Abstract
When murine erythroleukemia (MEL) cells are induced to differentiate by hexamethylene bisacetamide (HMBA), erythroid-specific genes are transcriptionally activated; however, transcriptional activation of these genes is severely impaired in cAMP-dependent protein kinase (protein kinase A)-deficient MEL cells. The transcription factor NF-E2, composed of a 45-kDa (p45) and an 18-kDa (p18) subunit, is essential for enhancer activity of the globin locus control regions (LCRs). DNA binding of NF-E2 and alpha-globin LCR enhancer activity was significantly less in HMBA-treated protein kinase A-deficient cells compared to cells containing normal protein kinase A activity; DNA binding of several other transcription factors was the same in both cell types. In parental cells, HMBA treatment and/or prolonged activation of protein kinase A increased the amount of NF-E2.DNA complexes without change in DNA binding affinity; the expression of p45 and p18 was the same under all conditions. p45 and p18 were phosphorylated by protein kinase A in vitro, but the phosphorylation did not affect NF-E2.DNA complexes, suggesting that protein kinase A regulates NF-E2.DNA complex formation indirectly, e.g. by altering expression of a regulatory factor(s). Thus, protein kinase A appears to be necessary for increased NF-E2.DNA complex formation during differentiation of MEL cells and may influence erythroid-specific gene expression through this mechanism.
Highlights
From the Wepartment of Medicine, University of California at San Diego, La Jolla, California 92093-0652 and the "IIHoward Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115
Transient Transfection of Erythroid Promoter / Enhancer Constructs into Parental and Protein Kinase A-deficient murine erythroleukemia (MEL) Cells-Since transcriptional activation of several erythroidspecific genes by hexamethylene bisacetamide (HMBA) is reduced in protein kinase A-deficient MEL cells compared to parental cells [5], we examined the activity of erythroid promoter/enhancer constructs in transient transfection assays in these cells
When paCAT was transiently transfected into MEL cells, chloramphenicol acetyltransferase (CAT) expression was the same in cells with severely reduced protein kinase A activity (PKI/C2 and RrmutlC3) as in cells with normal protein kinase A activity, and HMBA increased CAT expression 2- to 3-fold in both cell types (Fig. 1, hatched and cross-hatched bars represent cells cultured in the absence or presence of HMBA, respectively)
Summary
From the Wepartment of Medicine, University of California at San Diego, La Jolla, California 92093-0652 and the "IIHoward Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115. Paired in HMBA-induced differentiation, and that HMBA-induced increases in mRNA expression and transcription rates of several erythroid-specific genes are reduced in protein kinase A-deficient cells in proportion to their residual protein kinase A activity; mRNA expression and transcription rates of several other differentiation-associated and housekeeping genes are similar in HMBA-treated parental and protein kinase A-deficient cells [4, 5] These data suggest that protein kinase A is involved in regulating genes with erythroid-specific promoters during differentiation of MEL cells. The erythroid-specific transcription factor NF-E2 recognizes the sequence (T/C)GCTGA(G/C)TCA(CIT) found at DNase hypersensitive sites of the globin locus control regions (LCRs) and in the promoters of several erythroid genes [612] It is a member of the basic leucine zipper family of transcription factors and binds DNA as a dimer of a 45-kDa tissue-specific protein (p45) and an 18-kDa ubiquitously expressed protein (pI8) [8, 9,11]. Within the NF-E2 recognition site is the sequence TGA(G/C)TCA which is recognized by the transcription factor AP-l (Fos/Jun); several findings indicate that NF-E2 is the functional activator binding at the NF-E2/AP-l recognition sites of the globin LCRs and the porphobilinogen deaminase promoter, and that p45 is required for globin gene expression during erythroid cell differentiation [13,14,15,16,17,18]
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