Role In Granulocytic Differentiation Research Articles

Chromatin remodeling mediated by ARID1A is indispensable for normal hematopoiesis in mice

Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.

GADD45b Plays an Essential Role in the G-CSF Triggered Granulocytic Differentiation of Human Hematopoietic Cells

The mechanism of maturation arrest of bone marrow myeloid progenitors in severe congenital neutropenia (CN) patients is not fully elucidated. We found, that treatment of healthy individuals with G-CSF induces mRNA expression of GADD45b (Growth Arrest and DNA-Damage-inducible, beta) in CD33+ bone marrow myeloid progenitors. However, the expression of GADD45b was not activated in CD33+ cells of G-CSF treated CN patients. GADD45b functions as stress sensor downstream of G-CSF signaling and is essential in stress-induced murine myelopoiesis. Less is known about the function of GADD45b in the myeloid differentiation of human HSPCs. We hypothesized, that the inability of G-CSF to induce GADD45b expression might be a cause of diminished granulopoiesis in CN patients. To test this hypothesis, we inhibited GADD45b expression in CD34+ cells and iPSCs of healthy donors by introducing indels in exon 1 of the GADD45B gene using specific CRISPR/Cas9-gRNA ribonucleoprotein (RNP). We evaluated G-CSF-triggered myeloid differentiation of GADD45b-deficient iPSCs using embryoid body (EB)-based method and found that iPSCs cells present with severely diminished granulocytic differentiation upon GADD45b knockout, as assessed by FACS, CFU assay and morphological examination of cytospin slides. We also observed reduced G-CSF-mediated granulocytic differentiation of GADD45b-deficient CD34+ cells of healthy individuals in colony-forming units (CFU) assay and liquid culture differentiation followed by FACS analysis on day 7 and day 14.Importantly, rescue of GADD45b in HSPCs of one CN patient by lentivirus-based transduction of GADD45B cDNA restored defective granulocytic differentiation, as compared to control transduced cells. These data strongly support the essential role of GADD45b in G-CSF-mediated granulocytic differentiation. GADD45b rescue analysis of additional CN patients cells is ongoing.To study the mechanism of GADD45b activation upon G-CSF stimulation of hematopoietic cells, we performed in silico analysis of GADD45B promoter and found putative binding sites for G-CSF responsive hematopoietic transcription factors, including CEBPA, CEBPB, KLF4, STAT3 and STAT5. Using the dual luciferase reporter assay with 1.6 kb region of the GADD45B gene promoter, we found that KLF4, STAT5, CEBPA and CEBPB activate GADD45b expression in a dose-dependent manner. Intriguingly, CEBPA expression is severely diminished in myeloid cells of CN patients (Skokowa et al., 2006) and we assumed that G-CSF is not able to activate GADD45b expression in CN patients because of defective CEBPA. To study the mechanism by which GADD45b mediates myeloid differentiation, we performed RNA sequencing of WT or GADD45b-deficient CD34+ HSPCs treated or not with G-CSF. Interestingly, in GADD45b-deficient cells, G-CSF failed to induce mRNA expression of several genes essential for granulocytic differentiation and granulocyte functions including GLI1, CAMP/LL37, MMP8, CD16, LCN2, OLFM4, CX3CR1, SIGLEC5, as compared to WT cells. Reactome and Gene Set Enrichment Analysis (GSEA) of RNA-Seq data sets also revealed deregulation of the “myeloid CEBPA network”, “GLI proteins pathway” and “neutrophil degranulation pathway” in G-CSF-treated GADD45b-deficient CD34+ cells, as compared to control G-CSF-exposed cells. Of note, severely diminished expression of plasma CAMP/LL37 levels is a unique feature of CN patients (Y. Ye et al. 2015).In summary, our data suggest that GADD45b plays an essential role in granulocytic differentiation of human hematopoietic cells and inability of G-CSF to induce GADD45b expression in myeloid cells of CN patients may be a reason for the defective granulopoiesis. DisclosuresNo relevant conflicts of interest to declare.

LPS independent activation of the pro-inflammatory receptor Trem1 by C/EBP\u03b5 in granulocytes

C/EBPε is a critical transcriptional factor for granulocyte differentiation and function. Individuals with germline mutations of C/EBPε fail to develop normal granulocytes and suffer from repeated infections. In order to gain a global view of the transcriptional machinery regulated by C/EBPε, we performed whole-genome ChIP-Seq using mouse bone marrow cells. To complement the C/EBPε DNA binding analyses, RNA-Sequencing was done in parallel using sorted mature and immature granulocytes from WT and C/EBPε KO bone marrow. This approach led to the identification of several direct targets of C/EBPε, which are potential effectors of its role in granulocytic differentiation and function. Interestingly, Trem1, a gene critical to granulocyte function, was identified as a direct C/EBPε target gene. Trem1 expression overlaps very closely with expression signature of C/EBPε during hematopoietic development. Luciferase reporter and EMSA assays revealed that C/EBPε binds to the regulatory elements of Trem1 and regulates its expression during granulocytic differentiation. In addition, we provide evidence that inflammatory stimuli (LPS) can also control the expression of Trem1 independent of C/EBPε. Overall, this study provides comprehensive profiling of the transcriptional network controlled by C/EBPε during granulopoiesis and identifies Trem1 as one of its downstream effectors involved in eliciting an immune response.

Transcription factor C/EBPα-induced microRNA-30c inactivates Notch1 during granulopoiesis and is downregulated in acute myeloid leukemia

AbstractThe transcription factor CCAAT enhancer binding protein α (C/EBPα) is a master regulator in granulopoiesis and is frequently disrupted in acute myeloid leukemia (AML). We have previously shown that C/EBPα exerts its effects by regulating microRNAs (miRs) such as miR-223 and miR-34a. Here, we confirm miR-30c as a novel important target of C/EBPα during granulopoiesis. Thus, wild-type C/EBPα-p42 directly upregulates miR-30c expression, whereas C/EBPα-p30, found in AML, does not. miR-30c is downregulated in AML, especially in normal karyotype AML patients with CEBPA mutations. An induced C/EBPα knockout in mice leads to a significant downregulation of miR-30c expression in bone marrow cells. We identified NOTCH1 as a direct target of miR-30c. Finally, a block of miR-30c prevents C/EBPα-induced downregulation of Notch1 protein and leads to a reduced CD11b expression in myeloid differentiation. Our study presents the first evidence that C/EBPα, miR-30c, and Notch1 together play a critical role in granulocytic differentiation and AML, and particularly in AML with CEBPA mutations. These data reveal the importance of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML.

ER Stress-Induced Expression of CCAAT/Enhancer Binding Protein Gamma (C/EBPG) May Contribute to the Block in Granulocytic Differentiation in Severe Congenital Neutropenia

Abstract 8Severe congenital neutropenia (SCN) is a bone marrow failure syndrome characterized by an isolated block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are due to heterozygous mutations of ELANE, encoding neutrophil elastase (NE). We and others previously published data supporting a model in which the shared feature of the many different ELANE mutations is a propensity to misfold. Accumulation of misfolded mutant NE induces endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). However, the mechanisms by which UPR activation disrupts granulocytic differentiation are not clear.We previously presented results of RNA expression profiling of promyelocytes sorted from patients with SCN (n = 9), healthy donors (n = 5), healthy donors treated with G-CSF (n = 4), or patients with Shwachman Diamond syndrome (n = 5), an unrelated congenital neutropenia syndrome. A transcriptional signature consistent with UPR activation was observed only in SCN promyelocytes. Here we report, based on an unbiased analysis of the profiling data, that one of the most dysregulated genes in SCN promyelocytes is CCAAT/enhancer binding protein gamma (CEBPG). CEBPG is a member of the CCAAT/enhancer binding protein family that includes CEBP/alpha (CEBPA), which plays an obligatory role in granulocytic differentiation. Of note, Skokowa and colleagues previously reported reduced CEBPA expression in myeloid cells from patients with SCN (Nat Med 12:191, 2006). CEPBG is known to heterodimerize with CEBPA and inhibits its transcriptional activation of target genes. Based on these data, we hypothesize that UPR-induced expression of CEBPG may contribute to the block in granulocytic differentiation in SCN through antagonism of CEBPA.We first asked whether UPR activation directly induces CEBPG mRNA expression. Bone marrow from healthy donors was treated with a variety of UPR inducers (tunicamycin, thapsigargin, or dithiothreitol) for 24 hours, and real time PCR performed on sorted promyelocytes (CD16low, CD15+ CD14− CD9e− cells) was performed. CEBPG mRNA expression was induced 2–5 fold compared with untreated cells. Of note, no difference in CEBPA mRNA expression was observed after UPR activation. However, though confirmation is required, immunoblotting of CD15+ myeloid cells after UPR activation showed decreased expression of CEBPA protein, which is consistent with a prior report suggesting that UPR activation inhibits CEBPA translation (J Cell Mol Med. 14:1509–19). Thus, UPR activation may inhibit CEBPA function through two mechanisms: increased CEBPG antagonism and decreased CEBPA translation. We next asked whether enforced expression of CEBPG was sufficient to inhibit granulocytic differentiation. CD34+ hematopoietic progenitors from healthy donors were transduced with lentivirus expressing CEBPG or vector alone; the lentivirus also expressed green fluorescent protein (GFP) to track transduced cells. Transduced (GFP+) cells were sorted and then cultured on irradiated stromal cells in the presence of G-CSF for two weeks to induce granulocytic differentiation; CEBPG mRNA expression was increased approximately 20-fold compared to control transduced cells. Enforced expression of CEBPG resulted in a significant suppression of mature neutrophil production. The percentage of mature neutrophils in CEBPG-overexpressing cultures was 19.2 ± 0.04% compared with 42.2 ± 0.7% for vector-alone transduced cultures (P = 0.02, n =4). Together, these data suggest a model in which UPR activation inhibits granulocytic differentiation, at least in part, by inducing CEBPG expression and antagonizing CEBPA function. Disclosures:Boxer:Amgen: Equity Ownership; Alexion: Speakers Bureau; NIH: Research Funding; Up to Date: Patents & Royalties.

Proteomic study of granulocytic differentiation induced by apigenin 7-glucoside in human promyelocytic leukemia HL-60 cells

Nutritional factors is one of the most important regulators in the progression of cancer. Some dietary elements promote the growth of cancer but others, such as plant-derived compounds, may reverse this process. We tried to investigate yet another approach of cancer prevention through cancer cell differentiation, using a common non-mutagenic flavonoid apigenin 7-glucoside. HL-60 cells were treated with or without apigenin 7-glucoside. Cell proliferation was measured by MTT assay, and the cell cycle distribution was estimated by propidium iodide staining of DNA. To determine cellular differentiation, cell surface differentiation markers CD11b and CD14 were used. Two-dimensional gel electrophoresis was then performed to identify proteins that may be important in HL-60 cell differentiation following apigenin 7-glucoside treatment. Apigenin 7-glucoside inhibited HL-60 cell growth, dose- and time-dependently, but did not cause apoptosis. The distribution of cells at different stages in the cell cycle indicated an accumulation of treated cells in G(2)/M phase. Moreover, apigenin 7-glucoside induced granulocytic differentiation of HL-60 cells. Ten proteins that might play essential role in granulocytic differentiation were identified by proteomics. A complete understanding of the preventive effects of plant-based diet on cancer depends on the mechanisms of action of different plant components on processes. We hope these findings may contribute to the understandings of the different approaches for chemoprevention of cancer.

Granulocytic differentiation of HL-60 promyelocytic leukemia cells is associated with increased expression of Cul5

The human HL-60 promyelocytic leukemia cell line has been widely used as a model for studying granulocytic differentiation. All-trans retinoic acid (ATRA) treatment of HL-60 cells promotes granulocytic differentiation and is effective as differentiation therapy for patients with acute promyelocytic leukemia. The identification of genes that are transcriptionally regulated by ATRA has provided insight into granulocytic differentiation and differentiation therapy. The Asb-2 (ankyrin repeat SOCS box 2) gene has previously been identified as a transcriptional target in ATRA-treated HL-60 cells. The ASB-2 protein forms an E3 ubiquitin ligase complex with the proteins, Cul5, regulator of cullin 2 (ROC2), and elongin B and C. The purpose of this study was to determine if there is increased expression of Cul5 during granulocytic differentiation of HL-60 cells. To induce granulocytic differentiation, HL-60 cells were treated for 5 d with ATRA and differentiation was confirmed by examining superoxide anion production, nuclear morphology, and changes in the expression of CD11b, CD13, and CD15. Quantitative real-time RT-PCR was used to measure Cul5 mRNA expression and also the expression of other components of the E3 ubiquitin ligase (ASB-2, ROC2, elongin B and C). Granulocytic differentiation of HL-60 cells was associated with a 1.6-, 1.7-, and 23-fold statistically significant (P <or= 0.05) increase in mRNA expression for Cul5, ROC2, and ASB-2, respectively. No significant change was found in elongin B and C mRNA expression. Using Western blot analysis, the expression of Cul5 protein was increased 6.5-fold with granulocytic differentiation of the HL-60 cells. Increased expression of multiple components of the Cul5-containing E3 ubiquitin ligase complex with ATRA treatment of HL-60 cells indicates that this complex may play an important role in granulocytic differentiation.

HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

Diminished proteasomal degradation results in accumulation of Gfi1 protein in monocytes

AbstractGfi1 is a transcriptional repressor essential during myeloid differentiation. Gfi1−/− mice exhibit a block in myeloid differentiation resulting in the accumulation of an immature myelo-monocytic cell population and the complete absence of mature neutrophils. Even though mRNA levels of Gfi1 appear to be very low in monocytes, Gfi1 might play a role in the monocytic lineage as Gfi1−/− mice exhibit diminished monocyte-derived dendritic cells and disturbed cytokine production by macrophages in response to LPS. We show here that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Upon forced monocytic differentiation of U937 cells, Gfi1 mRNA levels dropped but protein levels increased due to diminished proteasomal turnover. Similarly, Gfi1 mRNA levels are low in primary monocytes whereas the protein is clearly detectable. Conversely, Gfi1 mRNA levels are high in granulocytes but the protein is swiftly degraded by the proteasome in these cells. Chromatin immunoprecipitation experiments showed that Gfi1 binds to the promoter of several granulocyte-specific genes in primary monocytes, including C/EBPα, neutrophil elastase, and Gfi1 itself. The binding of the repressor Gfi1 to these promoters correlated with low expression of these genes in monocytes compared with granulocytes. Our data fit a model in which Gfi1 protein levels are induced in primary monocytes, due to diminished proteasomal degradation, to repress genes that play a role in granulocytic differentiation.

Ectopic expression of interferon regulatory factor-1 potentiates granulocytic differentiation

Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of differentiation.

Ectopic expression of interferon regulatory factor-1 potentiates granulocytic differentiation.

Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of differentiation.