reduces the expression of MYOCD/SRF-dependent smooth muscle genes, suggesting that down-regulation of Sox9 is a prerequisite for MYOCD activity. We propose that the dynamic expression of Sox9 and the interaction between TSHZ3, SOX9 and MYOCD provide a mechanism that regulates the pace of progression of the myogenic program in the ureter. 

Retracted: No

PubPeer comments:
 2019 expression of concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0211924 Following publication of this article [1], several concerns were raised about the Western blots in Figs 1, 5, and 6. The authors confirmed that in preparing these figures they had spliced image fragments to remove empty lanes, rearrange the sample order, and in some cases to combine lanes from short and long exposures of a given blot. The Director of the Institute for Developmental Biology of Marseille discussed this matter with the corresponding author and examined the original data underlying the results in question. The Director concluded that the images were modified for the purpose of presentation, and that the scientific results presented in the article and underlying data are sound. thumbnailtDownload: PPT PowerPoint slide PNG larger image TIFF original image Fig 1. TSHZ3 and SOX9 physically interact in vitro and in vivo. (A-I) Mapping of the TSHZ3 interaction domain with SOX9. (A) Sequence analysis of the two Sox9 clones clA47 (amino acids 1u2013163) and clA45 (amino acids 1u2013168) showed that the selected interaction domain corresponds to amino acids 1 to 163 of SOX9 that contains part of the HMG domain. The SOX9DC construct contains the HMG domain but not the transactivation (TA) domain (B) Coimmunoprecipitation experiment shows SOX9DC interacting with TSHZ3 protein. (C) Schematic structure of the TSHZ3 full length (TSHZ3 fl) and TSHZ3 truncated proteins used in this study. N-TSHZ3 harbours the N-terminal half of TSHZ3 (amino acid: 1u2013483), C-TSHZ3 harbours the C-terminal half of TSHZ3 (amino acid: 484u20131081), TSHZ3 dZNF harbours N-terminal half of TSHZ3 (amino acid: 1u2013483) and mutated zinc finger motifs and, TSHZ3-trunc lacks the amino acids 1u2013182. AD = acidic domain; Znf = zinc finger domain; HD = homeodomain. (D) GST pulldown assays show that TSHZ3 interacts with in vitro translated SOX9. (E) TSHZ3-HA, N-TSHZ3-HA and SOX9-Flag localize to the nucleus in HEK293T transfected cells. Cells were counterstained with DAPI to detect nuclei. (F-I) HEK293T cells were transfected with HA-tagged TSHZ3 constructs and Flag-tagged SOX9 or control empty plasmids. Proteins were immunoprecipitated with a Flag antibody, followed by immunoblotting as indicated; in: input. https: doi.org 10.1371 journal.pone.0211924.g001 thumbnailtDownload: PPT PowerPoint slide PNG larger image TIFF original image Fig 5. TSHZ3 and MYOCD physically interact in vitro and in vivo. (A-E) Mapping of the TSHZ3 interaction domain with MYOCD. (A) GST pulldown assays show TSHZ3 constructs interacting with in vitro translated MYOCD. (B-E) HEK293T cells transfected with HA-tagged TSHZ3 constructs and Flag-tagged MYOCD or control empty plasmids and then immunoprecipitated with a Flag antibody, followed by immunoblotting as indicated; in: input. (F) 10T1 2 cells were cotransfected with a luciferase reporter controlled by the telokin promoter and TSHZ3 constructs. TSHZ3-trunc-HA lost the ability to suppress the transcriptional activity of MYOCD SRF (n = 8, mean u00b1 SEM). Asterisk indicates statistical significance as determined by a Wilcoxon test (p 0.05). https: doi.org 10.1371 journal.pone.0211924.g002 thumbnailtDownload: PPT PowerPoint slide PNG larger image TIFF original image Fig 6. TSHZ3 and SOX9 compete with SRF for binding to MYOCD. (A-C) HEK293T cells were transfected with vectors encoding HA-SRF, HA-SOX9, HA-TSHZ3, HA-TSHZ3-trunc with Flag-MYOCD or control empty plasmid as indicated. HA-tagged proteins were identified by their molecular weight. Flag-MYOCD was immunoprecipitated (IP) from nuclear extracts of the transfected cells and co-precipitating HA-tagged proteins were detected by Western blotting. https: doi.org 10.1371 journal.pone.0211924.g003 The authors provided the following explanations as to how the original figures were prepared; updated figures are included with this notice, and the original raw Western blot data underlying most of the affected figure panels are included in S1 File. Fig 1B: The original u03b1-HA panel was comprised of input data (lanes 1u20133) from a short exposure and IP Flag data (lanes 4u20136) from a longer exposure. In addition, an empty lane was removed from the u03b1-Flag Western blot panel. The new Fig 1B uses only the long exposure of the u03b1-HA blot, and the empty lane (lane 4) has not been removed in either panel. Fig 1D: In the original blot, the experimental samples were separated by empty lanes. In generating the published figure, the empty lanes were removed and experimental samples were spliced next to one another. The new figure presents the original image with the empty spacer lanes intact. Fig 1F: To generate the published figure, the authors removed empty lanes from the IP Flag lanes of the u03b1-HA blot. For the u03b1-Flag blot, the authors spliced together lanes to remove empty lanes and to combine data from different exposures. The images were cropped so as not to show unspecific bands in IP lanes. In the new figure, the authors present data from a replicate experiment. Raw images used to generate both the original figure and the revised figure are provided with this notice. Fig 1G: In the published figure, a blank lane was removed between the input and IP Flag lanes of both panels. Lanes 4u20136 in the u03b1-HA blot appear to have been inverted vertically in preparing the figure. The new figure presents the intact blot images, with the empty lanes in each blot (lane 4) intact. Fig 5A: In the published figure, lanes 3 and 4 were flipped horizontally. The new panel presents the same data in a direct representation of the original blot image. Note that the loading order in the new figure differs from that in the original version of the figure. Fig 5B: Concerns were raised regarding an irregularity in lane 6 of the u03b1-HA blot, and potential splicing between lanes 1 and 2 of the u03b1-Flag blot. An empty lane was also excised between the input and IP Flag lanes of the u03b1-Flag blot in the published figure. The original u03b1-HA blot is no longer available, and so the authors provide data from a replicate experiment in the new figure. Fig 5C: In the published figure, empty lanes were removed from each panel between lanes 3 and 4. The new figure represents the original blot with the empty lane intact in each panel. Fig 5D: In the published figure, an empty lane was removed between lanes 3 and 4. The new figure represents the original blot with the empty lane intact. Fig 6A: The published u03b1-HA panel is comprised of lanes from a short exposure (input lanes) and long exposure (IP Flag lanes) of the same experiment. The IP Flag lanes were inverted horizontally, and empty lanes between experimental samples were removed. The new figure presents the intact image from the long exposure, with empty lanes included. Note that the loading order for the new panel differs from the loading order of the u03b1-Flag blot shown in Fig 6A of the original publication. Figure S4A: The raw data image provided for the input lanes of the u03b1-Flag blot panel (S2 File) does not match the image in the published figure. Raw data supporting the u03b1-HA blot in this figure panel are in S3 File. Panels F and G in the original version of Fig 5 [1] were mislabeled and should have been labeled panels E and F. The corresponding figure legend in [1] likewise contained an error in referencing these panels. These errors have been corrected in the updated version of Fig 5 provided here; the corrected labels (E, F) align with the in-text citation to these figure panels in the Results section of [1]. Raw data supporting other results reported in the article are provided in S4u2013S20 Files. The underlying raw data are no longer available for the u03b1-Flag blot in Fig 5D, or for Figs 1H I, 2D E F, 4, 5G, 6B, 6C, 7C, S1, S2Q R, S3, S4B E, or S5. For Figure S5 in [1], the authors provided an updated figure that reports replication data. The new version of this figure is included with this notice as S21 File, and the raw data from the replication experiments are in S22u2013S25 Files. Given the unavailability of these data and the extent of concerns with the images in the published article [1], the PLOS ONE Editors issue this Expression of Concern. , 
 The Expression of Concern (February 2019) is more than a year old, and my initial report is almost 6 years old (May 2014). Here are illustrations from my May 2014 report to the journal: Figure 1B: possible splicing between two different exposures. Green arrows depict sharp background transitions suggestive of splicing. ![file] Figure 1D: The authors declared that empty lanes were removed, but the backgrounds appear different. ![file] Figure 1F: Sharp background transitions. The authors declared that empty lanes were removed: ![file] Figure 1G: Sharp background transitions. The authors declared that an empty lane was removed. A band might have been inserted upside down. ![file] , 
 Figure 5A: Two or three sharp background transitions were visible. *In the published figure, lanes 3 and 4 were flipped horizontally.* ![file] Figure 5B: sharp background transitions were visible. ![file] Figure 5C: sharp background transitions were visible. ![file] Figure 5D: sharp background transitions were visible. ![file] , 
 Figure 6A: Cuts and discontinuities were noted. The authors stated that *The published u03b1-HA panel is comprised of lanes from a short exposure (input lanes) and long exposure (IP Flag lanes) of the same experiment. The IP Flag lanes were inverted horizontally, and empty lanes between experimental samples were removed.* ![file] ,