Hydroxyurea responsiveness in β-thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity

β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) 'responders' who increase hemoglobin to therapeutic levels; (ii) 'moderate-responders' who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) 'non-responders' who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it h

Two novel GALNT3 mutations in familial tumoral calcinosis

Familial tumoral calcinosis(TC) is characterized by elevated serum ohosphate concentrations, normal or elevated 1.25(OH)(2) vitamin (D,) as well as periarticular and vascular calcifications. Recessive mutations in the mucin-like-glyco-syltransferase GalNAc transferase-3 (GALNT3) and the phosphaturic hormone fibroglast growth factor-23 (FGF23) have been shown to result in TC. in the present study, mutational analyses were performed on two patients with TC to determine the molecular basis of their diseases. Analysis of the first patient reavealed a novel, homozygous base insertion (1102_1103insT) inGALNT3 exon 5 that results in a frameshift and premature stop codon (E375X). The second patient had a novel homozygous transition (1460 g>a) in GALNT3 exon 7, which caused a nonsense mutation (W487X). Both mutations are predicted to markedly truncate the mature GALNT3 protein product. Although the patients carry GALNT3 mutations, these individuals presented with low-normal serum concentrations of onact biologically active FGF23 in TC, a comprehensive assesment of the reported TC mutations was also performed. In summary, we have detected novel GALNT3 mutations that result in familial TC, and show that disturbed serum FGF23 concentrations are present in our cases as well as in previously reported cases. These studies expand our current genetic understanding of familial TC, and support a pathophysiological association between GALNT3 and FGF23. (C) 2007Wiley-Liss, Inc

Hydroxyurea responsiveness in β-thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity

β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) 'responders' who increase hemoglobin to therapeutic levels; (ii) 'moderate-responders' who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) 'non-responders' who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it had little impact on cells from responders. Part of the gene program up-regulated by hydroxyurea in non-responders was already highly expressed in responders before hydroxyurea treatment. Baseline HbF expression was low in non-responders, and hydroxyurea treatment induced significant cell death. We conclude that cells from responders have adapted well to constitutive stress conditions and display a propensity to proceed to the erythroid differentiation progra

2009

ABSTRACT HU in vitro, HbF induction was comparable to the increase of HbF in peripheral blood of SCD patients following HU therapy in vivo

The mouse KLF1 Nan variant impairs nuclear condensation and erythroid maturation

Krüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >140 KLF1 variants, causing different erythroid phenotypes, have been described. The KLF1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the KLF1 Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the KLF1 Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the 782 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We propose that reduced expression of XPO7 is partially responsible for the erythroid defects observed in KLF1 Nan erythroid cells

Five friends of methylated chromatin target of protein-arginine- methyltransferase[Prmt]-1 (Chtop), a complex linking arginine methylation to desumoylation

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity

The DNA binding factor Hmg20b is a repressor of erythroid differentiation

Background: In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established. Design and Methods: To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene. Results: We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRASlike suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts. Conclusions: We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the downregulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins