GATA-1 Activity Research Articles

Activation of the Erythroid Transcriptional Program in Murine Adult Bone Marrow Takes Place during a Faster, Shorter S Phase and Is Dependent on S Phase Progression

We previously characterized an S phase-dependent commitment switch in mouse fetal liver. Specifically, the upregulation of CD71 in the transition from Subset 0 (S0, Lin-CD71medium) to Subset 1 (S1, Lin-CD71high) identifies cells in early S phase of the last colony-forming-unit-erythroid (CFUe) generation, undergoing key differentiation events, including onset of Erythropoietin (Epo) dependence, GATA-1 activation and reconfiguration of chromatin at the §-globin locus control region. This commitment switch requires S phase progression, and is regulated by the S-phase- dependent downregulation of the transcription factor PU.1, a GATA-1 antagonist. Of note, the specific cell cycle S phase in which this commitment switch takes place differs from S phase in previous cycles, in that it is shorter, with a 50% faster rate of nucleotide incorporation into DNA.Here, we investigate whether a similar commitment switch takes place in adult bone-marrow. Using fresh bone-marrow from mice in the basal state or following Epo injection, we determined colony-forming potential, transcriptional profiles and cell cycle status of Kit+Lin- subsets defined by cell surface markers that have been previously implicated as enriched in the megakaryocytic-erythrocytic lineages: CD105, CD150, CD55, CD71, in conjunction with a PU.1-GFP reporter mouse.Using quantitative RT-PCR together with flow-cytometric analysis and the SPADE algorithm we identified a linear erythroid developmental sequence of Epo-responsive Kit+Lin-CD55+ bone-marrow subsets, which gradually declined in PU.1, transiently increased CD150, and upregulated both CD105 and CD71. The loss of PU.1 coincided with increased GATA1 levels and the transcriptional activation of erythroid genes. CFUe activity peaked within Kit+Lin-CD55+ cells that were also CD105+, where colonies of other lineage potential were rare (Fig 1).Upregulation of CD71 in erythroid cells indicates the onset of Epo receptor signaling and Epo dependence. We found that in Kit+Lin-CD55+ bone-marrow in vivo, high levels of CD71 were highly correlated with S phase (Fig 2). Furthermore, the rate of incorporation of the nucleotide analogue BrdU was substantially higher in CD71 -high cells, suggesting a rapid, short S phase. To functionally examine the role of S phase, we isolated Kit+Lin-CD55+CD105+ cells that were CD71 low, and followed their differentiation in vitro. These cells upregulate CD71 within 10 to 16 hours, followed by upregulation of Ter119. Upregulation of CD71 was Epo dependent, and took place in S phase; further, it was reversibly inhibited by the DNA polymerase inhibitor, Aphidicolin, suggesting dependence on S phase progression.We conclude that adult bone marrow Kit+Lin-CD55+CD105+ are at the CFUe developmental stage, and undergo an Epo and S-phase dependent commitment switch that activates the erythroid transcriptional and differentiation program. S phase during this commitment switch is characterized by a fast DNA synthesis rate. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GATA1 Changes DNA-Binding Fashion in a Binding-Site-Specific Manner and Alters Transcriptional Activity during Erythropoiesis

GATA1 is a transcription factor that coordinately regulates multiple target genes during the development and differentiation of erythroid and megakaryocytic lineages through binding to GATA motif (A/T)GATA(A/G). GATA1 has four functional domains, i.e., two transactivation domains reside in amino- and carboxyl- terminus, which transactivate GATA1 target genes redundantly and/or cooperatively, and two zinc-finger domains in the middle of the protein. The two zinc finger domains of GATA1 have been characterized extensively and their links to human diseases have also been identified. Carboxyl-terminal side zinc (C)-finger is essential for the DNA binding of GATA1, whereas amino-terminal side zinc (N)-finger retains insufficient binding activity to the GATA motifs by itself, but contributes to stabilize the binding of C-finger to a double GATA site arranged in a palindromic manner. Of note, while this two-finger structure is conserved in six distinct vertebrate GATA factors, there exist GATA factors with single zinc finger in non-vertebrates, indicating that only the C-finger and following basic tail region are evolutionary conserved in both vertebrate and non-vertebrate GATA factors. In our transgenic rescue analyses, GATA1 lacking the N-finger (ΔNF-GATA1) supports, if not completely, the erythropoiesis in mice, but mice without C-finger (ΔCF-GATA1) die in utero showing similar phenotype to the mice with complete loss-of-GATA1-function. Therefore, roles that the N-finger plays have been assumed to be evolutionally acquired features during molecular evolution.In this study, we have examined GATA-motif configuration-specific modulation of GATA1 function by using composite GATA elements in which two GATA motifs aligned side-by-side, either tandem or palindromic. We have defined changes in the GATA1 binding and transactivation activity in accordance with the arrangement of cis -acting GATA motifs. While GATA1 binds to Single-GATA in a monovalent way via C-finger without the influence of N-finger, the N-finger appears to contribute to specific bivalent binding of GATA1 to Pal-GATA, i.e., the N- and C-fingers in a single GATA1 molecule individually bind to two GATA motifs aligned in a palindromic orientation. Showing very good agreement with the human case analyses, the transgenic expression of G1R216Q that lacks N-finger-DNA interaction potential hardly rescues the GATA1-deficient mice due to defects in definitive erythropoiesis, indicating that roles owed by R216 residue are vital for the GATA1 activity in vivo. The N-finger also contributes to GATA1 homodimer formation, which is a prerequisite for two GATA1 binding to two GATA motifs aligned in a tandem orientation. Each GATA1 C-finger in the dimeric GATA1 protein binds to each GATA motif in Tandem-GATA. In this regard, we previously found in a transgenic complementation rescue assay that mutant GATA1 molecule G13KA, which lacks the dimerization potential but possesses most of the other N- and C-finger functions, hardly rescues the GATA1-deficient mice from embryonic lethality, indicating that the GATA1 dimerization is important to attain full GATA1 activity. We surmise based on these observations that the configuration of cis -acting GATA motifs located in the regulatory regions of the GATA1 target genes critically influences the DNA-binding of GATA1 and controls transcription of the genes. DisclosuresNo relevant conflicts of interest to declare.

Rare and Common Germline Variants Contribute to Occurrence of Myelodysplastic Syndrome

Background: Majority of MDS cases appear to be sporadic in nature, but 10-15% have clear familial basis due to predisposing mutations in genes such as RUNX1, GATA2, CEBPA and DDX41. Contribution of germline variants in sporadic MDS is not studied. This study attempts to address the contribution of germline variants in MDS pathogenesis.Methods: We performed amplicon-based massively parallel sequencing (AmpliSeq custom panel adapted for Illumina HiSeq2500 sequencing) on all coding regions of 29 myeloid genes for 144 MDS samples. After identifying the variants in five genes (TET2, MET, GATA2, ASXL1, NOTCH1), we tested an additional 96 MDS samples including therapy-related myeloid neoplasm (T-MN) using a Sequenom assay. We also analyzed WES data for these variants in 178 AML samples and 758 normal controls and AmpliSeq data for ASXL1 and TET2 variants in 655 CML samples.Results: Collation of all coding variants in the 29 myeloid genes sequenced identified germline variants occurring in primary MDS at frequencies significantly higher than expected when compared to the normal population (ExAC and matched cohort were similar) (Table 1). These variants occurred in 5 genes (TET2, MET, GATA2, ASXL1 and NOTCH1) at increased frequencies of 1.5-16.6 fold. Numerous MDS samples had multiple variants (4 with 4 variants, 4 with 3 variants, 18 with 2 variants) while 70 had 1 variant.The 3 germline MET variants have been previously investigated in solid tumorigenesis and likely generate MET variant proteins that contribute to numerous cancer types including MDS. Interestingly, 7/17 (41%) MDS cases with germline MET variants also had other cancers including pancreatic, gastric and laryngeal cancers. Of the TET2 variants, Y867H and P1723S were concurrent in 5 MDS, 5 AML and 6 CML samples indicative of them being on the same allele (i.e. a haplotype). They were seen at higher than normal frequency in MDS and AML, but were not significantly enriched in CML. We are currently confirming their coexistence on the same allele and assaying for decreased TET2 activity to determine whether one or both variants contribute to the phenotype.Other variants identified in MDS include the rare GATA2 (P161A) variant which is present in 1% of the population and the nearby common GATA2 (A164T) allele (~20%). These were mutually exclusive in our cohort and were seen at 3.9 and 1.5-fold, respectively, above the expected population frequency. We generated the P161A variant using site-directed mutagenesis and assayed for GATA2 transactivation activity in HEK293 cells with a GATA2-responsive LYL1 promoter-Luciferase construct (Figure 1). We also included empty vector (EV), wildtype (WT) GATA2 and T354M which is the most common highly penetrant autosomal dominant mutation leading to familial MDS/AML. As expected, T354M displayed a marked decrease in transactivation ability when compared to WT. The P161A variant similarly displayed loss-of-function in this assay, but not to the same magnitude as T354M. This is consistent with the hypothesis that reduced GATA2 function predisposes to myeloid malignancy where decreasing GATA2 activity correlates with increasing risk of developing malignancy. In our study 10/36 (28%) cases harboring these variants were T-MN cases.Apart from MET (E168D) (11.4-fold), the 2 rare variants with highest frequency in MDS versus controls were ASXL1 (N986S) (16.6-fold) and NOTCH1 (R912W) (6.5-fold). ASXL1 is an epigenetic regulator often mutated in hematopoietic malignancy and aberrant NOTCH1 function has been associated with myeloid and lymphoid malignancies.Conclusions: We have identified common and rare germline variants in genes involved in myeloid malignancy that may contribute to MDS pathogenesis. It remains to be seen whether they contribute to initiation, maintenance and/or progression of MDS and other hematopoietic malignancies. This is the first study reporting higher frequency of germline variants in sporadic MDS cases. [Display omitted] [Display omitted] DisclosuresHiwase:Celgene Corporation: Research Funding.

Mechanical stretch-induced vascular hypertrophy occurs through modulation of leptin synthesis-mediated ROS formation and GATA-4 nuclear translocation

Background: Obesity and hypertension are associated with increased leptin production contributing to cardiovascular remodeling. Mechanisms involving mechanical stretch-induced leptin production and the cross talk between signaling pathways leading to vascular remodeling have not been fully elucidated.Methods and Results: Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch on leptin protein expression in vascular smooth muscle cells (VSMCs). Moreover, the involvement of reactive oxygen species (ROS), the RhoA/ROCK pathway, actin cytoskeleton dynamics and the transcriptional factor GATA-4 activation in mechanical stretch-induced vascular remodeling were investigated. Stretching the RPV for 1 or 24 h significantly increased leptin protein level and ROS formation in VSMCs, which was prevented by 1 h pretreatment with the ROCK inhibitor Y-27632 and the actin cytoskeleton depolymerization agent cytochalasin D. Moreover, Western blotting and immunohistochemistry revealed that mechanical stretch or treatment with 3.1 nmol/L leptin for 24 h significantly increased actin polymerization, as reflected by an increase in the F-actin to G-actin ratio. Increases in blood vessels’ wet weight and [3H]-leucine incorporation following a 24 h treatment with conditioned media from cultured stretched RPVs indicated RPV hypertrophy. This effect was prevented by 1 h pretreatment with anti-leptin antibody, indicating leptin’s crucial role in promoting VSMC hypertrophy. As an index of GATA-4 activation, GATA-4 nuclear translocation was assessed by immunohistochemistry method. Pretreating VSMC with leptin for 1 h significantly activated GATA-4 nuclear translocation, which was potently attenuated by the NADPH oxidase inhibitor apocynin, Y-27632, and cytochalasin D.Conclusion: Our results demonstrate that ROS formation, RhoA/ROCK pathway, and GATA-4 activation play a pivotal role in mechanical stretch-induced leptin synthesis leading to VSMC remodeling.

Transcriptional control of cell senescence

The transcription factor GATA4 promotes cell senescence.

Nanocurcumin Prevents Hypoxia Induced Stress in Primary Human Ventricular Cardiomyocytes by Maintaining Mitochondrial Homeostasis.

Hypoxia induced oxidative stress incurs pathophysiological changes in hypertrophied cardiomyocytes by promoting translocation of p53 to mitochondria. Here, we investigate the cardio-protective efficacy of nanocurcumin in protecting primary human ventricular cardiomyocytes (HVCM) from hypoxia induced damages. Hypoxia induced hypertrophy was confirmed by FITC-phenylalanine uptake assay, atrial natriuretic factor (ANF) levels and cell size measurements. Hypoxia induced translocation of p53 was investigated by using mitochondrial membrane permeability transition pore blocker cyclosporin A (blocks entry of p53 to mitochondria) and confirmed by western blot and immunofluorescence. Mitochondrial damage in hypertrophied HVCM cells was evaluated by analysing bio-energetic, anti-oxidant and metabolic function and substrate switching form lipids to glucose. Nanocurcumin prevented translocation of p53 to mitochondria by stabilizing mitochondrial membrane potential and de-stressed hypertrophied HVCM cells by significant restoration in lactate, acetyl-coenzyme A, pyruvate and glucose content along with lactate dehydrogenase (LDH) and 5' adenosine monophosphate-activated protein kinase (AMPKα) activity. Significant restoration in glucose and modulation of GLUT-1 and GLUT-4 levels confirmed that nanocurcumin mediated prevention of substrate switching. Nanocurcumin prevented of mitochondrial stress as confirmed by c-fos/c-jun/p53 signalling. The data indicates decrease in p-300 histone acetyl transferase (HAT) mediated histone acetylation and GATA-4 activation as pharmacological targets of nanocurcumin in preventing hypoxia induced hypertrophy. The study provides an insight into propitious therapeutic effects of nanocurcumin in cardio-protection and usability in clinical applications.

The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4.

Cellular senescence is a terminal stress-activated program controlled by the p53 and p16(INK4a) tumor suppressor proteins. A striking feature of senescence is the senescence-associated secretory phenotype (SASP), a pro-inflammatory response linked to tumor promotion and aging. We have identified the transcription factor GATA4 as a senescence and SASP regulator. GATA4 is stabilized in cells undergoing senescence and is required for the SASP. Normally, GATA4 is degraded by p62-mediated selective autophagy, but this regulation is suppressed during senescence, thereby stabilizing GATA4. GATA4 in turn activates the transcription factor NF-κB to initiate the SASP and facilitate senescence. GATA4 activation depends on the DNA damage response regulators ATM and ATR, but not on p53 or p16(INK4a). GATA4 accumulates in multiple tissues, including the aging brain, and could contribute to aging and its associated inflammation.

A novel mutation of GATA4 (K300T) associated with familial atrial septal defect

The GATA-binding protein 4 gene (GATA4) encodes a zinc-finger transcription factor that plays a key role in embryogenesis and cardiac development. Variants in the GATA4 gene have been implicated in several congenital heart diseases (CHD), such as the tetralogy of Fallot (ToF), atrial septal defect (ASD), ventricular septal defect (VSD), atrioventricular septal defect (AVSD), and dilated cardiomyopathy (DCM). We studied a four-generation Chinese ASD family and identified a novel GATA4 mutation (c.A899C, p.K300T) in all surviving affected members and two carriers with incomplete penetrance. Bioinformatics programs (PolyPhen-2, SIFT, and MutationTaster) predicted the mutation to be deleterious. The lysine at the mutation position was highly conserved from Drosophila to humans and was recognized as a methylation location in the GATA4 protein. The involvement of the lysine methylation in cardiogenesis by attenuating the transcriptional activity of GATA4 in mice has been previously examined. Our study broadens the mutation spectrum of the GATA4 gene and reveals for the first time a mutation at the methylation position of GATA4 associated with ASD.

TIEG1 Inhibits Angiotensin II-induced Cardiomyocyte Hypertrophy by Inhibiting Transcription Factor GATA4.

The transforming growth factor (TGF-β)-inducible early gene (TIEG1), a member of the Sp1/Krüppel-like family of transcription factors, plays an important role in regulating cell growth, differentiation, and apoptosis in many human diseases, including breast cancer, osteoporosis, and atherosclerosis. However, little is known about the role of TIEG1 in the heart. In this study, we investigated the role of TIEG1 in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and its underlying mechanism. Our results showed that TIEG1 expression was downregulated in Ang II-induced hypertrophic cardiomyocytes. Gene silencing of TIEG1 by RNA interference upregulated cellular surface area and ANF and BNP messenger RNA levels, whereas TIEG1 overexpression inhibited the expression of those genes. Mechanistically, TIEG1 could inhibit the expression and transcriptional activity of transcription factor GATA4 in cardiomyocytes, which was recognized as an important factor mediating cardiac gene transcription. In summary, our data disclose a novel role of TIEG1 as an inhibitor in Ang II-induced hypertrophic cardiomyocytes through GATA4 signal pathway.

Wogonoside induces growth inhibition and cell cycle arrest via promoting the expression and binding activity of GATA-1 in chronic myelogenous leukemia cells.

GATA-1, a zinc finger transcription factor, has been demonstrated to play a key role in the progression of leukemia. In this study, we investigate the effects of wogonoside, a naturally bioactive flavonoid derived from Scutellaria baicalensis Georgi, on cell growth and cell cycle in chronic myeloid leukemia (CML) cells, and uncover its underlying mechanisms. The experimental design comprised CML cell lines K562, imatinib-resistant K562 (K562r) cells, and primary CML cells, treated in vitro or in vivo, respectively, with wogonoside; growth and cell cycle were then evaluated. We found that wogonoside could induce growth inhibition and G0/G1 cell cycle arrest in both normal and K562r cells. Wogonoside promotes the expression of GATA-1 and facilitates the binding to methyl ethyl ketone (MEK) and p21 promoter, thus inhibiting MEK/extracellular signal-regulated kinase signaling and cell cycle checkpoint proteins, including CDK2, CDK4, cyclin A, and cyclin D1, and increasing p21 expression. Furthermore, in vivo studies showed that administration of wogonoside decreased CML cells and prolonged survival in NOD/SCID mice with CML cell xenografts. In conclusion, these results clearly revealed the inhibitory effect of wogonoside on the growth in CML cells and suggested that wogonoside may act as a promising drug for the treatment of imatinib-resistant CML.

Na+/H+ Exchanger Isoform 1 Induced Cardiomyocyte Hypertrophy Involves Activation of p90 Ribosomal S6 Kinase

Studies using pharmacological and genetic approaches have shown that increased activity/expression of the Na+/H+ exchanger isoform 1 (NHE1) play a critical role in the pathogenesis of cardiac hypertrophy. Despite the importance of NHE1 in cardiac hypertrophy, severe cerebrovascular side effects were associated with the use of NHE1 inhibitors when administered to patients with myocardial infarctions. p90 ribosomal S6 Kinase (RSK), a downstream regulator of the mitogen-activated protein kinase pathway, has also been implicated in cardiac hypertrophy. We hypothesized that RSK plays a role in the NHE1 induced cardiomyocyte hypertrophic response. Infection of H9c2 cardiomyoblasts with the active form of the NHE1 adenovirus induced hypertrophy and was associated with an increase in the phosphorylation of RSK (P<0.05). Parameters of hypertrophy such as cell area, protein content and atrial natriuretic mRNA expression were significantly reduced in H9c2 cardiomyoblasts infected with active NHE1 in the presence of dominant negative RSK (DN-RSK) (P<0.05). These results confirm that NHE1 lies upstream of RSK. Increased phosphorylation and activation of GATA4 at Ser261 was correlated with increased RSK phosphorylation. This increase was reversed upon inhibition of RSK or NHE1. These findings demonstrate for the first time that the NHE1 mediated hypertrophy is accounted for by increased activation and phosphorylation of RSK, which subsequently increased the phosphorylation of GATA4; eventually activating fetal gene transcriptional machinery.

Na(+)/H (+) exchanger isoform 1 induced osteopontin expression in cardiomyocytes involves NFAT3/Gata4.

Osteopontin (OPN), a multifunctional glycophosphoprotein, has been reported to contribute to the development and progression of cardiac remodeling and hypertrophy. Cardiac-specific OPN knockout mice were protected against hypertrophy and fibrosis mediated by Ang II. Recently, transgenic mice expressing the active form of the Na(+)/H(+) exchanger isoform 1 (NHE1) developed spontaneous hypertrophy in association with elevated levels of OPN. The mechanism by which active NHE1 induces OPN expression and contributes to the hypertrophic response remains unclear. To validate whether expression of the active form of NHE1 induces OPN, cardiomyocytes were stimulated with Ang II, a known inducer of both OPN and NHE1. Ang II induced hypertrophy and increased OPN protein expression (151.6 ± 28.19 %, P < 0.01) and NHE1 activity in H9c2 cardiomyoblasts. Ang II-induced hypertrophy and OPN protein expression were regressed in the presence of an NHE1 inhibitor, EMD 87580, or a calcineurin inhibitor, FK506. In addition, our results indicated that activation of NHE1-induced NFAT3 translocation into the nucleus and a significant activation of the transcription factor Gata4 (NHE1: 149 ± 28 % of control, P < 0.05). NHE1-induced activation of Gata4 was inhibited by FK506. In summary, our results suggest that activation of NHE1 induces hypertrophy through the activation of NFAT3/Gata4 and OPN expression.

CD41 marks the initial myelo-erythroid lineage specification in adult mouse hematopoiesis: redefinition of murine common myeloid progenitor.

Previous studies have predicted that reciprocal activation of GATA-1 and PU.1 regulates myelo-erythroid versus myelo-lymphoid lineage commitment in early hematopoiesis. Such PU.1-activating myelo-lymphoid progenitors exist within the lymphoid-primed multipotent progenitor (LMPP) population at the primitive Lineage(-) Sca-1(+) c-Kit(+) (LSK) stage. We here show that the counterpart of GATA-1-activating myelo-erythroid progenitor resides also at the LSK stage, expressing CD41 at a high level. Purified CD41(hi) LSK cells showed exceedingly strong and prolonged myelo-erythroid-restricted reconstitution, and primed myelo-erythroid gene expression with a more primitive molecular signature as compared to the original common myeloid progenitor (CMP). The CD41(hi) LSK cells more strongly contributed to emergent and malignant myelopoiesis than LMPPs, and produced the original CMP by downregulating Sca-1 and CD41, suggesting that they are the earliest CMPs. Thus, the hematopoietic developmental map should be revised by integrating the primary branchpoint comprised of the new, isolatable CD41(hi) CMP and the LMPP populations.

The regulation of troponins I, C and ANP by GATA4 and Nkx2-5 in heart of hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus.

Hibernation is an adaptive strategy used by various mammals to survive the winter under situations of low ambient temperatures and limited or no food availability. The heart of hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) has the remarkable ability to descend to low, near 0°C temperatures without falling into cardiac arrest. We hypothesized that the transcription factors GATA4 and Nkx2-5 may play a role in cardioprotection by facilitating the expression of key downstream targets such as troponin I, troponin C, and ANP (atrial natriuretic peptide). This study measured relative changes in transcript levels, protein levels, protein post-translational modifications, and transcription factor binding over six stages: euthermic control (EC), entrance into torpor (EN), early torpor (ET), late torpor (LT), early arousal (EA), and interbout arousal (IA). We found differential regulation of GATA4 whereby transcript/protein expression, post-translational modification (phosphorylation of serine 261), and DNA binding were enhanced during the transitory phases (entrance and arousal) of hibernation. Activation of GATA4 was paired with increases in cardiac troponin I, troponin C and ANP protein levels during entrance, while increases in p-GATA4 DNA binding during early arousal was paired with decreases in troponin I and no changes in troponin C and ANP protein levels. Unlike its binding partner, the relative mRNA/protein expression and DNA binding of Nkx2-5 did not change during hibernation. This suggests that either Nkx2-5 does not play a substantial role or other regulatory mechanisms not presently studied (e.g. posttranslational modifications) are important during hibernation. The data suggest a significant role for GATA4-mediated gene transcription in the differential regulation of genes which aid cardiac-specific challenges associated with torpor-arousal.

Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2

Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG.To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG.To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation.To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop.The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. DisclosuresShearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

Csnk2β Knockout during Hematopoiesis Results in Lethality at Mid/Late Gestation Mostly Due to Impaired Fetal Erythropoiesis

Background. CK2, a serine-threonine kinase composed of two catalytic (α) and two regulatory (β) subunits, has been clearly involved in several hematologic malignancies. This kinase regulates the PTEN/PI3K/AKT, Wnt/βcatenin, Hedgehog, JAK-STAT, cMyc and NF-κB signalling cascades, all of which are known to be of critical importance for hematopoietic stem cell (HSC) biology and normal hematopoiesis. However, the role played by CK2 during blood cell development has remained as yet unexplored.Aims and methods. CK2 function in hematopoiesis was investigated generating conditional knockout mice for CK2β by crossing Csnk2β-Flox/Flox mice with Vav1-CRE transgenic mice. Inactivation of Csnk2β started from 9.5 dpc during embryonic development. Histo-cytological methods, FACS analysis, colony-forming assays (CFA), signal transduction studies by western blotting and RT-PCR were employed to characterize the cellular and molecular phenotype. High throughput RNAseq analysis was also performed on purified Ter119-positive erythroid cells from Csnk2β knockout and Csnk2β control mice to identify differentially expressed CK2-dependent transcriptional targets.Results. Csnk2β knockout in hematopoiesis resulted lethal at mid-late gestation. Rarely some pups were found dead at birth. Macroscopic and phenotypic analysis during gestation revealed the appearance of pale and hydropic fetuses after 12.5 dpc. The majority of pups showed teleangiectasic vessels and haemorrhages. Fetal livers appeared smaller and paler. Cytological analysis and CFA studies unveiled a great depletion of hematopoietic elements belonging to both the erythroid, megakaryocytic and granulocytic-monocytic precursors. A more thorough analysis of the erythroid phenotype revealed that Csnk2β loss caused impairment/loss of red cell maturation at two developmental stages: the earlier stages of Megakaryocyte-Erythroid Precursors (MEP) and pro-erythroblasts and the later stages of terminal maturation (orthocromatic erythroblasts towards reticulocytes). Expression analysis of proteins/genes belonging to known hematopoietic and erythroid-regulating pathways showed perturbations in cell cycle regulatory molecules, cellular apoptosis, a marked reduction of total and phosphorylated Akt in Ser473 and Ser129, a decrease of GATA1 protein levels and a decrease of Hedgehog/Wnt target genes such as Gli-1 and Cyclin D1. Erythropoietin-dependent AKT activation and GATA1 phosphorylation was impaired by Csnk2β loss. Moreover, starting at 14.5 dpc, blood cells displayed a massive p53-dependent response, marked by high levels of p21 and a progressive clear apopototic phenotype. At 17.5 dpc residual hematopoietic cells in the fetal liver were represented by dying erythroid cells, immature myelo-monocytic precursors (expressing high CD11b and low Gr1 levels on the surface) and B-cells displaying an aberrant phenotype with low intensity of expression of B220 and CD19 on the surface. High throughput RNAseq analysis of Ter119-expressing fetal liver cells (erythroid lineage) obtained from 14.5 dpc pups revealed the upregulation of 145 transcripts and the downregulation of 68 transcripts. Among the most increased transcripts were the transcription factors Jun/AP1 and stress-related intermediaries and embryonal globin ε and ζ chains. Among the most decreased transcripts were sugar transporters, glycoproteins CD36 and CD59a, Duffy Blood Group Atypical Chemokine Receptor and component members.Conclusions. We found that Csnk2β plays a critical role in mouse blood development by regulating definitive hematopoiesis of all the hematopoietic cell lineages; however, Csnk2β was needed for the early and late erythropoiesis whilst its loss could be compatible with a certain extent of immature/altered myelo-monocytic and B cell development. Among the pathways found targeted by Csnk2β loss were the PI3K/Akt and the p53-p21 cascades. Our data also suggest that Csnk2β might have a role in the proper activation of the erythroid master regulator GATA1. Moreover, RNAseq analysis revealed that this kinase might have a broader impact during erythroid cell maturation by regulating the activity of critical stress related transcription factors, of molecules regulating energy-managing cellular processes and of mechanisms controlling the switch from embryonal to fetal erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Molecular Regulation of Adult Hematopoiesis By GATA-2

GATA-2 is a zinc finger-containing transcriptional regulator that plays important roles in embryonic and adult hematopoiesis. Mutations in human GATA2 are associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as immunodeficiency disorders that present with a profound loss of monocytes, dendritic cells and other myeloid lineage populations. Recent work reveals crucial roles for GATA-2 in definitive hematopoietic stem/progenitor cell activity, vascular integrity and lymphatic development. However, the molecular mechanisms by which GATA-2 controls adult hematopoiesis via hematopoietic-cell autonomous functions are largely unknown. To address this question, we generated a tamoxifen-inducible Gata2-deficient mouse strain by breeding Gata2flox/flox mice with Cre-ER transgenic animals. Following tamoxifen treatment, Cre-ER Gata2flox/flox mice show a rapid and profound loss of circulating neutrophils, monocytes and lymphocytes, concomitant with development of anemia. These results are consistent with the requirement for GATA-2 in hematopoietic stem/progenitor cells, and may also reflect GATA-2 function in endothelial cells within the vascular niche. To explore hematopoietic-specific GATA-2 activity, we generated bone marrow chimeric mice with hematopoietic-restricted Gata2-deficiency by transplanting Cre-ER Gata2flox/flox hematopoietic cells into wild type recipients. Cre-ER Gata2flox/flox bone marrow chimeras show rapid development of cytopenias upon tamoxifen exposure, suggesting a cell autonomous role for GATA-2 in maintaining adult hematopoiesis. Strikingly, hematopoietic progenitor cells rapidly lose c-Kit expression upon inducible Gata2 deletion. Chromatin immunoprecipitation and reporter assays suggest GATA-2 cooperates with C/EBPa in regulating kit transcription. Our study suggests conditional deletion of Gata2 restricted to the hematopoietic compartment provides a model for bone marrow failure associated with MDS and mutant GATA2 human immunodeficiencies that may enable further insight into the molecular network by which GATA-2 mediates definitive hematopoiesis.Supported by grants from NIH (AI098099) and the MD Anderson Center for Cancer Epigenetics.(SSW) and the MD Anderson Center for Cancer and Inflammation (HSL). DisclosuresNo relevant conflicts of interest to declare.

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.

Genetic dissection of Gata2 selective functions during specification of V2 interneurons in the developing spinal cord.

Motor activities are controlled by neural networks in the ventral spinal cord and consist in motor neurons and a set of distinct cardinal classes of spinal interneurons. These interneurons arise from distinct progenitor domains (p0-p3) delineated according to a transcriptional code. Neural progenitors of each domain express a unique combination of transcription factors (TFs) that largely contribute to determine the fate of four classes of interneurons (V0-V3) and motor neurons. In p2 domain, at least four subtypes of interneurons namely V2a, V2b, V2c, and Pax6(+) V2 are generated. Although genetic and molecular mechanisms that specify V2a and V2b are dependent on complex interplay between several TFs including Nkx6.1, Irx3, Gata2, Foxn4, and Ascl1, and signaling pathways such as Notch and TGF-β, the sequence order of the activation of these regulators and their respective contribution are not completely elucidated yet. Here, we provide evidence by loss- or gain-of-function experiments that Gata2 is necessary for the normal development of both V2a and V2b neurons. We demonstrate that Nkx6.1 and Dll4 positively regulate the activation of Gata2 and Foxn4 in p2 progenitors. Gata2 also participates in the maintenance of p2 domain by repressing motor neuron differentiation and exerting a feedback control on patterning genes. Finally, Gata2 promotes the selective activation of V2b program at the expense of V2a fate. Thus our results provide new insights on the hierarchy and complex interactions between regulators of V2 genetic program.

Carboxy terminus of GATA4 transcription factor is required for its cardiogenic activity and interaction with CDK4

GATA4-6 transcription factors regulate numerous aspects of development and homeostasis in multiple tissues of mesodermal and endodermal origin. In the heart, the best studied of these factors, GATA4, has multiple distinct roles in cardiac specification, differentiation, morphogenesis, hypertrophy and survival. To improve understanding of how GATA4 achieves its numerous roles in the heart, here we have focused on the carboxy-terminal domain and the residues required for interaction with cofactors FOG2 and Tbx5. We present evidence that the carboxy terminal region composed of amino acids 362–400 is essential for mediating cardiogenesis in Xenopus pluripotent explants and embryos. In contrast, the same region is not required for endoderm-inducing activity of GATA4. Further evidence is presented that the carboxy terminal cardiogenic region of GATA4 does not operate as a generic transcriptional activator. Potential mechanism of action of the carboxy terminal end of GATA4 is provided by the results showing physical and functional interaction with CDK4, including the enhancement of cardiogenic activity of GATA4 by CDK4. These results establish CDK4 as a GATA4 partner in cardiogenesis. The interactions of GATA4 with its other well described cofactors Tbx5 and FOG2 are known to be involved in heart morphogenesis, but their requirement for cardiac differentiation is unknown. We report that the mutations that disrupt interactions of GATA4 with Tbx5 and FOG2, G295S and V217G, respectively, do not impair cardiogenic activity of GATA4. These findings add support to the view that distinct roles of GATA4 in the heart are mediated by different determinants of the protein. Finally, we show that the rat GATA4 likely induces cardiogenesis cell autonomously or directly as it does not require activity of endodermal transcription factor Sox17, a GATA4 target gene that induces cardiogenesis non-cell autonomously.