Abstract
Abstract 2607
Pelger-Huët anomaly (PHA) is a disorder of neutrophil nuclear lobulation, in which mature human granulocytes have a mononuclear or bilobed nucleus (so-called pince-nez cells). PHA is a congenital human disorder, but nuclear hypolobation also arises as an acquired defect in pre-leukemic myelodysplastic syndromes. Lamin B receptor (LBR) is an inner nuclear membrane protein whose expression increases during myeloid differentiation, and loss of LBR expression causes PHA. We sought to examine the regulation of LBR in order to identify molecular mechanisms that contribute to neutrophil disorders, including myelodysplastic syndromes and acute myelogenous leukemia. Many hematopoietic-specific genes are regulated by the combinatorial activity of transcription factors, including the ETS factors, PU.1 and GABP (GA binding protein). GABP and PU.1 cooperate to regulate the expression of the leukocyte adhesion molecule CD18, and recently were shown to regulate the expression of the interleukin-7 receptor in developing B cells. GABP is an obligate heterotetramer that is composed of two structurally dissimilar proteins, GABPα and GABPβ. Our analysis of the Lbr gene promoter identified classic “GAGGAA” ets consensus sequences located proximal and distal to the Lbr transcription start site. Lbr promoter constructs containing either the proximal ets site or both the proximal and distal ets sites were not activated by PU.1, alone, following transfection into COS cells. However, these constructs were activated by co-expression of GABPα plus GABPβ, and combined expression of GABPα/β plus PU.1 further activated these constructs up to two-fold. This suggests that GABP and PU.1 cooperatively activate the Lbr gene promoter. Electrophoretic mobility shift assays (EMSA) using radiolabeled probes that include the distal or proximal putative ets sites and nuclear extracts from HEK-293 cells transfected with expression vectors for GABPα, GABPβ and PU.1, identified multiple low mobility bands that were competed by 100 fold excess of cold competitor probe, but not by an irrelevant control probe. Inclusion of anti-GABPα antibodies in the binding reaction disrupted mobility shifts of the probes, indicating that GABPα directly interacts with the Lbr promoter and may participate in the formation of a multimeric protein complex that binds the promoter. Similar results were observed with nuclear extracts from EML cells, which correspond to murine hematopoietic progenitor cells that can be induced to differentiate toward promyelocytic EPRO cells and thence to mature granulocytes. We examined protein expression of GABPα in HL-60 and EML/EPRO progenitor cells, and found that GABPα is highly expressed in uninduced progenitors but downregulated during either neutrophil or monocyte differentiation. We generated mice in which loxP recombination sites flank critical exons of Gabpa; in the presence of Cre recombinase the loxP sites undergo rearrangement and Gabpa is deleted. We bred these animals to mice that are transgenic for estrogen receptor (ER)-regulated Cre recombinase, and created a novel EML cell line from their bone marrow. Upon activating Cre expression with 4-hydroxytamoxifen, most EML cells died within 24 hours, as compared to control cells. This result is consistent with previous studies demonstrating that GABP is required for cell cycle progression, and suggests that GABP plays a critical role in myeloid cell survival. Together, our data indicate that the GABP tetramer binds to specific sequences of the Lbr promoter, and that GABP cooperates with PU.1 to drive Lbr expression during neutrophil differentiation. Analysis of promoter constructs with mutated ets sites in our reporter assays and mobility shift assays will further our knowledge about the importance of GABP/PU.1 complexes in Lbr gene regulation. EML cells that can undergo conditional deletion of Gabpa provide a powerful tool for analysis of the regulation of myeloid genes such as Lbr, and for the molecular mechanisms that cause disorders of myeloid maturation, including myelodysplastic syndromes and acute myelogenous leukemia.
Disclosures:
No relevant conflicts of interest to declare.
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