ALBERT

Description: The CACCC box is duplicated in the β globin gene promoter of humans and other mammals. While the function of the proximal element as a binding site for EKLF has already been well established, the role of the distal element remains unclear The distal CACCC box has been previously reported not to bind EKLF in vitro. A minor role of the distal CACCC element in β globin gene promoter function is suggested by the observation that naturally occurring β thalassemia mutations affecting the proximal CACCC box are far more severe than those affecting the distal element. Nevertheless recent evidences demonstrate: that EKLF does indeed bind to the distal CACCC motif, although with low affinity. that the CCTCACCC is required for maximal stimulation of the β-globin gene by EKLF and that silent β-thalassemia due to mutations of the distal CACCC box affects the binding and responsiveness to EKLF of the β-globin gene promoter. Our interest in the function of the distal CACCC element springs from the observation that β thalassemia mutation affecting the distal box show an age related pattern of expression being more severe in the childhood than in the adulthood. In order to get light inside the role of this element in the function of the β globin gene and in the γ to β hemoglobin switching we have analyzed the effect of mutations at the proximal and distal element “in vivo”. We have engineered, by site specific mutagenesis, the β-101 (distal CACCC element) and β-87 (proximal CACCC element) mutations inside the “minilocus “ γ-β construct. The minilocus construct has been widely used to study hemoglobin switching in vivo. This construct contains the full β-globin Locus Control Region (LCR), the Aγ globin gene, the β-globin gene and the 3′ hypersensitive site (HS) of the β-globin cluster. Three mice transgenic lines have been produced. The pattern of g versus β-globin switching has been analyzed during the development by S1 analysis and real time PCR. We have dissected the yolk sac at 10 days post conception (pc) to asses the embryonic stage of erythopoiesis; the fetal liver at 12, 14 and 16 days pc to asses the fetal stage or erythropoiesis when the g to b competitive switching take place; and the adult blood. Our results indicated that neither the β-101 nor the β-87 thalassemia mutations affect the competitive silencing of the b-globin gene in the yolk sac. During the fetal liver stage of erythropoiesis, were both human g and b human transgenes are expressed, the pattern of γ-β hemoglobin switching is striking different for the two different constructs. The b-87 minilocus γ-β construct shows a delayed switching patter mainly due to the low activation of the mutated β globin gene. The impairment of the expression of the β-87 globin gene became more severe during the fetal development compared to the control line. On the other hand the β-101 minilocus γ-β construct shows a γ-β hemoglobin switching pattern which is anticipated respect to the control line. In addition the effect of the β-101 mutation became less severe during the fetal development. These results highlight a possible role of the distal CACCC element in hemoglobin switching and in particular in the early stage of β-globin activation.

Description: The δ globin gene is the second adult β-like globin gene in humans and codes for the δ globin chain which forms together with the α globin chain Hemoglobin A2 (HbA2). HbA2 represents less than 3% of the total hemoglobin in normal individuals and it is typically increased in β thalassemia carriers. The δ globin gene is highly homologous to the β globin gene since it derives from a common ancestor. In our previous work we (as well as others) have demonstrated, in vitro, that the creation of the β globin proximal CACCC box consensus sequence, the binding site of the trascription factor EKLF, on the δ globin gene promoter is sufficient to enhance its expression to a considerable extent. Here we show that the δ globin gene promoter can be activated “ in vivo” in a transgenic mice model. We have produced transgenic mice lines with a DNA construct in which the wild type (wt) β globin gene promoter and either the wt or the proximal CACCC box containing δ globin gene promoter are linked in cis to the second hypersensitive site (HS2) of the Locus Control Region (LCR). The order of the different elements in our construct mimic the organization of the human β globin cluster were the δ globin gene is situated 5′ to the β and relatively closer to the LCR. The δ and β globin gene promoter are respectively linked to two different luciferase reporter genes, fireflies and renilla. All the transgenic lines produced are multicopy as assessed by southern blotting. The level of the expression of the two reporter genes has been assessed in the fetal liver at day 14 post coitum (pc). We have analyzed 3 out of 5 independent transgenic lines bearing the CACCC containing δ globin gene promoter construct, and 2 independent lines bearing the control construct (wt δ globin gene promoter). For each transgenic line we have analyzed two different litters. Transgenic fetuses have been identified by PCR. Fetal liver samples have been lisated and the crude protein extracted has been assayed for the luciferase versus renilla activity. Taking into account the different activity of the two reporter enzymes the wt δ globin gene promoter activity is 13% (+/−0,5) that of the wt β globin gene promoter in fetal liver (n = 18). On the other hand the transgenic lines bearing the construct with the CACCC box containing δ globin gene promoter show an activation up to 82% (+/−19) in respect to the wt β promoter, on average (n = 32) The difference in expression between the wt and the CACCC box bearing δ globin gene promoter is highly significant (ttest = 3.8X10−7 ). This observation is a step forwards to possible gene therapy strategies for hemoglobinopathies based on the reactivation of the endogenous δ globin gene. The δ globin chain could represent in fact a valid substituted of the δ globin chain in beta thalassemia patients. It is also well known that HbA2 is a powerful antisickling agent which could benefit sickle cell patients. Current gene therapy strategies for the hemoglobinopathies focus on the trasduction of the hematopoietic stem cells by viral vectors or on the correction of the endogenous β globin gene by homologous recombination. An alternative approach could be to introduce into the hematopoietic stem cells of an engineered transcription factor able to enhance δ globin gene transcription.

Description: Progressive iron overload is the most salient and ultimately fatal complication of β-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in β-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both β-thalassemia intermedia (th3/+) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/+ and th3/th3 mice, and that transfusion therapy can rescue mice affected by β-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/+ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/+ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of β-thalassemia.