Art27 Interacts with GATA4, FOG2 and NKX2.5 and Is a Novel Co-Repressor of Cardiac Genes

<div><p>Transcription factors play a crucial role in regulation of cardiac biology. FOG-2 is indispensable in this setting, predominantly functioning through a physical interaction with GATA-4. This study aimed to identify novel co-regulators of FOG-2 to further elaborate on its inhibitory activity on GATA-4. The Art27 transcription factor was identified by a yeast-2-hybrid library screen to be a novel FOG-2 protein partner. Characterisation revealed that Art27 is co-expressed with FOG-2 and GATA-4 throughout cardiac myocyte differentiation and in multiple structures of the adult heart. Art27 physically interacts with GATA-4, FOG-2 and other cardiac transcription factors and by this means, down-regulates their activity on cardiac specific promoters α-myosin heavy chain, atrial natriuretic peptide and B-type natriuretic peptide. Regulation of endogenous cardiac genes by Art27 was shown using microarray analysis of P19CL6-Mlc2v-GFP cardiomyocytes. Together these results suggest that Art27 is a novel transcription factor that is involved in downregulation of cardiac specific genes by physically interacting and inhibiting the activity of crucial transcriptions factors involved in cardiac biology.</p></div

FOG-2 physically interacts with Art27.

<p>(<b>A</b>) AH109 yeast were transformed with the respective bait and prey constructs and plated on synthetic dropout media lacking adenine, histidine, leucine and tryptophan and tested for X-GAL positive yeast growth. FOG-2 (amino acids 856<b>–</b>1156) failed to physically interact T-antigen (segment 1- negative control), Art27 and FOG-2 (856<b>–</b>1156) failed to autoactivate yeast growth (segment 2 and 3 respectively), Art27 and FOG-2 (856<b>–</b>1156) physically interact and promote yeast growth (segment 4) and as expected the physical interaction between p53 and T-antigen promoted yeast growth (segment 5 – positive control). (<b>B</b>) <i>In vitro</i> translated and ³⁵S radiolabeled Art27 protein was incubated with full length FOG-2/GST fusion protein or GST that was immobilised on glutathione sepharose beads. After extensive washing the proteins were resolved by electrophoresis and detected using a phosphorimager. ³⁵S labelled Art27 was retained only by the FOG-2/GST fusion protein (lane 3) indicating that they physically interact.</p

Art27 transcriptionally represses GATA-4 independently of FOG-2.

<p>293a cells were transfected with the various expression plasmids as indicated and relative transactivation was conducted using luciferase reporter assays. (<b>A</b>) Art27 and FOG-2 significantly diminish GATA-4 transactivation of the Bone Natriuretic Peptide luciferase (BNP-Luc) reporter independently and also additively when cotransfected. (<b>B</b>) GATA-4 V217G transactivation of the BNP-Luc reporter is significantly repressed by Art27 but not FOG-2.</p

Art27 knockdown de-represses endogenous cardiac genes.

<p>(<b>A</b>) Differentiation of P19CL6-Mlc2v-GFP cells was monitored by flow cytometry. The solid histogram represents GFP fluorescence of undifferentiated cells (Day 0). The solid and dotted lines show GFP fluorescence on differentiation Days 7 and 11, respectively. (<b>B</b>) Art27 knockdown relative to control. The level of Art27 mRNA was measured by qPCR in differentiated P19CL6-Mlc2v-GFP cells (Day 7) nucleofected with control siRNA (GFP siRNA) or specific Art27 siRNA. Nucleofection with Art27 siRNA resulted in a 10-fold reduction of Art27 mRNA. (<b>C</b>) Heatmap showing the relative expression of transcripts that were significantly upregulated (red) or downregulated (blue) in differentiated P19CL6-Mlc2v-GFP cells (Day 7) nucleofected with Art27 siRNA. Relevant cardiac transcripts are indicated.</p

FOG-2 protein binding partners from yeast-2-hybrid screen.

<p>FOG-2 protein binding partners from yeast-2-hybrid screen.</p

Art27 physically interacts and transcriptionally represses cardiac transcription factors.

<p>(<b>A</b>) <i>In vitro</i> translated and 35S radiolabeled GATA-4, GATA-1, GATA-6 or Nkx2.5 was incubated with full length Art27/GST fusion protein (GST-Art27) or GST-only (GST-control) that was immobilised on glutathione sepharose beads. After extensive washing and electrophoresis, phosphorimaging identified the presence of all factors tested with GST-Art27 but not with GST-control suggesting that they physically interact. (<b>B–I</b>) 293a cells were transfected with Art27 and various other cardiac transcription factors as indicated and luciferase transactivation assays were conducted with a variety of reporters. Art27 represses GATA-4 in a dose dependent manner on atrial natriuretic peptide luciferase (ANP-Luc) reporter (B), BNP-Luc (C), and alpha myosin heavy chain luciferase (αMHC) reporter (D). Art27 represses GATA-1 and GATA-6 on the BNP-luc reporter (E and F respectively) and GATA-1 on the glycoprotein 1b alpha luciferase (GP1bα-Luc) reporter (G). Art27 represses Nkx2.5 in a dose dependent manner on the ANP-Luc reporter (H). Art27 represses a GATA-4/Nkx2.5 synergistically transactivated ANP-Luc reporter (I). *, p<0.05, **, p<0.01, ***, p<0.001.</p

Art27 is a potential transcriptional regulator in cardiac developmental and adult tissues.

<p>(<b>A</b>) Northern blot on RNA derived from eight different heart tissues was probed for expression of GATA-4, FOG-2 and Art27. The membrane was stripped and re-blotted with the appropriate label. The housekeeping gene Gapdh was used as a loading control. Differential binding and/or absence of binding is indicative of probe specificity. (<b>B</b>) P19CL6 embryonal carcinoma cells with stably incorporated GFP under the control of the cardiac Mlc2v promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095253#pone.0095253-Moore1" target="_blank">[26]</a> were induced to undergo cardiomyocyte differentiation with 1% DMSO. At 5-day increments, mRNA was isolated to assess relative expression of cardiac transcription factors Art27, GATA-4, FOG-2 and Nkx2.5 compared to housekeeping gene Gapdh. These time points correspond to various stages of cardiomyocyte differentiation as determined by systematic scoring of GFP fluorescence and beating intensity. (<b>C</b>) 293a cells were transfected with the various expression plasmids as indicated and GAL₅LEXA₂-Luc reporter activity was assessed. GAL(DBD)-Art27 significantly diminishes reporter activity compared to GAL(DBD)-empty expression plasmid alone showing inherent transcriptional repression activity. GAL(DBD)-SUMO-1 was used as positive control and was observed to repress the activity of the reporter as expected <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095253#pone.0095253-Ross1" target="_blank">[49]</a> **p<0.01. FH, foetal heart, AH, adult heart, Ao, aorta, Ap, apex, LA, left atrium, RA, right atrium, LV, left ventricle, RV, right ventricle.</p

The ability of DC-rich CD117⁺ splenocytes to induce NK cells to increase their expression of IFN-γ in the presence of LPS is dependent on RasGRP4.

<p>(A-E) NK cells from WT mice were incubated with DC-rich CD117⁺ splenocytes from WT (□) or RasGRP4- null mice (■) in ratios of 1:1 and 1:2 for 24 and 48 h, respectively, in the presence of 1 μg/mL LPS. For controls, NK cells alone and CD117⁺ cells alone were exposed to LPS. The levels of IFN-γ (A), IL-6 (B), IL-10 (C), TNF/ TNFSF4 (D), and CCL2 (E) in the resulting supernatants were measured. The depicted data are the mean ± SD, and the results are shown from 3 experiments using different batches of NK cells and CD117⁺ splenocytes. * = p < 0.05, ** = p<0.005.</p

NK cells from RasGRP4-null mice produce significantly less IFN-γ than NK cells from WT mice due to the functional absence of the relevant accessory cell.

<p>(A) Splenocytes from RasGRP4-null and WT mice were isolated and incubated 16 h in the absence (left panels) or presence (right panels) of 1 μg/mL of LPS. The numbers of IFN-γ-expressing cells were measured using the intracellular flow cytometry assay. Fewer CD3^-/NK1.1⁺/IFN-γ⁺ cells were detected in the splenocytes from the RasGRP4-null mice relative to WT mice in this representative experiment. (B) Shown are the mean data ± SD, n = 7 of the percent IFN-γ⁺ NK cells for WT (□) and RasGRP4^-/- (■) mice. *** = p< 0.0005.</p

The CD117⁺/CD45R⁺/CD11c⁺ DCs in the spleens of naïve WT B6 mice express RasGRP4.

<p>DCs from the spleens of WT B6 and RasGRP4-null mice were stained for their surface expression of numerous proteins, including CD117, CD45R, and CD11c (A), and for their intracellular expression of RasGRP4 (B). An irrelevant immunoglobulin (Isotype IgG) was used as a negative control. Similar data was obtained in a second experiment.</p