Correction: Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation

<p>Correction: Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation</p

3-D model of the proposed Gβ₂ interaction with MEF2A and histone H4.

<p>(a) Multiple sequence alignment using the CLUSTAL W program revealed that the phosphopeptide motif (–LLpTPPG–) was conserved in the TFs that associated with Gβ₂ (MEF2A, STAT1, STAT3 and NFAT), but not in NFκB and GATA4. (b) Surface model of Gβ₂γ₁₂ based on Gβ₁γ₂ crystal coordinates. Gβ₂γ₁₂ is shown in gray, and the common site of interaction with cytoplasmic effectors (Gα and PLCβ) is shown in teal. The –LLpTPPG– peptide (shown as red ball and stick) anchors to the central core of the β-propeller structure and makes contact with the amino acid residues shown in green and purple. The purple side chains contacting the peptide are conserved charge interactions. The histone H4 tail peptide, shown in brown, may interact on the surface of the WD7 repeat. (c) Co-immunoprecipitation of Gβ₂ with the AngII-responsive TFs, NFAT, STAT1, and STAT3, but not with GATA4 and p65 NFκB. The nuclear fractions (100 µg) prepared from HEK-AT₁R cells treated with AngII (1 µM for 30 min) were subjected to pull-down with only ProtG (−) or with a Gβ₂ antibody and ProtG (+). The immunoblot on the right shows the abundance of the respective proteins in the immunoprecipitates (− and +) and input lysates for the − and + samples. (d) Gβ₂ interaction with selective AngII-responsive TFs, suggesting a role for Gβ₂ in genome wide transcription that eventually leads to changes in cellular functions.</p

Abundance of the G-protein β₂ subunit in the nucleus.

<p>(a) Mass spectrometry evidence for the differential nuclear translocation of Gβ₂. The Gβ₂-specific tryptic peptide (LLVSASQDGK) was monitored in control and AngII treated samples. The right hand corner in each panel gives identity of peptide by m/z ratio and the 100% abundance value of the peptide in that chromatogram. In the chromatogram shown, m/z ratio 509.5–511.5 identified the Gβ₂ peptide and the 100% abundance value of 1.3E6 after AngII treatment is 2.47-fold higher when compared to the 100% abundance value 5.5E5 of the control. Applying the same calculation, change in abundance of spiked-in control trypsin peptide, m/z 421.0–423 was 0.79. The actual fold change of Gβ₂ peptide was calculated, 2.47/0.79 = 3.13 in this chromatogram. (b) An increase in Gβ₂ in the nuclear fraction upon exposure of HASM cells to various prohypertrophic agonists (1 µM AngII for AT₁R, 1 µM 5-HT for 5-HT2AR; 10 µM isoproterenol for βAR and 1 µM dobutamine for β1AR). Nuclear fractions were immunoblotted for Gβ₂ and histone H1 as loading controls. See schematic for the relative levels of Gβ₂ in the nuclei of samples treated with various agonists compared with the untreated (UT) control.</p

Gβ₂-dependent global gene expression patterns.

<p>Altered gene expression patterns and gene networks that engage common biological processes are shown. Of the >47,000 transcripts monitored, 705 unique and annotated transcripts (2% of the transcriptome) were differentially affected by AngII stimulation in the Gβ₂i cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s016" target="_blank">Table S5</a>). Out of these, 299 transcripts were identical to the transcripts in the Gβ₂Sc control, indicating that these transcripts were regulated by Gβ₂-independent signals from AT₁R, and the remaining ≈400 transcripts were specifically regulated by Gβ₂. The false discovery rate was <3%. (a) Venn diagram: a total of 800 genes were modulated in Gβ₂Sc cells, and 705 genes were modulated upon Gβ₂ knockdown (Gβ₂i) in AT₁R-expressing cells treated with AngII (1 µM for 30 min). (b) The altered cell functions upon Gβ₂ knockdown. (c) The hierarchy of gene functions, Δ<i>p</i>-value, number of molecules involved and genes regulated by the Gβ₂-interacting TFs, MEF2A, NFAT, and STAT1/STAT3 (derived from the ‘Build Networks – Expand by one group interaction’ algorithm in MetaCore™). Shown in green are down regulated genes, in red are up regulated genes in an independent experiment. Additional promoter information is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s013" target="_blank">Table S2</a>.</p

G-protein β₂ and γ₁₂ subunits are components of the nuclear proteome.

<p>(a) Schematic for the isolation of intact nuclei from the cytosol and characterization of the nuclear proteome by mass spectrometry analysis. (b) Composition of the nuclear proteome of HEK-AT₁R cells 30 min after AngII activation of AT₁R (list of proteins is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s004" target="_blank">Fig. S3</a>). (c) CID spectrum of the Gβ₂-specific tryptic peptide; peptide coverage is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s012" target="_blank">Table S1</a>. (d) CID spectrum of the Gγ₁₂-specific tryptic peptide.</p

Multiphoton microscopy reveals cancer stem cell driven tumor propagation.

<p>Fractionated CSCs and non-stem tumor cells were labeled with different fluorescent proteins and transplanted into mice at a 10% cancer stem cell (YFP) to 90% non-stem tumor cell (CFP) ratio as shown in experimental design schematic (<b>A</b>). CSCs outgrew non-CSCs in vivo as shown in summary graph (<b>B</b>), which was calculated based on three-dimensional reconstructions of projection micrographs (<b>B, C</b>). Additionally, tumor populations did not intermingle in vivo (non-stem tumor population indicated by yellow oval). Fluorescent dextran (shown in purple) was injected into the circulation to illuminate blood vessels prior to imaging. Scale bar represents 100 µm.</p

CSCs and non-stem tumor cells prior to transplantation contain different fractions of stem-like and proliferating cells.

<p>Representative micrographs (<b>A</b>) and bar graph (<b>B</b>) of expanded cells prior to transplantation demonstrate Sox2 and PH3 expression (red) is higher in the CSC fraction of cells as compared with the non-stem tumor cells. Summary figure depicts marker expression from in vivo and in vitro analyses (<b>C</b>). Scale bar represents 50 µm. Data displayed as mean values +/− S.E.M. ***, p<0.001 and N.S. represents not significant (p>0.05) as assessed by one-way analysis of variance (ANOVA), nuclei counterstained with Hoechst 33342 (blue).</p

Agonist-activated nuclear translocation of Gβ₂ in intact cells.

<p>(a) The HEK-AT₁R, HASM and NRVM cells were treated with vehicle or 1 µM AngII for 30 min and fixed. Gβ₂ is shown in green. The nucleus (blue, stained with DAPI) shows green staining that corresponds to Gβ₂ in the nuclei. (b) Post-isolation viability and AngII response as assessed by calcium signals in AMVMs. The AMVMs that were paced at a frequency of 0.5 Hz displayed steady-state [Ca²⁺]_i transient signals. When AMVM pacing was stopped, the [Ca²⁺]_i signals ceased, and upon treatment with 1 µM AngII, the [Ca²⁺]_i signal resumed. (c) Beating AMVMs were treated with vehicle or 1 µM AngII for 30 min and fixed. α-Actinin-1 was labeled red and Gβ was labeled green. The far right-hand inset shows a magnified image (1000×) of a single nucleus. The nucleus displays green staining that corresponds to Gβ. Note: α-actinin-1 is a sarcomeric marker and does not translocate to the nucleus. (d) 3-D reconstruction of a mouse cardiac myocyte nucleus (confocal microscopy image). Green fluorescence represents Gβ₂, and blue represents DAPI staining. The top panel shows the localization of Gβ₂ (in Z-plane) from the top to the bottom of the myocyte nucleus. The lower panel shows an intact AMVM nucleus and a slice through the nuclear image that depicts a significant accumulation of Gβ₂ inside the nucleus of the AMVM cell upon AngII/AT₁R activation. Note: all images were acquired using a 63× objective (1.4 N.A.) at 0.232 µM/pixel in the plane resolution and 0.041 µM/pixel in the Z-axis resolution. The confocal image is a representative image of N = 3, and in each experiment, >50 cells were scored. Scale bars = 50 µm.</p

Interaction of Gβ₂ in the nuclear proteome and mechanism of modulation of MEF2A transcriptional activity.

<p>(a) Gβ₂ coimmunoprecipitates with α-actinin-4, HDAC5, MEF2A, and the histones H2B and H4. The nuclear fractions (100 µg) prepared from HEK-AT₁R cells treated with AngII (1 µM for 30 min) were subjected to pull-down with only ProtG (−) or with a Gβ₂ antibody and ProtG (+). The immunoblot on the right shows the abundance of the respective proteins in the immunoprecipitates (− and +) as well as input lysates for the − and + samples. Gel-C peptide index mining provided further supporting evidence for the provisional interactome of nuclear Gβ₂ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s012" target="_blank">Table S1</a>). (b) A significant increase in MEF2-luciferase activity (*p = 0.039) when AT₁R was exposed to AngII (bars 1 and 3 from right). The basal MEF2-luciferase activity was significantly (∼50%) attenuated in AT₁R-Gβ₂i cells when compared to wild-type AT₁R (bars 1 and 2; **p = 0.002). The RLU is normalized to co-expressed β-gal activity in each sample. Data were further normalized to basal MEF2-luciferase activity in wild-type AT₁R cells. Inset: No significant change was detected in MEF2A protein levels in the cell lysate. (c) A significant increase in MEF2-luciferase activity upon FLAG-Gβ₂ overexpression. (d) In Gβ₂-positive cells, immunoprecipitation with anti-TBP antibodies revealed the interaction of TBP with MEF2A and TAF. In the absence of Gβ₂ (Gβ₂i cells), TBP failed to co-immunoprecipitate MEF2A, but TAF was co-immunoprecipitated. The immunoblot on the right shows the abundance of the TAF, TBP, MEF2A and Gβ₂ proteins in lysates (INPUT; Gβ₂ and Gβ₂i). (e) Upon AT₁R activation with AngII, the transiently transfected myc-Gγ₁₂ translocated to the nucleus with endogenous Gβ₂ and associated with TBP and MEF2A. (f) Model depicting the modulation of MEF2A-dependent gene transcription by Gβ₂-associated proteins (MEF2, HDAC5, α-actinin-4, TBP and TAF). <b>Basal:</b> In this state, Gβ₂ forms a complex with MEF2A, HDAC5 and Actinin-4. Histones are deacetylated locally. This yields the basal transcription (for instance, that of MEF2-luciferase). <b>AngII:</b> MEF2A forms a complex with Gβ₂, which also interacts with the TBP-TAF complex. Incoming Gβ₂ displaces the existing repressor complex (i.e., the α-actinin-4-associated pHDAC is exported into the cytosol). In this state, the recruitment of HATs to the complex results in the acetylation of histones, synergy with the TBP complex, and activation of MEF2-luciferase transcription. <b>Gβ2i:</b> In this state, the absence of Gβ₂ leads to the cytosolic localization of the actinin-HDAC complex, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s009" target="_blank">Figure S8</a>. In addition, MEF2A cannot interact with TBP, which leads to a lack of synergy and the attenuation of basal transcription. Note: the schematic shows no change in the TBP and RNA polymerase complex.</p

Histological evaluation reveals tumors contained cancer stem cells and their descendants that had association with blood vessels.

<p>Tumors from the cell mixing experiments (n = 3) were evaluated to determine their composition. Subsequent evaluation of resulting tumors demonstrates that the majority of the cells within the tumor mass was of human origin and derived from CSC as confirmed by Tra-1-85 staining and YFP expression, shown in representative micrographs (<b>A</b>) and bar graph (<b>B</b>). Peripheral transplanted tumor cells (YFP positive CSCs and their descendants) were observed to have an association with blood vessels. Micrograph from multiphoton imaging and three-dimensional reconstruction (<b>C</b>) depict close association of tumor cells (green) with adjacent blood vessel (purple, illuminated by fluorescent dextran injection into the circulation prior to imaging). Histological examination of resulting tumors confirms close association of peripheral tumor cells to the vasculature using CD31 immunostaining (<b>D</b>; CD31 in red, tumor cells in green, nuclei in purple). Scale bar represents 50 µm. Data displayed as mean values +/- S.E.M. ***, p<0.001 as assessed by one-way analysis of variance (ANOVA).</p