Sox6 Directly Silences Epsilon Globin Expression in Definitive Erythropoiesis

Sox6 is a member of the Sox transcription factor family that is defined by the conserved high mobility group (HMG) DNA binding domain, first described in the testis determining gene, Sry. Previous studies have suggested that Sox6 plays a role in the development of the central nervous system, cartilage, and muscle. In the Sox6-deficient mouse, p(100H), ɛy globin is persistently expressed, and increased numbers of nucleated red cells are present in the fetal circulation. Transfection assays in GM979 (erythroleukemic) cells define a 36–base pair region of the ɛy proximal promoter that is critical for Sox6 mediated repression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that Sox6 acts as a repressor by directly binding to the ɛy promoter. The normal expression of Sox6 in wild-type fetal liver and the ectopic expression of ɛy in p(100H) homozygous fetal liver demonstrate that Sox6 functions in definitive erythropoiesis. The present study shows that Sox6 is required for silencing of ɛy globin in definitive erythropoiesis and suggests a role for Sox6 in erythroid cell maturation. Thus, Sox6 regulation of ɛy globin might provide a novel therapeutical target in the treatment of hemoglobinopathies such as sickle cell anemia and thalassemia

Transcriptional Regulation of the Mouse betamajor Globin Gene in Murine Erytroleukaemia Cells

There is considerable evidence that certain features of the mechanism of transcriptional regulation of the (betamajor globin gene are common to other erythroid-specific globin and non-globin genes (reviewed in chapter 1) . However, my work suggests that there are aspects of this regulation which are unique. My work on the binding of nuclear proteins to the mouse betamajor globin gene promoter has revealed a modular organisation of protein binding sites. These include two binding sites for the erythroid-specific factor, NF-E1 (at -65 and -210 bp) and binding by non-tissue-specific proteins to the CCAAT (-85 bp), CACCC (-95 bp) and Box 1 (-170 bp) motifs and to a nuclear factor 1 (NF-1) binding site (-255 bp). Mutational analysis of the mouse betamajor globin gene proximal promoter TATA, CCAAT and CACCC boxes by other researchers had shown that they are required for promoter function in erythroid and non-erythroid cells. Using site-directed mutagenesis, I have confirmed that the CACCC motif is required for promoter function when linked to a reporter gene and assayed in transient and stable transfection assay systems. Furthermore I have shown by mutagenesis that the NF-E1 site at -65 bp is necessary for promoter function. However mutation of the NF-E1, Box1 and NF-1 sites in the distal region of the promoter results in up-regulation of transcription from the promoter in uninduced MEL cells and it is proposed here that while the proximal promoter is essential for promoter function that the distal promoter has a negative regulatory function in uninduced MEL cells. This negative regulatory effect is derepressed when the MEL cells are induced to differentiate. In addition to the role of 5' promoter sequences in transcriptional regulation of the mouse betamajor globin gene, I have identified sequences 3' of the coding region of the gene which contain NF-E1 and Box 1 binding sites and which confer an up-regulatory effect on the transcription of a linked reporter gene from the mouse betamajor globin gene promoter. The up-regulatory effect of this enhancer is greater when the cells are induced to differentiate. Work with two different MEL cell lines, F4-12B2 and C88, and with two different inducing agents, DMSO and HMBA, suggests that there are differences in the responses of these cell lines to the same treatment and also that DMSO and HMBA act through different mechanisms to induce differen tiation

A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells

The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation

Control of erythroid differentiation: asynchronous expression of the anion transporter and the peripheral components of the membrane skeleton in AEV- and S13-transformed cells

Chicken erythroblasts transformed with avian erythroblastosis virus or S13 virus provide suitable model systems with which to analyze the maturation of immature erythroblasts into erythrocytes. The transformed cells are blocked in differentiation at around the colony-forming unit- erythroid stage of development but can be induced to differentiate in vitro. Analysis of the expression and assembly of components of the membrane skeleton indicates that these cells simultaneously synthesize alpha-spectrin, beta-spectrin, ankyrin, and protein 4.1 at levels that are comparable to those of mature erythroblasts. However, they do not express any detectable amounts of anion transporter. The peripheral membrane skeleton components assemble transiently and are subsequently rapidly catabolized, resulting in 20-40-fold lower steady-state levels than are found in maturing erythrocytes. Upon spontaneous or chemically induced terminal differentiation of these cells expression of the anion transporter is initiated with a concommitant increase in the steady- state levels of the peripheral membrane-skeletal components. These results suggest that during erythropoiesis, expression of the peripheral components of the membrane skeleton is initiated earlier than that of the anion transporter. Furthermore, they point a key role for the anion transporter in conferring long-term stability to the assembled erythroid membrane skeleton during terminal differentiation

The upstream area of the chicken α-globin gene domain is transcribed in both directions in the same cells

AbstractIt was demonstrated previously that in erythroid chicken cells an extended upstream area of the α-globin gene domain is transcribed in both directions as a part of ggPRX gene and a part of a full domain transcript of the α-globin gene domain. Here, we show that both DNA chains of the above-mentioned region are transcribed in the same cells and that the corresponding transcripts coexist in nuclei. The data obtained suggest that cells possess a molecular mechanism which in some cases prevents the formation of dsRNA and subsequent destruction of both transcripts in spite of the presence of complementary RNA chains in the cell nucleus