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

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

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 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

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

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