Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes

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Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

Transcriptional coregulators contribute to several processes involving nuclear receptor transcriptional regulation. The transcriptional coregulator androgen receptor-interacting protein 4 (ARIP4) interacts with nuclear receptors and regulates their transcriptional activity. In this study, we identified p62 as a major interacting protein partner for ARIP4 in the nucleus. Nuclear magnetic resonance analysis demonstrated that ARIP4 interacts directly with the ubiquitin-Associated (UBA) domain of p62. ARIP4 and ubiquitin both bind to similar amino acid residues within UBA domains; therefore, these proteins may possess a similar surface structure at their UBA-binding interfaces. We also found that p62 is required for the regulation of ARIP4 protein levels under nutrient starvation conditions. We propose that p62 is a novel binding partner for ARIP4, and that its binding regulates the cellular protein level of ARIP4 under conditions of metabolic stress

Replacing the HMG box of Sox9 abolished the inhibitory effect of Exp4 on Sox9 transcription.

<p>(A) Schematic representation of GAL4-fused Sox proteins (GAL4-Sox9) used in this study. The TA domain is shown as a light gray box (406–507 a.a.). (B) The effect of Exp4 on Sox9-mediated transcription of the Col2a1 reporter gene (Col2a1x3-tk-Luc). HEK293 cells were transiently transfected with the reporter gene and the indicated amounts (ng) of Sox9 or Exp4. The relative fold changes in the luciferase activities are shown: luciferase activity in the presence of 50 ng of Sox9 and the absence of Exp4 is set at 100. Values are the mean ± SD of at least three experiments. (C) The effect of Exp4 on GAL4-Sox9 mediated transcription of the GAL4 reporter gene (UAS_Gx4-TK-LUC). HEK293 cells were transiently transfected with the reporter gene and the indicated amounts (ng) of GAL4-Sox9 or Exp4. The relative fold changes in the luciferase activities are shown: luciferase activity in the presence of 50 ng of GAL4-Sox9 and the absence of Exp4 is set at 100. Values are the mean ± SD of at least three experiments.</p

Identification of Exp4 as a major interaction partner of Sox9.

<p>(A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.</p

Effect of Exp4 on Sox9 binding to its target gene.

<p>(A) The effect of Exp4 depletion on the recruitment of Sox9 to its endogenous target gene. U2OS cells were treated with control siRNA (open bar) or siRNA for Exp4 (solid bar). After a 48 h incubation, fixed chromatin was prepared from both groups of treated cells and subjected to immunoprecipitation with anti-Sox9 antibody. The <i>Col2a1</i> enhancer regions recovered in the two precipitated fractions were quantified by PCR. As a control, <i>GAPDH</i> genes recovered in the two precipitated fractions were quantified. The relative amount of enhancer DNA is shown: the amount of <i>Col2a1</i> enhancer DNA obtained from the control siRNA cells is set at 100. (B) The DNA sequence of the probe used for DNA pull-down contains a biotinylated 18 base-pair Sox9 binding sequence of the <i>Col2a1</i> enhancer region. (C) DNA pull-down assay with the indicated amounts (ng) of His-Sox9 and FLAG-Exp4. His-Sox9 and FLAG-Exp4 were incubated with a 5′ biotinylated double-stranded DNA probe. The complex was pulled down with streptavidin-agarose and the streptavidin-agarose bead bound fraction and flow-through fractions (FT) were analyzed by Western blotting.</p

Structural abnormalities induced in <i>Arx</i> KO mouse.

<p>Urogenital systems of wild type (Wild type XY) (A) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) (B) male mice at E18.5 are shown. Arrows indicate the testes. tes, testis; b, bladder; kid, kidney. Intratesticular testosterone levels of wild type (Wild type XY) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) testes were measured at E18.5 (C). The data are indicated as the mean ± SD. *, P<0.05. Expression of <i>Insl3</i> and <i>Vegfa</i> in wild type (Wild type XY) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) testes at E12.5 was determined by quantitative RT-PCR (D and E). The data were standardized using <i>β-actin</i> (Actb) and shown as the mean ± SD. *, P<0.05. Locations of the sections of the gonad are schematically shown with horizontal lines with numerals, 1 to 6 (F). The coelomic blood vessel is indicated with red line. Double immunofluorescent staining for LAMININ (green) and PECAM (red) was performed with the serial sections (1 to 6, corresponding to the numerals in (E)) with 100 µm interval of wild type and <i>Arx</i> KO mouse testes at E12.5 (G). Arrowheads indicate the coelomic blood vessel in wild type, while the corresponding structure could not be observed at this stage in the KO testis. Scale bars = 100 µm.</p

Expression of ARX in interstitial cells of fetal gonads.

<p>Expression of ARX was examined by immunohistochemistry using an anti-ARX antibody. Wild type male (XY) (A–D) and female (XX) (I–L) gonads of mouse fetuses at E11.5, E12.5, E14.5, and E18.5 were tested. Double immunofluorescent staining for ARX (green) and AD4BP/SF-1 (red) was performed with male (E–H) and female (M-P) gonads at the same stages. Dashed lines indicate the gonad-mesonephros border. Scale bars = 100 µm. Whole gonadal extracts (5 µg) prepared from mouse fetuses of both sexes at E11.5, E12.5, E13.5, E14.5, and E18.5 were subjected to western blot analysis using anti-ARX and anti-α-tubulin antibodies (Q). The location of 50 and 75 kDa protein markers are indicated. Arrowheads in L and P indicate middle part of ovary and mesovarium, respectively. Magnified views of E18.5 testis are shown (R-U). Sections are counterstained by DAPI (blue, T and U). Arrowheads in R-U indicate ARX-positive peritubular myoid cells and arrows in T and U indicate ARX-negative and DAPI-positive (blue) endothelial and unknown interstitial cells, respectively. Scale bars = 25 µm. ad, adrenal; tc, testis cord; bv, blood vessel.</p