Different Hemoglobin Switching Pattern of β-Thalassemia Mutations at the Proximal and Distal Human β-Globin CACCC Box.
Authors: Marongiu Maria F. , Porcu Susanna , Poddie Daniela , Drabeck Dubravka , DeWit Tom , Cao Antonio , Ristaldi Maria S. 
Published: April 2020

Abstract
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.