Gβγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts
2023; Elsevier BV; Volume: 299; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2023.103064
ISSN1083-351X
AutoresShahriar M. Khan, Ryan Martin, Andrew N. Bayne, Darlaine Pétrin, Kyla Bourque, Jace Jones-Tabah, Celia Bouazza, Jacob Blaney, Jenny Lau, Kimberly Martins-Cannavino, Sarah Gora, Andy Zhang, Sarah MacKinnon, Phan Trieu, Paul B. S. Clarke, Jean‐François Trempe, Jason C. Tanny, Terence E. Hébert,
Tópico(s)Ubiquitin and proteasome pathways
ResumoGβγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ signaling, we investigated the functional role of Gβγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gβ1γ dimer in the fibrotic response. Depletion of Gβ1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gβ1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gβγ with transcription complexes. Together, our findings suggest that Gβ1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gβγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gβγ signaling in other cell types. Gβγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ signaling, we investigated the functional role of Gβγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gβ1γ dimer in the fibrotic response. Depletion of Gβ1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gβ1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gβγ with transcription complexes. Together, our findings suggest that Gβ1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gβγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gβγ signaling in other cell types. In recent years, our understanding of the role of paracrine interactions between cardiomyocytes and cardiac fibroblasts in modulating the response to cardiac damage has expanded dramatically. Cardiac fibroblasts, in particular, respond dynamically following damage to the myocardium, characterized by differentiation into myofibroblasts, increased proliferation and migration to areas of damage (1Travers J.G. Kamal F.A. Robbins J. Yutzey K.E. Blaxall B.C. Cardiac fibrosis: the fibroblast awakens.Circ. Res. 2016; 118: 1021-1040Crossref PubMed Scopus (921) Google Scholar, 2Fu X. Khalil H. Kanisicak O. Boyer J.G. Vagnozzi R.J. Maliken B.D. et al.Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.J. Clin. Invest. 2018; 128: 2127-2143Crossref PubMed Scopus (346) Google Scholar, 3Dobaczewski M. Bujak M. Li N. Gonzalez-Quesada C. Mendoza L.H. Wang X.F. et al.Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction.Circ. Res. 2010; 107: 418-428Crossref PubMed Scopus (290) Google Scholar). 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While the fibrotic response initially aids in wound healing, a prolonged, activated fibrotic response worsens adverse cardiac remodeling, and accelerates progression to heart failure (1Travers J.G. Kamal F.A. Robbins J. Yutzey K.E. Blaxall B.C. Cardiac fibrosis: the fibroblast awakens.Circ. Res. 2016; 118: 1021-1040Crossref PubMed Scopus (921) Google Scholar, 12Weber K.T. Sun Y. Bhattacharya S.K. Ahokas R.A. Gerling I.C. Myofibroblast-mediated mechanisms of pathological remodelling of the heart.Nat. Rev. Cardiol. 2013; 10: 15-26Crossref PubMed Scopus (475) Google Scholar). Inhibiting aspects of the fibrotic response reduces adverse cardiac remodeling (2Fu X. Khalil H. Kanisicak O. Boyer J.G. Vagnozzi R.J. Maliken B.D. et al.Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.J. Clin. Invest. 2018; 128: 2127-2143Crossref PubMed Scopus (346) Google Scholar, 13Weber K.T. Diez J. Targeting the cardiac myofibroblast secretome to treat myocardial fibrosis in heart failure.Circ. Heart Fail. 2016; 9e003315Crossref Scopus (19) Google Scholar). Hence, deciphering how Ang II signaling regulates profibrotic gene expression is an important step toward understanding how these processes might be targeted therapeutically. Cardiac fibroblasts respond to increased Ang II levels through Ang II type I (AT1R) and type II (AT2R) G protein-coupled receptors (GPCRs). Of these, the AT1R is responsible for positively regulating the fibrotic response in cardiac fibroblasts (1Travers J.G. Kamal F.A. Robbins J. Yutzey K.E. Blaxall B.C. Cardiac fibrosis: the fibroblast awakens.Circ. Res. 2016; 118: 1021-1040Crossref PubMed Scopus (921) Google Scholar). The AT1R couples to multiple heterotrimeric G proteins composed of specific combinations of Gα and Gβγ subunits (14Namkung Y. LeGouill C. Kumar S. Cao Y. Teixeira L.B. Lukasheva V. et al.Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.Sci. Signal. 2018; 11eaat1631Crossref Scopus (80) Google Scholar). G proteins serve as signal transducers to relay extracellular ligands bound to GPCRs into activation of different intracellular signaling pathways (15Khan S.M. Sleno R. Gora S. Zylbergold P. Laverdure J.-P. Labbé J.-C. et al.The expanding roles of Gβγ subunits in G protein–coupled receptor signaling and drug action.Pharmacol. Rev. 2013; 65: 545-577Crossref PubMed Scopus (172) Google Scholar). Gβγ subunits, like the more extensively studied Gα subunits, modulate a wide variety of canonical GPCR effectors at the cellular surface such as adenylyl cyclases, phospholipases, and inwardly rectifying potassium channels (15Khan S.M. Sleno R. Gora S. Zylbergold P. Laverdure J.-P. Labbé J.-C. et al.The expanding roles of Gβγ subunits in G protein–coupled receptor signaling and drug action.Pharmacol. Rev. 2013; 65: 545-577Crossref PubMed Scopus (172) Google Scholar, 16Dupré D.J. Robitaille M. Rebois R.V. Hébert T.E. The role of Gβγ subunits in the organization, assembly and function of GPCR signaling complexes.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 31-56Crossref PubMed Scopus (210) Google Scholar, 17Smrcka A.V. G protein βγ subunits: central mediators of G protein-coupled receptor signaling.Cell. Mol. Life Sci. 2008; 65: 2191-3214Crossref PubMed Scopus (288) Google Scholar). However, compared with Gα-mediated events, Gβγ-mediated signaling is relatively understudied and is complicated by the existence of 5 Gβ and 12 Gγ subunits which combine in multiple ways to form obligate dimers (15Khan S.M. Sleno R. Gora S. Zylbergold P. Laverdure J.-P. Labbé J.-C. et al.The expanding roles of Gβγ subunits in G protein–coupled receptor signaling and drug action.Pharmacol. Rev. 2013; 65: 545-577Crossref PubMed Scopus (172) Google Scholar). Gβγ subunits also regulate a variety of noncanonical effectors in distinct intracellular locations and a number of studies have described roles for Gβγ signaling in the nucleus (15Khan S.M. Sleno R. Gora S. Zylbergold P. Laverdure J.-P. Labbé J.-C. et al.The expanding roles of Gβγ subunits in G protein–coupled receptor signaling and drug action.Pharmacol. Rev. 2013; 65: 545-577Crossref PubMed Scopus (172) Google Scholar, 18Campden R. Audet N. Hébert T.E. Nuclear G protein signaling: new tricks for old dogs.J. Cardiovasc. Pharmacol. 2015; 65: 110-122Crossref PubMed Scopus (29) Google Scholar). Nuclear Gβγ subunits modulate gene expression through interactions with a variety of transcription factors, such as adipocyte enhancer binding protein 1, the AP-1 subunit c-Fos, HDAC5 and MEF2A (19Park J.G. Muise A. He G.P. Kim S.W. Ro H.S. Transcriptional regulation by the γ5 subunit of a heterotrimeric G protein during adipogenesis.EMBO J. 1999; 18: 4004-4012Crossref PubMed Scopus (51) Google Scholar, 20Robitaille M. Gora S. Wang Y. Goupil E. Petrin D. Del Duca D. et al.Gβγ is a negative regulator of AP-1 mediated transcription.Cell Signal. 2010; 22: 1254-1266Crossref PubMed Scopus (26) Google Scholar, 21Spiegelberg B.D. Hamm H.E. Gβγ binds histone deacetylase 5 (HDAC5) and inhibits its transcriptional co-repression activity.J. Biol. Chem. 2005; 280: 41769-41776Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22Bhatnagar A. Unal H. Jagannathan R. Kaveti S. Duan Z.H. Yong S. et al.Interaction of G-protein betagamma complex with chromatin modulates GPCR-dependent gene regulation.PLoS One. 2013; 8e52689Crossref Scopus (13) Google Scholar). Furthermore, we have detected Gβ1 occupancy at numerous gene promoters in human embryonic kidney 293 (HEK 293) cells (23Khan S.M. Min A. Gora S. Houranieh G.M. Campden R. Robitaille M. et al.Gβ4γ1 as a modulator of M3 muscarinic receptor signalling and novel roles of Gβ1 subunits in the modulation of cellular signalling.Cell Signal. 2015; 27: 1597-1608Crossref PubMed Scopus (15) Google Scholar). While canonical Gβγ signaling has been implicated in both cardiac fibrosis and heart failure (24Kamal F.A. Travers J.G. Schafer A.E. Ma Q. Devarajan P. Blaxall B.C. G protein-coupled receptor-G-protein βγ-subunit signaling mediates renal dysfunction and fibrosis in heart failure.J. Am. Soc. Nephrol. 2017; 28: 197-208Crossref PubMed Scopus (35) Google Scholar, 25Travers J.G. Kamal F.A. Valiente-Alandi I. Nieman M.L. Sargent M.A. Lorenz J.N. et al.Pharmacological and activated fibroblast targeting of Gβγ-GRK2 after myocardial ischemia attenuates heart failure progression.J. Am. Coll. Cardiol. 2017; 70: 958-971Crossref PubMed Scopus (43) Google Scholar), how nuclear Gβγ signaling impacts these events is currently unknown. Here, to understand the potential role of individual Gβγ subunits, we knocked down Gβ1 and Gβ2 as exemplars of Gβ subunits highly expressed in these cells and we then characterized how nuclear Gβ1, in particular, is a key regulator of AT1R-driven transcriptional changes. Finally, we describe a novel interaction between Gβγ subunits and RNA polymerase II (RNAPII) which regulates the cardiac fibrotic response to Ang II activation of AT1R. The Gβ family is comprised of five members which, with the exception of Gβ5, exhibit high levels of sequence and structural similarity (15Khan S.M. Sleno R. Gora S. Zylbergold P. Laverdure J.-P. Labbé J.-C. et al.The expanding roles of Gβγ subunits in G protein–coupled receptor signaling and drug action.Pharmacol. Rev. 2013; 65: 545-577Crossref PubMed Scopus (172) Google Scholar, 26Tennakoon M. Senarath K. Kankanamge D. Ratnayake K. Wijayaratna D. Olupothage K. et al.Subtype-dependent regulation of Gβγ.Cell Signal. 2021; 82: 109947Crossref PubMed Scopus (16) Google Scholar). Despite these similarities, Gβγ isoforms differ considerably with respect to their associated receptors and signaling pathways (23Khan S.M. Min A. Gora S. Houranieh G.M. Campden R. Robitaille M. et al.Gβ4γ1 as a modulator of M3 muscarinic receptor signalling and novel roles of Gβ1 subunits in the modulation of cellular signalling.Cell Signal. 2015; 27: 1597-1608Crossref PubMed Scopus (15) Google Scholar, 26Tennakoon M. Senarath K. Kankanamge D. Ratnayake K. Wijayaratna D. Olupothage K. et al.Subtype-dependent regulation of Gβγ.Cell Signal. 2021; 82: 109947Crossref PubMed Scopus (16) Google Scholar, 27Yim Y.Y. Betke K.M. McDonald W.H. Gilsbach R. Chen Y. Hyde K. et al.The in vivo specificity of synaptic Gβ and Gγ subunits to the α2A adrenergic receptor at CNS synapses.Sci. Rep. 2019; 9: 1718Crossref PubMed Scopus (12) Google Scholar, 28Greenwood I.A. Stott J.B. The Gβ1 and Gβ3 subunits differentially regulate rat vascular Kv7 channels.Front. Physiol. 2019; 10: 1573Crossref PubMed Scopus (4) Google Scholar). We initially focused on Gβ1 and Gβ2 as they exhibit the highest expression in primary rat neonatal cardiac fibroblasts (RNCFs) as determined by RNA-seq (29Shu J. Liu Z. Jin L. Wang H. An RNAsequencing study identifies candidate genes for angiotensin II-induced cardiac remodeling.Mol. Med. Rep. 2018; 17: 1954-1962PubMed Google Scholar) and RT-qPCR (Fig. S1). These Gβ isoforms were efficiently depleted by siRNA transfection in these cells at both the mRNA and protein levels (Fig. 1, A and B). To determine whether specific Gβ isoforms were required to initiate signalling cascades proximal to AT1R activation, we assessed the relative roles of Gβ1 and Gβ2 in AT1R-dependent Ca2+ mobilization. Following receptor activation, Gβγ subunits regulate intracellular Ca2+ mobilization through activation of phospholipase (PLC)-β (30Park D. Jhon D.Y. Lee C.W. Lee K.H. Rhee S.G. Activation of phospholipase C isozymes by G protein βγ subunits.J. Biol. Chem. 1993; 268: 4573-4576Abstract Full Text PDF PubMed Google Scholar) and we have previously demonstrated Gβ isoform specificity for PLCβ signaling in HEK 293F cells (23Khan S.M. Min A. Gora S. Houranieh G.M. Campden R. Robitaille M. et al.Gβ4γ1 as a modulator of M3 muscarinic receptor signalling and novel roles of Gβ1 subunits in the modulation of cellular signalling.Cell Signal. 2015; 27: 1597-1608Crossref PubMed Scopus (15) Google Scholar). To assess AT1R-dependent intracellular Ca2+ mobilization, we used the cell-permeable Ca2+ dye Fura 2-acetoxymethyl ester. Following AT1R activation, we observed a rapid increase in intracellular Ca2+ mobilization in control RNCFs (Fig. 1C, black, empty triangles; quantified area under the curve (AUC) in Fig. 1D). Knockdown of Gβ1 did not appear to alter Ca2+ mobilization following stimulation with Ang II (mean ± S.E.M., 8.1 ± 7.0% decrease, red bar). However, knockdown of Gβ2 resulted in a significant 31.6 ± 9% decrease in Ca2+ release (Fig. 1, C and D), suggesting a selective role for Gβ2-containing Gβγ dimers in mediating receptor-proximal signalling downstream of AT1R activation. The effect size is consistent with Gβγ-mediated activation of PLCβ, compared to the much larger effects of Gαq activation per se. We next investigated potential isoform-specific roles for Gβγ in fibrotic events further downstream of AT1R activation, such as gene expression. We examined changes in the levels of 84 genes involved in the fibrotic response using Qiagen RT2 Profiler PCR arrays. Gene expression changes were assessed following 75 min (acute) or 24 h (chronic) Ang II treatment; control siRNA RNCFs were compared to Gβ1 or Gβ2 knockdown conditions. We determined expression changes at individual genes (Table 1) and collective fold changes in expression across all the genes on the array (Fig. 2). After 75 min of Ang II treatment, we observed a significant increase in the expression of a small number of fibrotic genes under both control siRNA [connective tissue growth factor (Ctgf), Edn1, Serpine1, Tgfb3, thrombospondin 1] or Gβ1 knockdown (Cav1, Ctgf, Edn1, Itgb8, Serpine1, and Tgfb3) conditions. An additional three genes reproducibly showed increased expression under control siRNA conditions but did not meet our statistical cutoff due to variable induction (Table 1; listed as "Trending"- this was defined as fold change greater than 1.5 but with a p value above 0.05). This was true of ten (six significant and four trending) genes in Gβ1 knockdown cells treated with Ang II and of four genes in the untreated knockdown cells, implying that Gβ1 knockdown led to increased expression of fibrotic genes (Table 1). Following 24 h Ang II treatment, the effects of Ang II and Gβ1 knockdown became more pronounced. Gβ1 knockdown led to increases in number of genes with significantly induced expression, both in the presence and absence of Ang II stimulation. It also led to a dramatic increase in induced genes in the "Trending" category. In the absence of Ang II, there were 0 "Trending" genes in control versus 25 in Gβ1 knockdown cells; in the presence of Ang II there were 31 and 42 induced "Trending" genes in control and Gβ1 knockdown cells, respectively (Table 1). This corresponded with significant increases in expression across all genes on the array caused by Gβ1 knockdown in the absence of Ang II and an enhanced average gene expression change induced by Ang II in Gβ1 knockdown cells as compared to controls (Fig. 2B). The increased expression of fibrotic genes following Gβ1 knockdown suggests that Gβ1 negatively modulates the Ang II transcriptional response.Table 1Summary of fibrosis RT-qPCR array resultsTimesiRNATreatmentIncreased gene expressionDecreased gene expressionSignificantTrendingTrending75 minControlDMEM0001 μM Ang II5CtgfEdn1Serpine1Tgfb3Thbs130Gnb1DMEM1Hgf411 μM Ang II6Cav1CtgfEdn1Itgb8Serpine1Tgfb34224 hControlDMEM0001 μM Ang II7CtgfEdn1Itga2Itgb3Ltbp1Serpine1Tgfb3310Gnb1DMEM2Cxcr4Mmp82511 μM Ang II13Col1a2CtgfEdn1IlkItga2Itgb3Itgb8Ltbp1Mmp8PdgfaSerpine1Tgfb3Timp2420Gnb2DMEM0811 μM Ang II11CtgfEdn1IlkItga2Itgb3Itgb6Itgb8Ltbp1Serpine1Tgfb3Timp2331This table summarizes gene expression changes measured using the Qiagen RT2 Profiler PCR Array at 75 min and 24 h Ang II stimulation. Listed are genes with fold changes ≥ 1.5 or ≤ 0.5 compared to DMEM/siRNA control conditions at the respective time point. This includes genes with significant (p < 0.05) changes in expression and genes that showed fold changes ≥1.5 without reaching statistical significance (labeled as "Trending"). Statistical significance of the gene expression changes for the indicated genes was assessed by performing two-way ANOVA on normalized Ct values for each gene individually, followed by post hoc t test comparisons with Bonferroni correction. Data is representative of three independent biological replicates.Abbreviation: DMEM, Dulbecco's modified Eagle's medium. Open table in a new tab This table summarizes gene expression changes measured using the Qiagen RT2 Profiler PCR Array at 75 min and 24 h Ang II stimulation. Listed are genes with fold changes ≥ 1.5 or ≤ 0.5 compared to DMEM/siRNA control conditions at the respective time point. This includes genes with significant (p < 0.05) changes in expression and genes that showed fold changes ≥1.5 without reaching statistical significance (labeled as "Trending"). Statistical significance of the gene expression changes for the indicated genes was assessed by performing two-way ANOVA on normalized Ct values for each gene individually, followed by post hoc t test comparisons with Bonferroni correction. Data is representative of three independent biological replicates. Abbreviation: DMEM, Dulbecco's modified Eagle's medium. Disruption of AT1R signaling by Gβ2 knockdown also resulted in increased average gene expression of genes on the array in the absence of Ang II but to a lesser extent than that observed for Gβ1 knockdown (Fig. 2B). Moreover, average Ang II-induced gene expression in Gβ2 knockdown cells was similar to the siRNA control and the number of individual genes on the array whose expression was altered >1.5-fold was similar in Gβ2 knockdown and siRNA control conditions (Table 1). Thus, Gβγ signaling through Ca2+ release is not absolutely required for AT1R-mediated transcriptional changes. Instead, Gβγ is also required to modulate processes driven by other signaling pathways and dampen the fibrotic response until such signals rise above a threshold. Thus, the dampening of the fibrotic gene expression program seems to be a specific function of Gβ1. Next, using a global proteomic strategy, we assessed the fibrotic response at the protein level in response to Ang II stimulation under control and Gβ1-siRNA conditions. For these experiments, RNCFs were treated with vehicle or Ang II for 6 h to capture events between induction and full expression of the fibrotic response. This treatment time was chosen to be intermediate between 75 min, when Ang II effects on gene expression are first detected, and the appearance of the full-blown fibrotic response after 24 h of stimulation. Total protein was extracted from the samples and processed for quantitative, whole-proteome MS as described (31Doellinger J. Schneider A. Hoeller M. Lasch P. Sample preparation by easy extraction and digestion (SPEED) - a universal, rapid, and detergent-free protocol for proteomics based on acid extraction.Mol. Cell. Proteomics. 2020; 19: 209-222Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). As expected, Gβ1 (GNB1) was identified as significantly reduced in the Gβ1-siRNA samples (Fig. 3A, left panel). Under basal conditions, knockdown of Gβ1 resulted in increased expression of 19 proteins and reduced expression of 81 proteins (Fig. 3A). Gene ontology (GO) analysis revealed that these are not simply fibrotic genes, highlighting a generalized effect of Gβ1 knockdown (Figs. 3A and S2). Under control siRNA conditions, Ang II treatment significantly increased expression of ten proteins, including the fibrotic proteins Ctgf and Serpine1 (both of which were represented on the fibrosis qPCR array in Fig. 2). This result is consistent with activation of the fibrotic response (Fig. 3B). We presume that the response was only partial, as a full response requires a longer treatment time to develop (32Sadoshima J. Izumo S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype.Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1302) Google Scholar). Gβ1 knockdown combined with Ang II treatment resulted in a more robust fibrotic response than that seen under vehicle conditions (Fig. 3C). GO analysis of Gβ1 knockdown highlighted a clear activation of pathways implicated in the fibrotic response, including "positive regulation of collagen biosynthetic process," at a time when there is little response when Gβ1 is present (Fig. 3C and Supplementary Data Spreadsheet 1). The latter finding supports the notion that Gβ1-containing Gβγ subunits normally play an inhibitory role in regulating the fibrotic response. Also of note, knockdown of Gβ1 led to increased expression of Gβ2. It is possible that having more of the Gβ isoform that is responsible for proximal receptor-mediated signaling might also increase the fibrotic response but no increase in calcium signalling was detected when Gβ1 was knocked down (Fig. 1, C and D). As the negative role of Gβ1 in the fibrotic response did not seem to be related to an effect on receptor-proximal signaling, we tested whether it might involve a downstream gene-regulatory function. Gβγ is known to interact with transcription factors and occupies gene promoter regions (19Park J.G. Muise A. He G.P. Kim S.W. Ro H.S. Transcriptional regulation by the γ5 subunit of a heterotrimeric G protein during adipogenesis.EMBO J. 1999; 18: 4004-4012Crossref PubMed Scopus (51) Google Scholar, 20Robitaille M. Gora S. Wang Y. Goupil E. Petrin D. Del Duca D. et al.Gβγ is a negative regulator of AP-1 mediated transcription.Cell Signal. 2010; 22: 1254-1266Crossref PubMed Scopus (26) Google Scholar, 21Spiegelberg B.D. Hamm H.E. Gβγ binds histone deacetylase 5 (HDAC5) and inhibits its transcriptional co-repression activity.J. Biol. Chem. 2005; 280: 41769-41776Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 22Bhatnagar A. Unal H. Jagannathan R. Kaveti S. Duan Z.H. Yong S. et al.Interaction of G-protein betagamma complex with chromatin modulates GPCR-dependent gene regulation.PLoS One. 2013; 8e52689Crossref Scopus (13) Google Scholar). Thus, we assessed the possibility of genome-wide Gβ1 recruitment and changes in RNAPII occupancy following brief (75-min) Ang II treatment in cardiac fibroblasts. We performed chromatin immunoprecipitation (IP) for heterologously expressed FLAG-Gβ1 and endogenous Rpb1 followed by next generation sequencing. We focused on genes with RNAPII peaks identified by the peak calling software macs2 (https://github.com/macs3-project/MACS) and annotated with HOMER (33Heinz S. Benner C. Spann N. Bertolino E. Lin Y.C. Laslo P. et al.Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.Mol. Cell. 2010; 38: 576-589Abstract Full Text Full Text PDF PubMed Scopus (6990) Google Scholar, 34Zhang Y. Liu T. Meyer C.A. Eeckhoute J. Johnson D.S. Bernstein B.E. et al.Model-based analysis of ChIP-seq (MACS).Genome Biol. 2008; 9R137Crossref Scopus (9436) Google Scholar). The same Gβ1 knockdown conditions that increased the number of fibrotic genes upregulated in response to Ang II (Fig. 2 and Table 1) also increased the number of genes occupied by RNAPII, as measured by ChIP-seq following Ang II treatment (Fig. 4A). To identify groups of genes with similar FLAG-Gβ1 and RNAPII occupancy patterns, we performed k-means clustering with genes for which RNAPII peaks were identified in any treatment condition. Two K-means clusters were identified (104 genes in cluster 1 and 806 in cluster 2) with distinct occupancy patterns (Fig. 4, B and C). In cluster 1, FLAG-Gβ1 occupancy increased within the gene body in response to Ang II. A similar but weaker tendency was also observed in cluster 2 (Fig. 4B). The increased FLAG-Gβ1 occupancy in cluster 1 corresponded to Gβ1-dependent changes to Ang II-induced RNAPII occupancy alterations. First, Ang II treatment led to increased RNAPII occupancy throughout the gene body under siRNA control conditions (Fig. 4C). In the absence of Ang II, Gβ1 knockdown increased RNAPII occupancy near transcription start sites which corresponds with increased gene expression under these conditions (Fig. 4C). Lastly, there was greater RNAPII occupancy when Ang II treatment was combined with Gβ1 knockdown than in the absence of knockdown (Fig. 4C). Similar RNAPII occupancy patterns were observed in cluster 2, suggesting that Gβ1 also plays a regulatory role along these genes and our FLAG-Gβ1 chromatin IP sequencing (ChIP-seq) was not sensitive enough to reliably detect Gβ1. We also assessed the functional pathways enriched in cluster 1, through GO term enrichment. The top four significant GO terms identified corresponded to cellular processes such as inflammation, fibroblast activation and apoptosis, indicating that Gβ1 is recruited to genes involved in processes essential to fibrosis (Fig. 4D). Occupancy by FLAG-Gβ1 or RNAP II on 2 individual genes are also shown (Thbs1, Fig. 4E) and (Ctgf, Fig. 4F) are also shown. The increased number of genes with RNAPII occupancy in the Ang II and Gβ1 knockdown condition suggested that Gβ1 occupancy impairs RNAPII recruitment. As such, we would expect cluster 1 genes to be more enriched in genes with RNAPII occupancy under Ang II and Gβ1 knockdown condition than Ang II and siRNA control conditions. Therefore, we performed a Fisher's
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