Mechanistic Deficits Of a Leukemogenic GATA-2 Mutant

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3668-3668
Author(s):  
Koichi Ricardo Katsumura ◽  
Chenxi Yang ◽  
Jing Zhang ◽  
Lingjun Li ◽  
Kirby D Johnson ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Phosphorylation Site ◽  
Spectrometric Analysis ◽  
Ras Signaling ◽  
Dependent Manner ◽  
Subnuclear Localization ◽  
Transactivation Activity ◽  
N Terminus ◽  
Fold Activation

Abstract Recent studies have demonstrated a role for the master regulator of hematopoiesis GATA-2 in MonoMAC Syndrome, a human immunodeficiency disorder associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Though GATA2 coding region and cis-regulatory element mutations underlie MonoMAC syndrome, many questions remain unanswered regarding how GATA-2 is controlled physiologically and how it is dysregulated in pathological contexts. We dissected how a T354M mutation in the GATA-2 DNA binding zinc finger, which is frequently detected in MonoMAC syndrome and familial MDS/AML, alters GATA-2 activity. The T354M mutation reduced GATA-2 chromatin occupancy, induced GATA-2 hyperphosphorylation, and disrupted GATA-2 subnuclear localization. These molecular phenotypes also characterized an additional familial MDS/AML-associated GATA-2 mutant (Δ355T). T354M hyperphosphorylation and ectopic subnuclear localization were detected in hematopoietic and non-hematopoietic cell lines. We developed a new model system in mouse aortic endothelial (MAE) cells to quantitate GATA-2 activity to regulate endogenous target genes. T354M exhibited significantly reduced activity in this assay (GATA-2: 200-fold activation; T354M: 7.7-fold activation). Mass spectrometric analysis of the phosphorylation states of GATA-2 and T354M revealed that the T354M mutation enhanced phosphorylation at several GATA-2 residues. Analysis of single phosphorylation site mutants indicated that only mutation of S192 (S192A) abolished T354M-induced hyperphosphorylation. The S192A mutation attenuated phosphorylation of sites within wild-type GATA-2 and reduced transactivation activity (50% decrease, p < 0.01). A distinct 60 amino acid (aa) region within the GATA-2 N-terminus was required for T354M hyperphosphorylation and ectopic subnuclear localization. Deletion of this sequence decreased GATA-2 transactivation activity (60 aa deletion: 85% decrease, p < 0.01; 10 aa deletion: 45% decrease, p < 0.05). GATA-1 lacks an analogous subnuclear targeting sequence, and accordingly, a GATA-1(T263M) mutant, which corresponds to the GATA-2(T354M) mutant, localized normally and was not hyperphosphorylated. However, a GATA-1 chimera containing the GATA-2 subnuclear targeting sequence localized to ectopic subnuclear foci in a T263M-dependent manner. The GATA-2 N-terminus endowed GATA-1 with the capacity to induce GATA-2 target genes. By contrast, a GATA-2 chimera containing the GATA-1 N-terminus exhibited normal subnuclear localization. Thus, the leukemogenic T354M mutation utilizes the GATA-2-specific subnuclear targeting sequence to disrupt the normal subnuclear localization pattern, and this disruption is associated with S192-dependent hyperphosphorylation. In addition to its involvement in AML, GATA-2 interfaces with RAS signaling to promote the development of non-small cell lung cancer. We discovered that RAS signaling promotes S192-dependent GATA-2 hyperphosphorylation and ectopic subnuclear localization and propose that GATA-2 is an important component in oncogenic RAS-dependent leukemogenesis, which is being formally tested using innovative mouse models. In summary, dissecting the mechanistic deficits of a leukemogenic GATA-2 mutant revealed unexpected insights into mechanisms underlying physiological GATA-2 function and GATA-2-dependent hematologic pathologies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aurora-A Abrogation of p53 DNA Binding and Transactivation Activity by Phosphorylation of Serine 215

2004 ◽  
Vol 279 (50) ◽  
pp. 52175-52182 ◽  
Author(s):  
Qiyuan Liu ◽  
Satoshi Kaneko ◽  
Lin Yang ◽  
Richard I. Feldman ◽  
Santo V. Nicosia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Phosphorylation Site ◽  
Dependent Manner ◽  
Aurora A ◽  
Γ Irradiation ◽  
Mitotic Kinase ◽  
Transactivation Activity

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003)Curr. Opin. Cell Biol.15, 164–171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such asp21Cip/WAF1andPTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e.phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and γ-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004)Nat. Genet.36, 55–62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function.

Controlling Hematopoiesis through Sumoylation-Dependent Regulation of a GATA Factor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1467-1467
Author(s):  
Hsiang-Ying Lee ◽  
Kirby D. Johnson ◽  
Tohru Fujiwara ◽  
Meghan E. Boyer ◽  
Shin-Il Kim ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Target Gene ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Nuclear Periphery ◽  
Transcriptional Networks ◽  
Gata Factor ◽  
Megakaryoblastic Leukemia ◽  
N Terminus

Abstract Abstract 1467 Poster Board I-490 GATA factors function via distinct modes to establish transcriptional networks that control fundamental developmental processes including hematopoiesis. Whereas the master regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 activity at endogenous loci is poorly understood. GATA-1 is sumoylated at K137, which resides in the N-terminus, but how the N-terminus contributes to GATA-1 function remains unclear. Expression of a GATA-1 mutant lacking amino acids 1-83 of the N-terminus is linked to the development of acute megakaryoblastic leukemia [Wechsler et al. (2002) Nat. Genet. 32, 148], and deletion of the N-terminus preferentially deregulates a subset of target genes [Johnson et al. (2006) PNAS. 103, 15939]. We demonstrate that sumoylation at K137 promotes transcriptional activation only at a subset of its target genes – those requiring the cell type-specific coregulator, Friend of GATA-1 (FOG-1). Interestingly, a GATA-1 mutation that disrupts FOG-1 binding (V205G) and K137 mutations yielded similar phenotypes, although FOG-1 was not required for K137 sumoylation. Both V205 and K137 mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent target gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes localized at the periphery independent of maturation. These results illustrate how sumoylation of a critical developmental regulator selectively controls its function at specific loci, and members of a target gene ensemble with distinct coregulator and posttranslational modification requirements reside in different subnuclear compartments. Disclosures: No relevant conflicts of interest to declare.

Bcr-Abl Represses IRF8 Gene Transcription in a Stat5-Dependent Manner

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3497-3497
Author(s):  
Elizabeth Hjort ◽  
Weiqi Huang ◽  
Elizabeth A. Eklund
Keyword(s):  
Gene Transcription ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Myeloproliferative Neoplasm ◽  
Proximal Promoter ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Interleukin Receptors

Abstract Abstract 3497 The interferon consensus sequence binding protein is a member of the interferon regulatory factor family of transcription factors (referred to as Icsbp or Irf8). The first described functions for Icsbp involved regulation of phagocyte and B-cell effector genes, including genes encoding components of the phagocyte NADPH-oxidase, Toll-like receptors and interleukin receptors. However, subsequent studies in murine models and human disease indicated that Icsbp also functions as a myeloid leukemia suppressor. For example, decreased Icsbp expression is found in chronic myeloid leukemia (CML) in association with uncontrolled disease, drug resistance and progression to blast crisis (BC). Decreased Icsbp expression is also found in the bone marrow of subjects with some subtypes of acute myeloid leukemia (AML). Consistent with this clinical correlative data, IRF8−/− mice exhibit a myeloproliferative neoplasm that is similar to CML and progresses to BC over time. However, the mechanism for decreased Icsbp expression in leukemia is not known, although preliminary studies indicate that DNA-methylation of the IRF8 locus is not altered. Therefore, in these studies, we investigate the effects of Bcr-abl on IRF8 transcription. This is clinically relevant, because previous studies in our laboratory identified a set of Icsbp-target-genes that contribute to the pathogenesis of CML. We find that Bcr-abl decreases expression of Icsbp mRNA and protein in a kinase dependent manner. Since it is unlikely that Bcr-abl directly binds to the promoter to regulate gene transcription, we hypothesized that Bcr-abl regulates IRF8 through an intermediary transcription factor. In this study, we determine that Stat5 negatively regulates IRF8 transcription through a proximal promoter cis-element. We also find that Stat5 repression activity is necessary for Bcr-abl dependent regulation of IRF8. Bcr-abl is known to phosphorylate and activate Stat5 in CML. In our studies, we find that Stat5 protein (but not mRNA) is also increased in Bcr-abl+ cells. Stat5 is a known substrate for calpain; a serine protease. We previously demonstrated that Icsbp regulates calpain protease activity through repression of the gene encoding Gas2; an endogenous calpain inhibitor. Consistent with this, our current studies demonstrate that Stat5 protein stability is increased in Bcr-abl+ cells in an Icsbp/Gas2/calpain-dependent manner. These results identify novel mechanisms by which Bcr-abl-kinase activity controls a positive feedback loop that leads to decreased Icsbp expression and stabilization of Stat5 protein. These studies suggest that targeting Gas2/calpain might be a novel therapeutic approach to CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IGF2BP3 As an N6-Methyladenosine Reader Promotes Acute Myeloid Leukemia Progression By Regulating the Stability of Target RNA

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4322-4322
Author(s):  
Nan Zhang ◽  
Jianchuan Deng ◽  
Fuling Zhou
Keyword(s):  
Acute Myeloid Leukemia ◽  
Genetic Risk ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Clinical Samples ◽  
Dependent Manner ◽  
High Genetic Risk ◽  
Acute Myeloid

Abstract Background: N6-methyladenosine (m6A) is the most common post-transcriptional modification of eukaryotic mRNA. Recent evidence suggests that dysregulated m6A-associated proteins and m6A modifications play a pivotal role in the initiation and progression of diseases such as cancer. Here, we identified that IGF2BP3 is specifically overexpressed in acute myeloid leukemia (AML), which constitutes a subtype of this malignancy associated with poor prognosis and high genetic risk. Methods: Bioinformatics analysis of public databases was performed to screen the differentially expressed m6A regulators in AML. Clinical samples were collected to detect the expression of IGF2BP3 in AML by RT-qPCR. The effects of IGF2BP3 on the proliferation, apoptosis and cycle of AML cells were detected by CCK-8 and flow cytometry. RNA-seq was used to identify target genes of IGF2BP3 by integrating analysis with RIP-Seq, iCLIP-Seq and MeRIP-Seq data sets. Results:High expression of IGF2BP3 is closely associated with poor prognosis of AML and is higher in patients with high genetic risk group. IGF2BP3 was the lowest expressed in AML-M3 and the highest expressed in RUNX1 mutant type. IGF2BP3 is required for maintaining AML cell survival in an m6A-dependent manner, and knockdown of IGF2BP3 suppressed dramatically induces apoptosis, reduces proliferation and impaired leukemic capacity AML cells in vitro and in vivo. Mechanistically, IGF2BP3 interacts with RCC2 mRNA and stabilizes the expression of m6A-tagged RNA. Conclusions:We provided compelling evidence to demonstrate that m6A reader IGF2BP3 contributed to tumorigenesis and poor prognosis of AML, which can serve as a target to develop therapeutics for cancer treatment. Disclosures No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

The Flt3 ITD Accelerates An Already Established Myeloid Leukemia and Alters Chemotherapy Response In Vitro and In Vivo in a p53 Dependent Manner.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1719-1719
Author(s):  
Timothy Pardee ◽  
Johannes Zuber ◽  
Scott Lowe
Keyword(s):  
Myeloid Leukemia ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Disease Onset ◽  
High Dose ◽  
Chemotherapy Response ◽  
Dependent Manner ◽  
Response To Chemotherapy

Abstract Abstract 1719 Poster Board I-745 Acute myeloid leukemia (AML) is an aggressive disease with heterogeneous genetics and variable prognosis. The presence of an internal tandem duplication within the FLT3 gene (Flt3 ITD) is a marker for poor prognosis and has been linked to anthracycline resistance in cell lines and primary patient samples in vitro. The effect of this mutation on response to chemotherapy in vivo has not been examined and its effect on response to cytarabine is not known. In this study we use a genetically defined mouse model of AML to examine the effects of the Flt3 ITD on response to cytarabine and the anthracycline doxorubicin in vitro and in vivo. In vitro the Flt3 ITD conferred resistance to doxorubicin and the combination of doxorubicin and cytarabine but sensitivity to cytarabine alone in comparison to the identical leukemia without the Flt3 ITD. In vivo the presence of the Flt3 ITD provided an advantage in leukemic engraftment and accelerated disease onset. This advantage could be partially reversed by treatment of the animals with cytarabine but not by treatment with doxorubicin. Surprisingly, in vivo the Flt3 ITD conferred a marked increase in sensitivity to cytarabine when compared to the parental leukemia without this mutation. In contrast to the parental leukemia, the addition of doxorubicin to cytarabine provided no advantage over cytarabine alone. When the DNA damage response was assessed the presence of the Flt3 ITD resulted in an increase in the levels of p53 following treatment with either doxorubicin or cytarabine. Induction of the p53 target genes p21 and MDM2 was also increased. Surprisingly, the Flt3 ITD had no effect on disease onset or chemotherapy response in vitro or in vivo in the setting of p53 null AML. These data when taken together demonstrate that the Flt3 ITD confers a mixed sensitivity and resistance to standard chemotherapy and provides an engraftment advantage in a manner that depends on an intact p53 allele. This may at least in part explain the rarity of dual p53 null and Flt3 ITD positive AML. Furthermore, these data suggest that patients with Fl3 ITD positive AML may benefit more from treatment with high dose Ara-C then with combinations containing an anthracycline. Disclosures No relevant conflicts of interest to declare.

Two Distinct Transactivation Domains of GATA1 Contribute Megakaryopoiesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3384-3384
Author(s):  
Hiroshi Kaneko ◽  
Eri Kobayashi ◽  
Ritsuko Shimizu ◽  
Masayuki Yamamoto
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Salient Feature ◽  
Luciferase Reporter ◽  
Transactivation Domain ◽  
Transactivation Activity ◽  
Transactivation Domains ◽  
C Terminus ◽  
N Terminus

Abstract Abstract 3384 Transcription factor GATA1 orchestrates erythroid/megakaryocytic cell differentiation by coordinately regulating the expression of multiple genes encoding proteins responsible for the proliferation, differentiation and survival of progenitors. Mice lacking GATA1 expression die in utero due to insufficient primitive erythropoiesis. GATA1 retains three functional domains; N-terminus (NT) transactivation domain (TAD) and N-terminus and C-terminus zinc finger domains that are both critical for DNA-binding and interaction with co-factors. We previously showed that each domain of GATA1 confers distinguishable properties to GATA1 during primitive and definitive hematopoiesis in vivo. Especially, GATA1 mutant lacking NT domain (GATA1s) is sufficient to rescue the GATA1-deficient mice from embryonic lethality, but the rescued mice suffered from anemia and thrombocytopenia. Showing very good agreement with this finding, inherited mutation yielding GATA1s was found in a family with dyserythropoiesis and dysmegakaryopoiesis. Whereas previous biochemical studies argued that NT domain was a unique TAD in GATA1, these findings suggest that alternative TAD domain is vital for the GATA1 activity in vivo. In this study, we focus on the C-terminus (CT) region of GATA1. The transactivation activity of GATA1 lacking the CT region (G1-DCT) was significantly reduced to approximately 30% of the wild-type GATA1, which was similar to the GATA1s or NT deletion level. Practically no transactivation activity was found in the GATA1 lacking both NT and CT domains. We also examined transactivation potential of the NT and CT fused with GAL4-DBD. CT-GAL4-DBD fusion protein showed Luciferase reporter activity over 20-fold higher than the GAL4-DBD. Furthermore, retrovirally transduced G1-DCT failed to control the hyperproliferation of megakaryocyte progenitors lacking endogenous GATA1, similar to the salient feature reported for GATA1s. These results thus indicate that the coordinated function of CT- and NT-TADs of GATA1 is required in the megakaryopoiesis. GATA1s mutation was found in majority cases of Down syndrome patients with transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Since CT domain is rich in Ser, Thr and Pro residues, while the NT domain is an acidic transactivation domain rich in Glu, we surmise that GATA1 regulates target gene expression by utilizing two distinct transactivation domains. The imbalance in gene expression in megakaryocytic progenitors, which is caused by GATA1s loosing the function of NT-TAD, may be the key aspect for the understanding of the onset of TMD and AMKL. Disclosures: No relevant conflicts of interest to declare.

Pleiotrophin Improves Survival Following Radiation-Induced Myelosuppression and Mediates HSC Expansion Via Induction Of Ras Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1205-1205
Author(s):  
Heather A Himburg ◽  
Phuong L. Doan ◽  
Mamle Quarmyne ◽  
Mai Nakamura ◽  
Nelson J. Chao ◽  
...  
Keyword(s):  
Total Body Irradiation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Erk Signaling ◽  
Ras Signaling ◽  
Heparin Binding ◽  
Total Body ◽  
Deficient Mice

Abstract Discovery of the mechanisms through which the bone marrow microenvironment stimulates hematopoietic regeneration following myelosuppression could lead to therapies to accelerate hematopoietic reconstitution in patients receiving chemotherapy, total body irradiation and stem cell transplantation. We have previously shown that treatment with pleiotrophin (PTN), a heparin-binding growth factor which is secreted by BM endothelial cells (ECs), causes a 10-fold expansion of murine long term-HSCs in culture (Himburg et al. Nat Med 2010). More recently, we demonstrated that PTN-deficient mice have a >10-fold deficit in LT-HSCs and hematopoietic regenerative capacity compared to PTN+/+ mice, suggesting an important role for PTN in maintaining the HSC pool in vivo (Himburg et al. Cell Reports 2012). In keeping with this, 100% of PTN-deficient mice died prior to day +30 following 700 cGy total body irradiation (TBI) compared to 30% mortality in irradiated, PTN+/+ mice (P<0.0001). In order to determine the therapeutic potential of PTN as a systemically deliverable agent to accelerate hematopoietic recovery following myelosuppression, we irradiated C57Bl6 mice with 700 cGy TBI followed by every other day dosing with PTN or saline through day +14. At day +30, 80% of the PTN-treated mice were alive compared to 30% survival in the irradiated, saline-treated group (P=0.03). Interestingly, systemic administration of PTN was equally effective at promoting the survival of irradiated mice when administered as late as 96 hrs post-TBI, suggesting that PTN promotes HSC regeneration after injury. Mechanistically, PTN treatment significantly decreased HSC apoptosis following ionizing radiation exposure (P=0.007), which may explain, at least in part, the mitigative effects of PTN treatment on survival from radiation injury. Furthermore, we have discovered that PTN treatment strongly induces elements of the Ras/MEK/ERK signaling cascade in HSCs, including phosphorylation of Grb2 and ERK1/2, while also increasing levels of the ERK1/2 target genes, phospho-Erf1 and Fra-1. Consistent with this finding, PTN-deficient mice have significantly decreased levels of phospho-ERK1/2 in BM HSCs, suggesting that deficient Ras/MEK/ERK signaling may explain, at least in part, the HSC deficit observed in PTN-deficient mice. Importantly, pharmacologic inhibition of Ras or MEK1/2 proteins or genetic inhibition of KRas in BM KSL cells significantly abrogated PTN-mediated expansion of BM KSL cells and CFU-GEMMs in culture (P<0.01 for total KSL cells and CFU-GEMMs). Taken together, these results suggest that PTN-mediated expansion of HSCs may be dependent upon activation of the Ras/MEK/ERK pathway and provide the basis for further studies to delineate the role of this pathway in mediating PTN effects on the HSC pool in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

Transcription Factor Bach1 and Bach2 Operate Erythro-Myeloid Competitive Differentiation By Responding to Environmental Changes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2649-2649
Author(s):  
Hiroki Kato ◽  
Ari Itoh-Nakadai ◽  
Mitsuyo Matsumoto ◽  
Risa Ebina-Shibuya ◽  
Yuki Sato ◽  
...  
Keyword(s):  
Target Genes ◽  
Environmental Changes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Myeloid Differentiation ◽  
Dependent Manner ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Expression Levels

Abstract Hematopoietic system is maintained by the differentiation and proliferation of hematopoietic stem/progenitor cells (HSPCs) and their commitment to the mature blood cells should be tightly controlled by gene regulatory networks (GRNs) governed by transcription factors (TFs). To keep the homeostasis, GRNs should respond to the environmental changes, such as infection. However, the precise mechanism of such a system remains to be elucidated. TFs Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements containing AP-1 site (Oyake et al., 1996). We have previously shown that Bach1-/-Bach2-/-(DKO; double knockout) mice show erythropoiesis disorders with increased myelopoiesis from common myeloid progenitors (CMPs), which is an erythro-myeloid bifurcation point (ASH2015; Abstract ID# 81562) (Akashi et al., 2000). Since this phenotype is similar to that of LPS treated mice (O'Connell et al., 2008), we hypothesized that Bach factors work as sensors for infection. First, to evaluate the cell-intrinsic function of Bach factors, WT or DKO bone marrow cells were depleted of mature differentiated cells and transplanted to lethally irradiated WT mice. After 8 weeks, DKO donor cells showed greater myelopoiesis and lesser lymphogenesis compared to WT, suggesting Bach factors are necessary to suppress myelopoiesis to the appropriate level in regenerating hematopoiesis. To reveals the function of Bach factors in HSPCs from other aspect, LSKs (Lin-Sca1+c-kit+) were infected with retroviruses expressing Bach1-IRES-eGFP or Bach2-IRES-eGFP and transplanted to lethally irradiated WT mice. Cells derived from transgene induced LSKs were monitored by GFP fluorescence. After 2 weeks, Bach1 overexpressing LSKs did not show any difference in erythropoiesis and myelopoiesis. This might be explained by the high Bach1 expression levels in HSPCs according to the previous report (Lara-Astiaso et al., 2014). On the other hand, Bach2 overexpressing LSKs showed increased erythropoiesis and decreased myelopoiesis, suggesting that Bach2 regulates the erythro-myeloid lineage specification as expected by the observations of DKO mice. To assess the function of Bach factors under infection, we used M1 murine myeloid leukemia cells that differentiate to macrophage-like cells by LPS stimulation. LPS stimulation reduced expressions of Bach1, Bach2 and erythroid gene Gata1, and induced those of myeloid genes such as Cebpb and Csf1rin a dose-dependent manner. To determine if down-regulation of Bach factors is necessary for myeloid differentiation, Bach1 or Bach2 were transgenically overexpressed in M1 cells. Both of the M1 cells overexpressing Bach1 or Bach2 showed lower expression levels of myeloid marker CD11b compared to control under LPS stimulation. Thus, reductions of the expression of Bach factors in response to LPS were necessary for appropriate myeloid differentiation. To identify the direct target genes of Bach factors, Bach1 or Bach2 ChIP-seq data of M1 cells (Ebina-Shibuya et al., 2016) were merged with results of expression profile of WT and DKO CMPs. Several myeloid or inflammatory genes such as Cebpb, Fcgr1 and Tlr4 were identified as putative repressed target genes and several erythroid or lymphoid genes such as Klf1, Rag1 and Rag2 were identified as putative activated target genes. In addition, when Bach1 or Bach2 ChIP-seq data were merged by that of C/EBPb, which also possesses AP-1 site as its target motif, obtained from ENCODE database (ENCSR000AIB), we found that there were several co-localized regions of Bach and C/EBPb near the myeloid genes such as Cebpa, Il6 and Fcgr1. These observations suggest that Bach factors repress myeloid genes by competitively working with C/EBPb at same genomic regions. This is particularly interesting in the light of the latest findings showing the Bach2 function on AP-1 site in lymphoid cells (Sidwell et al., 2016). These results reveal a novel mechanism by which how the differentiation of erythro-myeloid bifurcation is controlled by responding to environmental changes. Bach factors regulate erythro-myeloid competitive differentiation by promoting and repressing erythroid and myeloid differentiation, respectively. We suggest that infection promote myelopoiesis at the expense of erythropoiesis by reducing the expression of Bach factors. Therefore, Bach factors may function as sensors for environmental changes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Examining the CREB Transcriptional Program in Erythropoiesis

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 933-933
Author(s):  
Jacky Chung ◽  
Barry H. Paw ◽  
Harvey Lodish ◽  
Alireza Ghamari ◽  
Alan B. Cantor
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Gene Signature ◽  
Cell Maturation ◽  
Dependent Manner ◽  
Red Cell ◽  
Transcriptional Program ◽  
Cellular Functions ◽  
Creb Phosphorylation ◽  
Pka Pathway

Abstract The erythropoietin (EPO) signal transduction network regulates a plethora of critical cellular functions during red blood cell maturation including proliferation and iron metabolism. Recently, it has been discovered that EPO signaling also controls heme metabolism through activation of the protein kinase A (PKA) pathway. The activation of PKA by EPO leads to the mitochondrial phosphorylation of the terminal heme enzyme, ferrochelatase (FECH), to ramp up heme production. In addition to FECH, a second principal PKA target is the CREB transcription factor, leading to the hypothesis that the EPO-PKA signaling pathway also regulates gene expression during red cell maturation. Here, we tested this hypothesis using biochemical and bioinformatics approaches. We found that CREB becomes phosphorylated in human and murine erythroid cells in an EPO-dependent manner. Using pharmacologic inhibitors, we confirmed that CREB phosphorylation is downstream of JAK2 and PKA signaling. Moreover, we found enrichment of a previously identified CREB-gene signature within the erythroid transcriptional program. To date, our work suggests that CREB plays a role during red cell development as an effector of EPO signaling. Further work will be required to understand the function of CREB target genes. Disclosures No relevant conflicts of interest to declare.

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