The general transcription initiation factor TFIID contains the TATA-binding protein (TBP) and TBPassociated factors (TAFs) implicated in the function of gene-specific activators. Previous studies have indicated that a hamster cell line (ts13) with a point mutation in the TAF II 250/CCG1 (TAF II 250) gene shows temperature-sensitive expression of a subset of genes and arrests in late G 1 at 39.5°C. Here, we report the identification of cell cycle-specific (G 1 -specific) genes that appear to be regulated directly through TAF II 250 both in vivo and in vitro. Transcription rates of several cell cycle-regulatory genes were determined by run-on assays in nuclei from ts13 cells grown at permissive (33°C) and nonpermissive (39.5°C) temperatures. Temperaturedependent differences in transcription rates were observed for cyclin A, D1, and D3 genes. In transienttransfection assays, the human cyclin D1 promoter fused to a luciferase reporter showed a temperaturedependent reduction in activity in ts13 cells but not in parental BHK cells. In in vitro assays, upstream sequence-dependent transcription from the human cyclin D1 promoter was significantly reduced in ts13 nuclear extracts preincubated at 30°C but not in similarly treated BHK nuclear extracts, and transcription in the ts13 extract was restored by addition of an affinity-purified human TFIID. Preincubation of the ts13 nuclear extracts did not affect the function of several GAL4-activation domain fusion proteins (GAL4-VP16, GAL4-p65, and GAL4-p53) on either the adenovirus major late or cyclin D1 core promoter bearing GAL4 sites, further indicating that the effect of the TAF II 250 mutation is both core promoter and activator specific.TFIID is a general initiation factor which binds to core promoter elements of class II genes and nucleates the assembly of RNA polymerase II and other general initiation factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) into a functional preinitiation complex (reviewed in references 6 and 53). Consistent with its key role in transcription initiation, TFIID was also implicated as a target for gene-specific activators in the earliest studies of activation mechanisms. These studies showed both qualitative and quantitative effects of activators on TFIID binding that in turn correlated with enhanced recruitment and function of other general initiation factors (1,23,34,71). Resolution of the polypeptide structure of TFIID, comprised of both a small TATA-binding polypeptide and a large number (Ͼ13 in human) of TATA-binding protein (TBP)-associated factors (TAFs) (reviewed in reference 6), has enhanced our understanding of the mechanisms involved. Various activators have been shown to interact directly with TBP or with specific TAFs (reviewed in references 6 and 67), altering either the binding of TFIID (56) or the conformation of the resulting promoter complex (23, 47). The significance of such interactions for activator-specific function has been indicated by a variety of approaches using mutated factors (17,26,28, 38) and partial TFIID complex...

Na,K-ATPase al subunit gene (ATPlA1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATPAL1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Spl binding site), from -102 to -61, and from -58 to -49 (including an Spl consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATPL1A regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.

We have identified a positive regulatory cis-acting element of the adhesion molecule on glia (AMOG)/Na,K-ATPase beta 2 subunit gene as GAGGCGGGG at position -87 to -79 by transient transfection assay using B103 cells (rat neuroblastoma cell line). The newly identified AMOG regulatory element (AMRE) enhanced the promoter activity in a mutually compensating manner with the Sp1 element at position -147 to -142. AMRE acts as a positive regulatory element not only in B103 cells but also in 3Y1 (rat embryo cell line) cells to roughly the same extent and in MDCK (canine kidney cell line) cells to a lesser extent. AMRE also enhances other gene promoters, such as myelin basic protein (MBP) and herpes simplex virus (HSV) thymidine kinase (TK) gene promoters. The element is not a typical enhancer element because when it is introduced downstream of the HSV TK promoter, it has no enhancing activity.

No abstract

No abstract