Cloning And Characterization Of A Hydroxyurea-inducible Gene

Rodgers, Griffin

Abstract

Hydroxyurea (HU) has been shown to augment the production of fetal hemoglobin (HbF) and for this reason is being used in the treatment of sickle cell anemia. It has been assumed that HU promote HbF production indirectly by perturbing the maturation of erythroid precursors. However, the molecular mechanism(s) involved in how HU regulates γ-globin expression remains unclear. By using a two-phase liquid erythroid culture system in conjunction with mRNA differential display, a HU-inducible small GTP-binding protein gene, designated SAR, was successfully cloned and its biological function is being investigated. We found the SARgene is localized to chromosome 10. SAR tagged with a GFP fusion protein is co-expressed with anti-calreticulin in the ER complex in K562 cell.? ? Stable SAR expression in K562 cells increased γ-globin mRNA expression and resulted in macrocytosis and cells that appeared immature. SAR-mediated induction of γ-globin also inhibited K562 cell growth by causing arrest in G1/S, apoptosis, and delay of maturation, cellular changes consistent with the previously known effects of HU on erythroid cells. SAR also enhanced both gamma- and beta-globin transcription in primary bone marrow CD34+ cells, with a greater effect on gamma-globin than on beta-globin. SAR exhibits a tight correlation with gamma gene in stable transfected K562 cell, AC133+ cell cultured with EPO and K562 treated with HbF production inducers. Several pathways that contribute to the SAR induction of the gamma globin gene are being investigated. SAR upregulated GATA-2, p21, inhibited PI3 kinase and phosphorylated ERK. These data reveal a novel role of SAR distinct from its previously known protein trafficking function. SAR may participate in both erythroid cell growth and γ-globin production by regulating PI3 kinase/ERK and GATA-2/p21-dependent signal transduction pathways. The research data has been summarized and published in Blood 106:3256, 2005..? ? Based on the SAR unique biological function in gamma globin induction, SAR seems play a central role in fetal erythropoiesis. By using RNA interference technology, we are able to knock down SAR in different cells and examine related signal pathway which may illuminate how SAR regulates erythrocyte maturation and proliferation. . Our data revealed that SAR microRNA is able to down regulate HbF cell population 15-22%, block HU induced Hb F35-40% in K562 cells, and abolished HU mediated S phase cell arrest in HEK 293 cells (61-65%). SAR micro RNA also inhibited the effects of HU on PI3 Kinase protein expression, and AKT, ERK dephosphorylation. We suggest that SAR may participate in both erythroid cell growth and gamma-globin production by regulating PI3-kinase/ERK and GATA-2/p21 dependent signal transduction pathways.? ? ? Our preliminary analysis indicates that HU inducibility is mediated at the transcriptional level, and is localized to elements in the SAR1A promoter.
The aim of this study was to assess whether polymorphisms in the SAR1A gene promoter are associated with Hb F levels or HU therapeutic responses. We studied 386 sickle cell disease patients consisting of 269 adults treated with or without HU and 117 newborns with sickle cell disease identified from a newborn screening program. Twenty point mutations, including one nonsynonymous variant, were identified in SAR1A, including nine previously reported in SNP databases. A difference in genotype frequencies was observed between adults and newborns for rs2310991 in the 5UTR (odd ratio OR = 1.9, 95% confidence interval CI = 1.1-3.2, P=0.009) and +31 T>C in 5UTR (odd ratio OR 9.8 1.3-73.9; confidence interval CI 95%; P<0.001). Three previously unknown SNPs in the upstream 5UTR (-809 C>T, -502 G>T and -385 C>A) were significantly associated with the HbF response in Hb SS patients treated with HU (P<0.05). These data suggest that variation within SAR1A regulatory elements might contribute to inter-individual differences in regulation of HbF expression and patient responses to HC in SCD.? ? We further dissected the SAR promoter region to identify HU-responded elements and cognate DNA binding proteins that may confer HU inducibility. Structural deletion analysis revealed that the elements between -41 and +3 region is sufficient to drive SAR promoter activity and is responsible for HU inducibility. Mutation of NF-KB binding site within this region could significantly decrease SAR promoter activity; and with this mutation, HU lost its ability to enhance SAR promoter activity; moreover, Gel shift and super gel shift assays clearly indicate that NFKB protein binds to SAR promoter. Together these results suggest that NFKB binding site on SAR promoter is necessary for SAR transcription and responsible for HU mediated SAR induction.