Abstract
Using nuclear extracts prepared from rat liver it was demonstrated that binding of a transcription factor to site II of the D-site binding protein promoter could be induced by dephosphorylation of these extracts. Competition band shifts and supershift assays reveal this protein to be the general transcription factor Sp1. Phosphorylation of Sp1 appears to occur as a result of terminal differentiation of the liver. Proteins from both 1-day-old rat liver and adult liver undergoing regeneration have less of the phosphorylated form of Sp1 present with consequent increased DNA binding activity. Sp1 is similarly phosphorylated in brain, kidney, and spleen with phosphatase treatment of the extracts significantly increasing the level of DNA binding activity. Dephosphorylation of Sp1 results in a 10-fold increase in the affinity of Sp1 for its cognate site. Two-dimensional gel electrophoresis reveals that approximately 20% of the detectable protein appears to be in the phosphorylated form in adult liver extracts. Another protein with similar characteristics also appears to be present in the liver. Decreasing Sp1 DNA binding activity by phosphorylation may be an important mechanism for regulating gene expression, and possibly bringing about growth arrest during terminal differentiation.
Highlights
The regulation of terminal differentiation is a complex process involving both the induction of specific genes and growth arrest of the differentiating cells
Sp1 has been implicated in the function of the retinoblastoma control element (RCE) (24, 25) and cotransfection of a retinoblastoma gene expression vector is able to modulate the transactivation of responsive genes by Sp1
In order to determine if post-translational modification, phosphorylation was responsible for these changes, dephosphorylation of normal liver nuclear extracts using calf intestinal alkaline phosphatase was carried out
Summary
The regulation of terminal differentiation is a complex process involving both the induction of specific genes and growth arrest of the differentiating cells. Sp1 is a member of the C2-H2 zinc finger family (12) and was one of the first transcription factors to be cloned (13) It is involved in the regulation of a wide variety of different genes, including the early promoter of SV40 (14), genes involved in proliferative response (15–17), extracelluar matrix protein genes (18, 19), housekeeping genes (20 –22), and a number of growth factor genes (17, 23). Sp1 has been implicated in the function of the RCE (24, 25) and cotransfection of a retinoblastoma gene expression vector is able to modulate the transactivation of responsive genes by Sp1 These findings have linked Sp1 function to that of the retinoblastoma protein, suggesting that Sp1 may be involved in modulating transcription in response to the growth state of the cell.
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