RESUMO
Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and ß-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.
Assuntos
Fator 4 Ativador da Transcrição/genética , Hemoglobina Fetal/genética , Proteínas Repressoras/genética , eIF-2 Quinase/genética , Anemia Falciforme/sangue , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Elementos Facilitadores Genéticos , Eritroblastos/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Especificidade da Espécie , Talassemia beta/sangue , Talassemia beta/genética , Talassemia beta/terapia , gama-Globinas/biossíntese , gama-Globinas/genéticaRESUMO
N-oxide derivatives 5(a-b), 8(a-b), and 11(a-c) were designed, synthesized and evaluated in vitro and in vivo as potential drugs that are able to ameliorate sickle cell disease (SCD) symptoms. All of the compounds demonstrated the capacity to releasing nitric oxide at different levels ranging from 0.8 to 30.1%, in vivo analgesic activity and ability to reduce TNF-α levels in the supernatants of monocyte cultures. The most active compound (8b) protected 50.1% against acetic acid-induced abdominal constrictions, while dipyrone, which was used as a control only protected 35%. Compounds 8a and 8b inhibited ADP-induced platelet aggregation by 84% and 76.1%, respectively. Both compounds increased γ-globin in K562â¯cells at 100⯵M. The mechanisms involved in the γ-globin increase are related to the acetylation of histones H3 and H4 that is induced by these compounds. In vitro, the most promising compound (8b) was not cytotoxic, mutagenic and genotoxic.
Assuntos
Anemia Falciforme/tratamento farmacológico , Descoberta de Drogas , Histonas/metabolismo , Oxidiazóis/farmacologia , gama-Globinas/biossíntese , Ácido Acético/antagonistas & inibidores , Ácido Acético/farmacologia , Acetilação , Anemia Falciforme/metabolismo , Relação Dose-Resposta a Droga , Humanos , Células K562 , Estrutura Molecular , Óxido Nítrico/metabolismo , Oxidiazóis/síntese química , Oxidiazóis/química , Relação Estrutura-AtividadeRESUMO
Hereditary persistence of fetal hemoglobin (HPFH) is characterized by increased levels of Hb F during adult life. Nondeletional forms of HPFH are characterized by single base mutations in the (A)gamma and (G)gamma promoters, resulting in an increase of Hb F ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the (A)gamma -195 (C>G) mutation that causes the Brazilian type of HPFH (HPFH-B). To better understand this mechanism, we have developed HPFH-B transgenic mice. mRNA levels of human gamma-globin of -195 transgenic mice were clearly higher when compared with control transgenic mice bearing a wild type sequence of the gamma promoter. Thus, our data indicate that the -195 mutation is the unique cause of elevation of Hb F in Brazilian HPFH. These results could provide us with an opportunity to study the modifying effects of the Hb F in the phenotype of sickle cell disease and beta-thalassemia (beta-thal).