To identify new genes functionally involved in erythropoiesis during development and maturation, we analysed by DNA microarray three cell populations of different maturity during mouse fetal liver development (E11.5 - E13.5). Among genes whose expression increases in parallel with erythroid maturation there is Sox6, a member of the Sry–related transcription factors family. Sox6 is known to play a major role in erythropoiesis: its ablation in mouse causes a strong relative increase of the expression of embryonic (εy) versus other non-alpha globin genes in late gestation, and a high number of circulating nucleated and misshapen erythrocytes. To study the role of Sox6 in human erythropoiesis, we overexpressed it by lentiviral vector transduction both in the K562 erythroleukemic cell line and in human primary CD34+ cord blood cells . Sox6 induces significant differentiation in both models as shown by morphological and FACS analysis. Moreover, several erythroid specific transcripts are greatly increased , i.e. mRNAs for enzymes controlling the heme-biosynthetic pathway, for transcription factors and for all globins (although the ratio between epsilon and gamma-globin is decreased), suggesting that Sox6 is a general positive regulator of erythroid genes expression. Despite their erithroleukemic origin, K562 overexpressing Sox6 grow at a very low rate when compared with control cells, and die in culture within about ten days after transduction. Sox6 overexpression causes in fact a strong increase of SOCS3 (suppressor of cytokine-signaling) transcript, which is known to block Jak2 signalling. Since erythroblasts undergo cell-cycle withdrawal during terminal differentiation, our working hypothesis is that Sox6 may induce the erythroid terminal maturation program through a block in the cell-cycle progression, possibly by acting as a repressor of the Jak signalling pathway. We then used the Sox6 consensus from the εy-globin promoter to perform a bioinformatic genome-wide search for similar evolutionarily conserved motifs, and 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. Moreover, the binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by a strong downregulation of the endogenous Sox6 transcript. Finally, we demonstrated that Sox6 expression, in human erythroid cultures and in mouse bone marrow cell populations, peaks at the erythroblast stage and decreases along with erythroid differentiation. Together these observations suggest that the negative Sox6 autoregulation mediated by the double Sox6 binding site within its own promoter, might be relevant to control the Sox6 transcriptional downregulation observed in late erythroid maturation

(2009). The Sox6 transcription factor: its role in human and murine erythroid differentiation and mechanisms for its regulation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).

The Sox6 transcription factor: its role in human and murine erythroid differentiation and mechanisms for its regulation

CANTU', CLAUDIO
2009-11-26

Abstract

To identify new genes functionally involved in erythropoiesis during development and maturation, we analysed by DNA microarray three cell populations of different maturity during mouse fetal liver development (E11.5 - E13.5). Among genes whose expression increases in parallel with erythroid maturation there is Sox6, a member of the Sry–related transcription factors family. Sox6 is known to play a major role in erythropoiesis: its ablation in mouse causes a strong relative increase of the expression of embryonic (εy) versus other non-alpha globin genes in late gestation, and a high number of circulating nucleated and misshapen erythrocytes. To study the role of Sox6 in human erythropoiesis, we overexpressed it by lentiviral vector transduction both in the K562 erythroleukemic cell line and in human primary CD34+ cord blood cells . Sox6 induces significant differentiation in both models as shown by morphological and FACS analysis. Moreover, several erythroid specific transcripts are greatly increased , i.e. mRNAs for enzymes controlling the heme-biosynthetic pathway, for transcription factors and for all globins (although the ratio between epsilon and gamma-globin is decreased), suggesting that Sox6 is a general positive regulator of erythroid genes expression. Despite their erithroleukemic origin, K562 overexpressing Sox6 grow at a very low rate when compared with control cells, and die in culture within about ten days after transduction. Sox6 overexpression causes in fact a strong increase of SOCS3 (suppressor of cytokine-signaling) transcript, which is known to block Jak2 signalling. Since erythroblasts undergo cell-cycle withdrawal during terminal differentiation, our working hypothesis is that Sox6 may induce the erythroid terminal maturation program through a block in the cell-cycle progression, possibly by acting as a repressor of the Jak signalling pathway. We then used the Sox6 consensus from the εy-globin promoter to perform a bioinformatic genome-wide search for similar evolutionarily conserved motifs, and 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. Moreover, the binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by a strong downregulation of the endogenous Sox6 transcript. Finally, we demonstrated that Sox6 expression, in human erythroid cultures and in mouse bone marrow cell populations, peaks at the erythroblast stage and decreases along with erythroid differentiation. Together these observations suggest that the negative Sox6 autoregulation mediated by the double Sox6 binding site within its own promoter, might be relevant to control the Sox6 transcriptional downregulation observed in late erythroid maturation
OTTOLENGHI, SERGIO
Molecular biology, Hematopoiesis, Gene regulation
BIO/18 - GENETICA
English
Scuola di Dottorato in Medicina Traslazionale e Molecolare
MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 45R
22
2008/2009
(2009). The Sox6 transcription factor: its role in human and murine erythroid differentiation and mechanisms for its regulation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/8374
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