Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.

Abstract

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and Hpal sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic LAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease Im footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.Site-specific promoter methylation causes gene inactivation in a number of viral and nonviral eucaryotic systems (for reviews, see references 10, 11, and 36). For example, in vitro methylation at specific sites interferes with expression of the a-globin gene (7, 37), the herpes simplex thymidine kinase gene (5), and the adenine phosphoribosyltransferase gene (26). DNA methylation has been implicated in the regulation of intracisternal A-particle (IAP) gene expression. Expression of IAP proviral elements in various normal and transformed mouse cells is inversely correlated with their methylation state (22,29,34) (Fig. 1). We show here by site-directed mutagenesis studies that only two of these sites (sites b and c; Fig. 1) are involved in the methylation effect.Methylation may influence LTR function by altering the interaction between one or more regulatory proteins and their binding sites. In this study, we show that the exonuclease III footprint of the HhaI-methylated LTR in the presence of crude nuclear extract is perturbed over one of the methylation sites (site c; Fig. 1). We also show that a purified DNA-binding protein (EBP-80) specific for the LTR region containing this site fails to bind to an oligonucleotide containing the methylated form of the HhaI site c. The MIA14 LTR methylated at the HhaI site c is a less effective * Corresponding author.promoter of transcription in vitro than is its unmethylated counterpart and responds poorly to added EBP-80. These results provid...

Ku Represses the HIV-1 Transcription

“…Furthermore, it has been suggested that Ku may act as a transcriptional repressor in the context of the expression of retroviral elements either endogenous such as the intracisternal A particle (20) or exogenous as the GR strain of MMTV (36) and the HTLV (21). Although, to our knowledge, a putative role for Ku in lentiviral, and in particular for HIV-1 transcription, has not been considered to date, we noticed that the expression of a reporter gene was increased in Ku80-deficient cells that had been transduced with an HIV-derived vector in the course of a study addressing a possible role of Ku during the early steps of HIV-1 replication (25).…”

“…Second, Ku may recruit and activate the kinase at specific sites (4). Putative Ku-specific binding sites were proposed in a variety of genes such as c-myc r (16), the transferrin receptor (17), collagen III (18), the U1 snRNA (19), and also in retroviral sequences, notably in the long terminal repeat (LTR) sequences of intracisternal A particle (20), HTLV-1 (21,22), and mouse mammary tumor virus (MMTV) (4). In the latter two cases, these sequences were suggested to be involved in the negative regulation of transcription.…”

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

“…22 Biochemical analyses of Ku demonstrated that it bound in vitro to DNA termini in the DNA structure without apparent sequence specificity. [23][24][25] Ku has also been shown to possess DNA-dependent adenosine triphosphatase 26 and helicase 27 activities. In vitro, heterodimerization of active Ku requires the cotranslation of both subunits.…”

The Intracisternal A-Particle Proximal Enhancer-Binding Protein Activates Transcription and Is Identical to the RNA- and DNA-Binding Protein p54nrb/NonO

“…The cis elements and their interacting transcription factors identified so far include the Enh1 and Enh2 elements located between nucleotides (nt) Ϫ164 and Ϫ110, which bind to EBP-80 of murine myeloma cells (13,14), and sites located at the 5Ј end of the LTR between nt Ϫ210 and Ϫ168, which bind to 28-and 46-kDa proteins of murine PCC3 embryonal carcinoma (EC) cells (40). In our analyses of the mechanism by which IAP expression becomes activated, we have found that IAP gene expression in murine F9 EC cells, which resemble early embryonic cells, is restricted, whereas upon differentiation of these cells into parietal endoderm-like cells, IAP expression becomes highly activated.…”