Acetylation Of GATA4 Research Articles

GATA-1 and BCL11A Collaboratively Regulate Erythroid Gene Expression through HDAC1 Dependent Manner

Transcription factor GATA-1 plays important roles in hematopoiesis through interaction with other key transcription factors and epigenetic modulators. Previously, we established that GATA-1 can physically interact with HDAC1 at two arginine residues of the linker region between two zinc fingers. Arginine to alanine mutation at these sites blocks the HDAC1 interaction and results in constitutive GATA-1 acetylation, and reduced GATA-1 chromatin binding at the gene regulatory regions. In addition, the loss of HDAC1 interaction causes the blockage of erythroid and megakaryocytic differentiation in mice. Having established the importance of GATA-1 and HDAC1 interaction in GATA-1 function, next we aim to further investigate whether HDAC1-associated proteins are involved in regulating the function of GATA-1. We performed LC-MS/MS analysis using GATA-1 wild type and mutant expressing cells and identified that BCL11A was one of the major proteins that associates with GATA-1 through HDAC1. BCL11A is a master regulator of γ-globin gene silencing through association with the NuRD complex and blocking of NFYA recruitment to γ-globin gene promoters. The role of BCL11A in regulating gene expression during erythroid differentiation is not well studied. HDAC1 mediated GATA-1 and BCL11A association was validated by pull down and immunoprecipitation assay. Next, the BCL11A and HDAC1 interacting site was determined. The N-terminal region of BCL11A is found to be important for HDAC1 interaction by pull down assay. Through analysis of the secondary structure of BCL11A N-terminal, L12/K14 are predicted to be important for protein-protein interaction. Indeed, L12A/K14A mutation significantly reduced HDAC1 interaction as well as HDAC1 mediated GATA-1 and NuRD complex association. To assess the effects of the L12A/K14A mutation of BCL11A on gene expression, we performed RNA-seq analysis using BCL11A knock down (KD) or L12A/K14A mutant overexpressed in HUDEP-2 cells. The result shows that the L12A/K14A mutation de-repressed γ-globin expression and positively affects genes for erythrocyte development that is similar to the expression profile of BCL11A KD. Therefore, L12/K14 residues of BCL11A might have an important role in γ-globin silencing and erythropoiesis by mediating HDAC1 associated GATA-1 and NuRD complex. The Cut&Run assay was performed to study the recruitment of BCL11A, GATA-1, HDAC1, NuRD complex, and NFYA in WT, BCL11A KD, and Flag-BCL11A L12A/K14A mutant cells. There are over 7000 GATA-1 and BCL11A co-binding peaks detected in wild type cells. Within these peaks, about 2000 peaks are at BCL11A binding motif site. GATA-1, HDAC1 and NuRD binding were abolished at these sites in BCL11A KD or L12A/K14A mutant cells, while BCL11A binding was not affected. There are about 3000 peaks at GATA-1 binding motif sites. While GATA-1 binding is not affected at these sites in BCL11A KD or L12A/K14A mutant cells, BCL11A, HDAC1 and NuRD complex binding were significantly reduced. This study demonstrates that there are two modes of BCL11A and GATA-1 co-recruitment: BCL11A can recruit GATA-1 and GATA-1 can recruit BCL11A to their specific binding motifs, respectively. Interestingly, these two modes regulate differential gene targets. BCL11A recruits GATA-1 at genes mainly involved in chromatin organization and cell cycle, while GATA-1 recruits BCL11A to genes mainly involved in transcription, erythrocyte and megakaryocyte differentiation. At the β-globin locus, BCL11A KD or L12A/K14A mutation reduced GATA-1 and NuRD binding and enhanced NFYA binding at the γ-globin promoter, suggesting that BCL11A cooperates with GATA-1, HDAC1, and NuRD complex and prevents NFYA binding at the distal binding region to suppress γ-globin expression. At LCR hypersensitive site 2 (HS2) region, we also observed a reduction of GATA-1 and NuRD recruitment with BCL11A KD or L12A/K14A mutation while GATA-1 recruitment at other promoters or LCR regions were not changed, suggesting that the HS2 region may be involved in reactivating γ-globin in BCL11A KD or L12A/K14A mutation cells. Future study will continue to analyze how BCL11A KD or mutation affects BCL11A and GATA-1 co-binding and target gene expression, and NFYA or other factors recruitment. In summary, our study shows that BCL11A interacts with GATA-1 in a HDAC1 dependent manner and this interaction is important in regulating fetal globin expression and erythroid development.

PS-BPB02-8: CURCUMIN, AN INHIBITOR OF P300-HAT ACTIVITY, PREVENTS HYPERTENSION-INDUCED CARDIAC HYPERTROPHY WITH PRESERVED SYSTOLIC FUNCTION IN DAHL RATS

Background: Hypertension causes left ventricular hypertrophy (LVH) with a preserved systolic function. While LVH might be a compensatory process, its presence of LVH is associated with an increased incidence of cardiovascular events and heart failure. Recently, we found that curcumin, a major curcuminoid isolated from Curcuma longa, inhibits the histone acetyltransferase (HAT) activity of p300 as well as the p300-induced acetylation of histones and a hypertrophy-responsive transcription factor, GATA-4, in cultured cardiomyocytes. Furthermore, curcumin prevents deterioration of the systolic function in rat heart failure models in vivo. However, whether curcumin prevents the development of hypertension-induced LVH at an early stage of hypertension is unclear. Objective: To solve this problem, we have utilized a salt-sensitive Dahl rat (DS) model of hypertension. Methods: We randomized 6-week-old DS (n = 19) and control salt-resistant Dahl rats (DR, n = 10) to curcumin or the vehicle group. Then, these rats were given a high-salt diet and subjected to daily oral treatment with 50 mg/kg/day of curcumin or its vehicle (1% Gum arabic) for 6 weeks. Results: There were no differences between curcumin and vehicle in any data examined before treatment (6 weeks of age). The high-salt diet-induced a similar degree of hypertension in these two groups of DS compared with DR. At 12 weeks of age, transthoracic echocardiography in the vehicle group showed that the LV wall thickness, LV mass, and LV fractional shortening were significantly higher in DS compared with DR, and the LV systolic and diastolic dimensions were significantly smaller in DS than DR. Curcumin treatment significantly (p < 0.01) decreased the LV wall thickness in DS but not DR. Curcumin also significantly (p < 0.01) decreased the LV mass in DS, but not in DR. However, LV fractional shortening in DS was similar between the two groups. Curcumin also prevented the increases in hypertension-induced myocardial cell hypertrophy and perivascular fibrosis. Furthermore, curcumin significantly attenuated the up-regulation of hypertrophy-response gene transcriptions as well as acetylation of GATA4 but not phosphorylation of ERK1/2 and GATA4 in the heart. Conclusions: The natural compound, curcumin, inhibits the development of hypertension-induced concentric LVH without affecting the systolic function through inhibition of GATA4-acetylation. Thus, this compound might be applicable to patients with early-stages hypertensive heart diseases.

Abstract P362: Gata4 Dimerization May Be A Target For Heart Failure Therapy

Introduction: Cardiac hypertrophy is regulated by activation of GATA4. Although GATA4 post-translational modification such as acetylation by p300 is well examined, the details of the activation mechanism of GATA4 are still unclear. The purpose of this study is to investigate whether GATA4 dimerization involved in transcriptional activation and cardiomyocyte hypertrophic responses. Methods and Results: A GST pull-down assay using GST fusion GATA4 full-length and deletion mutants demonstrated that GATA4 308-326, including the acetylation site, was required for the dimerization of GATA4. A DNA pull down assay showed that the C-zinc finger motif (256-295) and the acetylation site were required for the DNA binding capacity of GATA4. IP-WB using nuclear extract from HEK293T cells expressing FLAG- or HA-tagged GATA4 showed that co-expression of p300 increased the formation of the homo-dimer as well as acetylation of GATA4. The GATA4 homo-dimer was disrupted by both acetyl-deficient GATA4 and HAT-deficient p300. This result indicates that acetylation of GATA4 is important for dimerization of GATA4. Overexpression of the deletion mutant containing a GATA4 308-326 (G4D) prevented p300-induced GATA4 dimerization but not the p300 binding nor acetylation of GATA4. ChIP assay and DNA pulldown assay showed that G4D did not inhibit the p300-induced DNA binding of GATA4. In cardiomyocytes, the G4D inhibited phenylephrine-induced ANF and ET-1 promoter activities and cardiomyocyte hypertrophy. To perform the X-ray crystal structure analysis, recombinant GATA4 fragment including GATA4 308-326 was highly purified. The X-ray diffraction data of obtained crystals was collected. Resolution of the crystal was 3.1Å, which was insufficient for phase determine. To obtain a high-quality crystal, GATA4 fragment was crystallized in international space station, in collaboration with JAXA. Resolution of the crystal was 3.16Å, which was similar to the best data before obtained. Conclusions: These results suggest that GATA4 dimerization may play an important role in hypertrophy-response gene transcription. It is expected to elucidate the GATA4 dimerization mechanism and targeted this dimerization will lead to the development of a noble heart failure therapy.

Retraction Statement.

Retraction Statement.

HDAC1 is required for GATA-1 transcription activity, global chromatin occupancy and hematopoiesis.

The activity of hematopoietic factor GATA-1 is modulated through p300/CBP-mediated acetylation and FOG-1 mediated indirect interaction with HDAC1/2 containing NuRD complex. Although GATA-1 acetylation is implicated in GATA-1 activation, the role of deacetylation is not studied. Here, we found that the FOG-1/NuRD does not deacetylate GATA-1. However, HDAC1/2 can directly bind and deacetylate GATA-1. Two arginine residues within the GATA-1 linker region mediates direct interaction with HDAC1. The arginine to alanine mutation (2RA) blocks GATA-1 deacetylation and fails to induce erythroid differentiation. Gene expression profiling and ChIP-seq analysis further demonstrate the importance of GATA-1 deacetylation for gene activation and chromatin recruitment. GATA-12RA knock-in (KI) mice suffer mild anemia and thrombocytopenia with accumulation of immature erythrocytes and megakaryocytes in bone marrow and spleen. Single cell RNA-seq analysis of Lin− cKit+ (LK) cells further reveal a profound change in cell subpopulations and signature gene expression patterns in HSC, myeloid progenitors, and erythroid/megakaryocyte clusters in KI mice. Thus, GATA-1 deacetylation and its interaction with HDAC1 modulates GATA-1 chromatin binding and transcriptional activity that control erythroid/megakaryocyte commitment and differentiation.

Deacetylase-independent function of SIRT6 couples GATA4 transcription factor and epigenetic activation against cardiomyocyte apoptosis.

SIRT6 deacetylase activity improves stress resistance via gene silencing and genome maintenance. Here, we reveal a deacetylase-independent function of SIRT6, which promotes anti-apoptotic gene expression via the transcription factor GATA4. SIRT6 recruits TIP60 acetyltransferase to acetylate GATA4 at K328/330, thus enhancing its chromatin binding capacity. In turn, GATA4 inhibits the deacetylase activity of SIRT6, thus ensuring the local chromatin accessibility via TIP60-promoted H3K9 acetylation. Significantly, the treatment of doxorubicin (DOX), an anti-cancer chemotherapeutic, impairs the SIRT6–TIP60–GATA4 trimeric complex, blocking GATA4 acetylation and causing cardiomyocyte apoptosis. While GATA4 hyperacetylation-mimic retains the protective effect against DOX, the hypoacetylation-mimic loses such ability. Thus, the data reveal a novel SIRT6–TIP60–GATA4 axis, which promotes the anti-apoptotic pathway to prevent DOX toxicity. Targeting the trimeric complex constitutes a new strategy to improve the safety of DOX chemotherapy in clinical application.

P1608Inhibition of GATA4 dimerization suppress hypertrophic responses

Abstract Introduction Hypertrophic signals eventually reach the nuclei of cardiomyocytes, change patterns of gene expression, and cause the development of heart failure. During the development of heart failure, intrinsic histone acetyltransferase called p300 induce GATA4 acetylation. Acetylated GATA4 increases its DNA binding, up-regulates cardiac hypertrophic response genes, and lead to heart failure. A zinc finger protein, GATA4 is the transcription factor that expression level is high in heart. It has been reported that GATA1, the same GATA family, regulates transcriptional activity through its homo-dimerization. However, GATA4 homo-dimerization and its relationship to hypertrophic responses are still unknown. Purpose To clarify the relationship between GATA4 homo-dimerization and transcriptional activity and investigate whether inhibition of this homo-dimerization become therapeutic target for cardiac hypertrophy. Methods GST pull-down and DNA pull-down assay were performed using GST fusion full length and deletion mutants of GATA4 and biotin-conjugated ET-1 promoter probe including a GATA element. Recombinant C-zinc finger domain (256–326), including C-zinc finger motif (256–295) and acetylation site (308–326) was cross-linked using glutaraldehyde and subjected to silver staining. An expression plasmid with three GATA4-acetylation site mutant-conjugated with nuclear localization sequence (3xG4D) was constructed. Immunoprecipitation and western blotting were performed using nuclear extract from HEK293T cells expressing p300, GATA4, and 3xG4D. Luciferase assay was using ANF and ET-1 promoter sequences. Neonatal rat cultured cardiomyocyte expressed 3xG4D and then stimulated with phenylephrine (PE) for 48 hours. Next cardiomyocytes stained with α-actinin antibody and measured the cell surface area. Results The acetylation site of GATA4 was required for the dimerization of GATA4. But, C-zinc finger motif (256–295) and the acetylation site were required for the DNA binding. Recombinant C-zinc finger domain formed not only a homo-dimer but also a multimer. Co-expression of p300 increased the formation of homo-dimer as well as the acetylation of GATA4 in HEK293T cells. The GATA4 homo-dimer was disrupted by acetyl-deficient GATA4 or HAT-deficient p300 mutant. Overexpression of 3xG4D prevented the dimerization of GATA4, but not acetylation of GATA4. The result of luciferase assay showed that overexpression of 3xG4D prevented p300/GATA-induced ANF and ET-1 promoter activities. Furthermore, overexpression of 3xG4D inhibited phenylephrine-induced cardiomyocyte hypertrophy. Conclusions These results suggest that GATA4 dimerization may play an important role in hypertrophy-response gene activation. Thus, it is likely that inhabitation of GATA4 dimerization become therapeutic target for cardiac hypertrophy.

P4998Novel GATA4 binding proteins, RbAp48/46, regulate cardiomyocyte hypertrophy with depending on the phosphorylate State of GATA4

Abstract Introduction Cardiac hypertrophy is being recognized as a critical event during the development of heart failure. A zinc finger protein GATA4 associates with an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. Here, we show that Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46) are novel components of p300/GATA4 complex. Both RbAp48 and 46 form a repressor complex with HDACs and has been implicated in chromatin remodeling and transcriptional repression. However, the precise functional relationships among p300, GATA4, RbAp48, and RbAp46 remain unknown. Hypothesis We assessed the hypothesis that RbAp48/46 form a functional complex with p300/GATA4 and regulated hypertrophic responses in cardiomyocytes. Methods and results IP-WB using nuclear extract from rat heart demonstrated that GATA4 formed a complex with RbAp48, RbAp46, HDAC1, and HDAC2. GST pull down assay using recombinant proteins showed that GATA4 physically interacted with both RbAp48 and RbAp46 but not HDAC1 and HDAC2. Deletion mutant assay revealed that N-terminal domain of GATA4 interacted with RbAp48/46. In HEK293T cell, overexpression of RbAp48/46 recruited HDAC1/2 to GATA4, inhibited p300-induced GATA4 acetylation and suppressed p300/GATA4-dependent ANF and ET-1 promoter activations. Conversely, the knockdown of RbAp48/46 reversed these changes. Although overexpression of HDAC1/2 did not change p300/GATA4-induced these promoter activities, co-expression of HDAC1 or HDAC2 with RbAp48/46 enhanced RbAp48/46-mediated inhibitory actions. In cardiomyocytes, overexpression of RbAp48/46 significantly inhibited phenylephrine (PE)-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and cardiomyocyte hypertrophy. The knockdown of RbAp48/46 reversed these changes. Moreover, the knockdown of HDAC1/2 deteriorated PE-induced hypertrophy-responsive events and did not exhibit RbAp48/46-induced inhibitory actions. Finally, MEK1/ERK-mediated S105 phosphorylation of GATA4 by PE stimulus induced the dissociation of RbAp48/46 with GATA4, the increase of p300-induced GATA4-acetylation, the synergistic activation of ANF and ET-1 promoters with p300/GATA4, and the decrease of RbAp48/46 recruitments onto the GATA element of the ANF promoter. Conversely, PD98059, a MEK1 inhibitor, treatment inhibited GATA4-phosphorylation and these changes. Conclusion In this study, we demonstrate that RbAp48/46 mediate the binding between GATA4 and HDAC1/2 and regulate p300/GATA4 axis. The phosphorylation of S105 GATA4 has a critical role on the dissociation of GATA4/RbAp48/46/HDAC repressor complex, the formation of 300/GATA4 activator complex, and the increase of GATA4 acetylation and hypertrophic responses. These findings suggest that RbAp48/46 may regulate hypertrophic responses involved in modulating the posttranslational modification crosstalk of GATA4. Acknowledgement/Funding This work was supported by JSPS KAKENHI Grant.

Acetylation of GATA4 on Lysine Residue K313 Promotes Osteoblastic Cells Growth

GATA4, a protein related to osteoblast differentiation and mineralization, whose acetylation is essential for cardiac defects. Here, we aimed to explore the functional impacts of GATA4 acetylation on osteoporosis (OS). GATA4 acetylation in hFOB1.19 and 293T cells was detected after exposure of HDAC inhibitors (TSA and SAHA). Co-immunoprecipitation was conducted to determine which HATs and HDACs was involved in the modulation of GATA4 acetylation/deacetylation, and to identify the acetylation site. The transcriptional activity of GATA4 was measured in the presence or absence of cycloheximide. Furthermore, hFOB1.19 cells viability and apoptosis were evaluated after transfection with acetylation-defective mutant of GATA4. As a result, GATA4 acetylation was identified as a pivotal event in hFOB1.19 cells. GATA4 can be acetylated by P300/CBP, and the acetylation site was on lysine residue K313. Besides, the acetylation of GATA4 can be impaired by HDAC1, rather than by HDAC2-5. GATA4 acetylation contributed to the stability and transcription of GATA4. Moreover, GATA4 acetylation activated CCND2 transcription, and mutation of GATA4 on K-313 reduced cell viability and increased a mitochondria-dependent apoptosis in hFOB1.19 cells. Our data suggest that GATA4 exists as an acetylated protein in hFOB1.19 cells. Acetylation regulates the stability and transcription of GATA4, and activates CCND2 transcription, which may explain the growth-promoting functions of GATA4 in hFOB1.19 cells.

The GATA4 Acetylation Site Plays a Key Role in the Development of Cardiomyocyte Hypertrophy

The GATA4 Acetylation Site Plays a Key Role in the Development of Cardiomyocyte Hypertrophy

GATA-1 Deacetylation and Interaction with HDAC1 Is Critical for GATA-1 Mediated Gene Transcription

The transcription factor GATA-1 is an essential factor for globin gene transcrption and is required for erythroid and megakaryocytic cell differentiation and maturation. GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 chromatin binding activity. However, it is not clear whether GATA-1 acetylation can be reversed by a deacetylase. It is showed that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) associated NURD corepressor complexes through binding to FOG-1. However, we found that the NURD complex does not deacetylate GATA-1. We discovered that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for the interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) blocks the interaction of HDAC1, but doesn't affect its DNA binding in vitro. Importantly, the mutation does not affect the interaction with FOG-1, indicating that GATA-1 direct interaction and indirect association via FOG-1 with HDAC1 are separate events. To further understand the role of the HDAC1-GATA-1 direct interaction in gene transcription and erythropoiesis, we introduced the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Interestingly, upon estradiol induction, GATA-1 2RA does not promote b-globin gene transcription and erythroid differentiation of G1E cells, although GATA-1 2RA is highly acetylated. Chromatin immunoprecipitation assay (ChIP) shows that GATA-1 2RA binds poorly to HS3 and β-globin promoter. Similar binding defect is also detected on GATA-1 promoter, indicating defective GATA-1 recruitment on chromatin. Interestingly, HDAC1 binding to these regions are also significantly reduced, suggesting HDAC1-GATA-1 interaction may be important for GATA-1 deacetylation, as well as stabilizing GATA-1 binding. In order to investigate the effect of GATA-1 2RA in vivo, we generated a GATA-1 2RA knock in mice. The knock in mice are viable but suffered from anemia and thrombocytopenia. β-globin expression reduced at least 50% in knock in mice compare to wild type litter mate. To further identify and compare gene expression profiles regulated by HDAC1 direct or indirect associated GATA-1 during erythroid differentiation, we performed RNA sequencing assays to study the effects of GATA-1 2RA in gene expression in comparison with wild type GATA-1 or GATA-1-V205M (a mutation abolished binding with FOG-1). Expression of GATA-1 2RA largely affects gene expression profile in both GATA-1 activated and repressed genes compared to cells expressing wild type GATA-1. The gene expression pattern in 2RA cells also is largely different from cells expressing GATA-1 V205M, indicating the direct and indirect interaction with HDAC1 may mediate differential functions. Our results indicate that HDAC1 is required for GATA-1 recruitment and GATA-1 mediated transcription regulation. Thus, this study unveils a novel regulation of GATA-1 by its direct interaction with HDAC1. DisclosuresNo relevant conflicts of interest to declare.

Abstract 34: Acetylation of GATA 4 Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes

Objective: Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2c and Tbx5), has recently been demonstrated, suggesting a promising statregy for cardiac regeneration. However, the efficiency of direct reprogramming is usually relatively low and requires extensive epigenetic redesigning, although the underlying mechanism are largely unknown. Methods: In a recent study, we created a novel mutation in rat GATA 4 by replacing lysine residue with glutamine at position 299 i.e. (K299Q), to mimic constitutive acetylation and examined whether constitutive acetylation of GATA4, when compared with wild type GATA4, further enhance GMT-mediated direct reprogramming efficiency of induced cardiomyocytes in vitro and accordingly ventricular function after myocardial infarction in rat, in vivo . Results: We found that acetylated GATA 4 (K299Q), in the presence of Mef2c and Tbx5 upregulated cardiac-specific markers, suppressed fibroblast genes, in rat cardiac fibroblasts (RCFs) more efficiently when compared with Mef2c, Tbx5 plus wild type GATA4. FACS analyses revealed that G(K299Q) MT induced significantly more cardiomyocyte marker cardiac troponin T (cTnT) expression compared with GMT alone. Mechanistic studies demonstrated that the K299Q substitution, resulting in enriched p300 occupancy at the GATA 4 promoter, induced acetylation of Histine 3, decreased HDAC expression. In addition, substitution augmented the increase in an acetylated form of GATA-4 and its DNA binding and transcriptional activity, compared with wildtype GATA 4. In agreement with upregulated cTNT gene expression in vitro , echocardiographic analysis demonstrate that the acetylated G(K299Q) MT vectors have improved effect in enhancing ventricular function than GMT vectors from postinfarct baselines as compared to negative control [G(K299Q) MT, 15.6% ± 2.7%; G(WT)MT, 12.8% ± 1.7%; GFP, -2.3% ± 1.1%]. Conclusions: Collectivily, these data indicate that acetylated GATA4 (K299Q) significantly increases reprogramming efficiency of induced cardiomyocytes (iCMs), in vitro and in vivo, and provide new insight into the molecular mechanism underlying cardiac regeneration.

Up-regulation of IRF-3 expression through GATA-1 acetylation by histone deacetylase inhibitor in lung adenocarcinoma A549 cells

Interferon regulatory factor 3 (IRF-3) is an important transcription factor for interferon genes. Although its functional activation by viral infection has been widely explicated, the regulatory mechanism of IRF-3 gene expression in cancer cells is poorly understood. In this study, we demonstrated treatment of lung adenocarcinoma A549 cells with trichostatin A (TSA) and valproic acid (VPA), two different classes of histone deacetylase inhibitors, strongly stimulated IRF-3 gene expression. Truncated and mutated IRF-3 promoter indicated that a specific GATA-1 element was responsible for TSA-induced activation of IRF-3 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TSA treatment increased the binding affinity of GATA-1 to IRF-3 promoter. Using immunoprecipitation assay and immunoblotting, we demonstrated that TSA increased the level of acetylated GATA-1 in A549 cells. In summary, our study implied that TSA enhanced IRF-3 gene expression through increased GATA-1 recruitment to IRF-3 promoter and the acetylation level of GATA-1 in lung adenocarcinoma A549 cells.

Bnip3 Binds and Activates p300: Possible Role in Cardiac Transcription and Myocyte Morphology.

Bnip3 is a hypoxia-regulated member of the Bcl-2 family of proteins that is implicated in apoptosis, programmed necrosis, autophagy and mitophagy. Mitochondria are thought to be the primary targets of Bnip3 although its activities may extend to the ER, cytoplasm, and nucleus. Bnip3 is induced in the heart by ischemia and pressure-overload, and may contribute to cardiomyopathy and heart failure. Only mitochondrial-dependent programmed death actions have been described for Bnip3 in the heart. Here we describe a novel activity of Bnip3 in cultured cardiac myocytes and transgenic mice overexpressing Bnip3 in the heart (Bnip3-TG). In cultured myocytes Bnip3 bound and activated the acetyltransferase p300, increased acetylation of histones and the transcription factor GATA4, and conferred p300 and GATA4-sensitive cellular morphological changes. In intact Bnip3-TG hearts Bnip3 also bound p300 and GATA4 and conferred enhanced GATA4 acetylation. Bnip3-TG mice underwent age-dependent ventricular dilation and heart failure that was partially prevented by p300 inhibition with curcumin. The results suggest that Bnip3 regulates cardiac gene expression and perhaps myocyte morphology by activating nuclear p300 acetyltransferase activity and hyperacetylating histones and p300-selective transcription factors.

Abstract 12566: Receptor for Activated Protein Kinase C1 Competes With GATA4 for p300 and Represses Hypertrophic Responses in Cardiomyocyte

Introduction: The zinc finger protein GATA4 is one factor involved in transcriptional regulation during myocardial cell hypertrophy, and forms a complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activity of GATA4, as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo . We recently identified Receptor for Activated Protein Kinase C1 (RACK1), a multi-functional scaffold protein, as a novel GATA4-binding partner, and revealed that RACK1 regulates the p300/GATA4-dependent transcriptional pathway and hypertrophic responses in cardiomyocytes. However, the molecular mechanism by which RACK1 suppresses p300/GATA4-dependent gene transcription remains unknown. Hypothesis: We hypothesized that RACK1 reduces the phenylephrine (PE)-induced increase of the interaction between GATA4 and p300 in cardiacmyocytes. Methods and Results: To examine the effect of RACK on the binding activity of p300 to GATA4, expression plasmids encoding these proteins were used to co-transfect HEK293 cells. Nuclear extracts from these cells were subjected to immunoprecipitation, followed by Western blotting. Overexpression of RACK1 inhibited p300-induced GATA4 acetylation and the association with p300/GATA4. Based on chromatin immunoprecipitation and reporter assays, RACK1 inhibited not only p300-induced DNA binding activity of GATA4 on atrial natriuretic factors (ANF) and endothelin-1 (ET-1) promoters but also p300/GATA4-induced ANF and ET-1 promoter activities. In cultured neonatal rat ventricular cardiomyocytes, overexpression of RACK1 significantly inhibited PE-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and myocardial cell hypertrophy. Also, PE decreased binding between GATA4 and RACK1. Finally, PE stimulation increased tyrosine phosphorylation of RACK1. This phosphorylation was inhibited by a tyrosine kinase inhibitor, Dasatinib. Conclusions: The findings demonstrated that RACK1 disrupted the p300/GATA4 complex and inhibited hypertrophic responses in cardiomyocytes. Tyrosine phosphorylation of RACK1 may trigger the dissociation of RACK1 from p300/GATA4.

Inhibition of Cardiomyocyte Hypertrophy by Protein Arginine Methyltransferase 5

Protein arginine methyltransferase 5 (PRMT5), a protein arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues within target proteins, has been implicated in many essential cellular processes ranging from the regulation of gene expression to cell proliferation and differentiation. PRMT5 is highly expressed in the heart; the functional role of PRMT5 in the heart, however, remains largely elusive. In the present study, we show that PRMT5 specifically interacts with GATA4 in both co-transfected HEK293T cells and neonatal rat cardiomyocytes by co-immunoprecipitation. Importantly, this interaction leads to the arginine methylation of GATA4 at positions of 229, 265, and 317, which leads to an inhibition of the GATA4 transcriptional activity, predominantly through blocking the p300-mediated acetylation of GATA4 in cardiomyocytes. Moreover, overexpression of PRMT5 substantially inhibited the acetylation of GATA4 and cardiac hypertrophic responses in phenylephrine-stimulated cardiomyocytes, whereas knockdown of PRMT5 induced GATA4 activation and cardiomyocyte hypertrophy. Furthermore, in response to phenylephrine stimulation, PRMT5 translocates into the cytoplasm, thus relieving its repression on GATA4 activity in the nucleus and leading to hypertrophic gene expression in cardiomyocytes. These findings indicate that PRMT5 is an essential regulator of myocardial hypertrophic signaling and suggest that strategies aimed at activating PRMT5 in the heart may represent a potential therapeutic approach for the prevention of cardiac hypertrophy and heart failure.

EDAG positively regulates erythroid differentiation and modifies GATA1 acetylation through recruiting p300.

Erythroid differentiation-associated gene (EDAG) has been considered to be a transcriptional regulator that controls hematopoietic cell differentiation, proliferation, and apoptosis. The role of EDAG in erythroid differentiation of primary erythroid progenitor cells and in vivo remains unknown. In this study, we found that EDAG is highly expressed in CMPs and MEPs and upregulated during the erythroid differentiation of CD34(+) cells following erythropoietin (EPO) treatment. Overexpression of EDAG induced erythroid differentiation of CD34(+) cells in vitro and in vivo using immunodeficient mice. Conversely, EDAG knockdown reduced erythroid differentiation in EPO-treated CD34(+) cells. Detailed mechanistic analysis suggested that EDAG forms complex with GATA1 and p300 and increases GATA1 acetylation and transcriptional activity by facilitating the interaction between GATA1 and p300. EDAG deletion mutants lacking the binding domain with GATA1 or p300 failed to enhance erythroid differentiation, suggesting that EDAG regulates erythroid differentiation partly through forming EDAG/GATA1/p300 complex. In the presence of the specific inhibitor of p300 acetyltransferase activity, C646, EDAG was unable to accelerate erythroid differentiation, indicating an involvement of p300 acetyltransferase activity in EDAG-induced erythroid differentiation. ChIP-PCR experiments confirmed that GATA1 and EDAG co-occupy GATA1-targeted genes in primary erythroid cells and in vivo. ChIP-seq was further performed to examine the global occupancy of EDAG during erythroid differentiation and a total of 7,133 enrichment peaks corresponding to 3,847 genes were identified. Merging EDAG ChIP-Seq and GATA1 ChIP-Seq datasets revealed that 782 genes overlapped. Microarray analysis suggested that EDAG knockdown selectively inhibits GATA1-activated target genes. These data provide novel insights into EDAG in regulation of erythroid differentiation.

Abstract 9686: Novel GATA4 Binding Proteins, RbAp48/46 Regulate Post-Translational Modification During Hypertrophic Responses in Cardiomyocytes

Introduction: A zinc finger protein GATA4 associates with not only ERK1/2 but also an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. We recently reported that Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48/46) are novel components of the p300/GATA4 complex and form a repressor complex with HDACs in cardiomyocytes. However, precise functional regulation by RbAp48/46 during hypertrophic responses is still unknown. Hypothesis: We assessed the hypothesis that RbAp48/46 suppressed cardiac hypertrophic responses by regulating post-translational modification of GATA4 in cardiomyocytes. Methods and Results: Overexpression of RbAp48/46 inhibited phenylephrine (PE)-induced acetylation of GATA4, activation of atrial natriuretic factors (ANF) and endotheline-1 (ET-1) promoters, and increase in cell size in cultured cardiomyocytes. Conversely, knockdown of RbAp48/46 by shRNA augmented such responses. In the absence of HDAC1/2, RbAp48/46 failed to suppress cardiac hypertrophy. GST pull down assay revealed that RbAp48/46 interacted with a portion near the S105 phosphorylation site within N-terminal domain of GATA4. Immunoprecipitation and Western Blot demonstrated that a constituted active form of MEK1 induced S105 phosphorylation of GATA4, the dissociation between RbAp48/46 and GATA4, the increase in p300-dependant acetylation of GATA4, and p300/GATA4-dependant synergistic activations of ANF and ET-1 promoters. Such inductions did not occur in phosphorylation-mutated GATA4. Stimulation of cardiomyocytes with PE increased S105 phosphorylation of GATA4 and the dissociation of GATA4 from RbAp48/46 and HDAC1/2, and decreased recruitments of RbAp48/46 onto GATA element of the ANF promoter. These changes are suppressed by PD98059, a MEK1 inhibitor. Conclusion: These findings demonstrate that RbAp48 and RbAp46 form repressor complex with GATA4 as well as HDAC1/2, release p300 from GATA4, and inhibit hypertrophic responses in cardiomyocytes. The phosphorylation of GATA4 may be a trigger of the disassociation between RbAp/HDAC complex and GATA4, and of subsequent formation of p300/GATA4 complex and hypertrophic gene activation.

Abstract 9477: Retinoblastoma Protein-associated Protein 48 and 46 Form Repressor Complex With p300/GATA4 And Inhibit Hypertrophic Responses in Cardiomyocytes

Introduction: A zinc finger protein GATA4 associates with an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46) form a repressor complex with HDACs and are implicated in chromatin remodeling and transcriptional repression. We showed that RbAp48 and 46 are novel component of the p300/GATA4 complex. However, precise functional relationships among p300, GATA4 and RbAp48/46 and their roles in hypertrophic responses remain unknown. Hypothesis: We have assessed the hypothesis that RbAp48/46 regulate hypertrophic responses in cardiomyocytes by modulating functions of the p300/GATA4 complex. Methods and Results: Immunoprecipitation followed by Western blotting in HEK293T cells demonstrated that overexpression of RbAp48/46 inhibited p300-induced GATA4 acetylation and the association between p300 and GATA4. Conversely, knockdown of RbAp48/46 by siRNA reversed these changes. RbAp48/46 inhibited not only p300-induced DNA binding activity of GATA4 onto the atrial natriuretic factor (ANF) and endotheline-1 (ET-1) promoters, but also p300/GATA4-induced ANF and ET-1 promoters activities. In cardiomyocytes, overexpression of RbAp48/46 significantly inhibited phenylephrine (PE)-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and myocardial cell hypertrophy. On the other hand, knockdown of both RbAp48 and 46 by shRNA reversed these changes. PE decreased the binding between GATA4 and RbAp48/46, and the recruitment of RbAp48/46 onto the GATA element of the ANF promoter. Although RbAp48/46 were co-localized with GATA4 in the nucleus of cardiomyocytes at the basal state, PE stimulus induced the export of RbAp48/46 to cytoplasm. LMB, a inhibitor of chromosomal region maintenance 1 (CRM1), inhibited nuclear export of RbAp48/46. Conclusion: These findings demonstrate that RbAp48/46 interact with GATA4, disasociate GATA4 from p300, and inhibit its acetylation as well as hypertrophic responses in cardiomyocytes. The nuclear export of RbAp48/46 by hypertrophic stimuli may trigger hypertrophy-responsive transcription by releasing these inhibitions.

Protein kinase D-HDAC5 signaling regulates erythropoiesis and contributes to erythropoietin cross-talk with GATA1

AbstractIn red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5−/− progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin–independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.