The Myc Intron-Binding Polypeptide Associates with RFX1 In Vivo and Binds to the Major Histocompatibility Complex Class II Promoter Region, to the Hepatitis B Virus Enhancer, and to Regulatory Regions of Several Distinct Viral Genes

Abstract

We demonstrated that MIF-1, identified initially as a binding activity that associated with the intron I element of the c-myc gene, consists of two polypeptides, the myc intron-binding peptide (MIBP1) and the major histocompatibility class II promoter-binding protein, RFX1. Using a polyclonal antiserum directed against either oligonucleotide affinity-purified MIBP1 or a peptide derived from RFX1, we showed that MIBP1 and RFX1 are distinct molecules that associate in vivo and are both present in DNA-protein complexes at the c-myc (MIF-1) and major histocompatibility complex class II (RFX1) binding sites. We have also found that MIBP1 and RFX1 bind to a regulatory site (termed EP) required for enhancer activity of hepatitis B virus. In addition, we have identified MIF-1-like sequences within regulatory regions of several other viral genes and have shown that MIBP1 binds to these sites in cytomegalovirus, Epstein-Barr virus, and polyomavirus. We have also demonstrated that the MIF-1 and EP elements can function as silencers in the hepatocarcinoma HepG2 and the cervical carcinoma HeLa cell lines. These findings indicate that MIBP1 and EP/RFX1 can associate in vivo and may regulate the expression of several distinct cellular and viral genes.The product of the c-myc oncogene has been implicated in diverse cellular processes, including cell proliferation, differentiation, and tumorigenesis. The observation that c-Myc can heterodimerize with other cellular proteins to transactivate gene expression in vitro has suggested that c-Myc may function to modulate genes involved in cell growth and differentiation pathways (3,4,14). The regulation of the c-myc gene involves a complex interplay of cis-and trans-acting elements, and several nuclear proteins that interact with regulatory sequences found in the 5Ј upstream and exon I untranslated regions of the c-myc gene have been identified (16,31). In addition, a 20-bp region of intron I was defined as a binding site for a phosphoprotein, initially designated MIF-1, and it was demonstrated that binding to this site was abolished by a point mutation present in the corresponding region of a translocated Burkitt's lymphoma c-myc gene (34,35). Adjacent binding sites in c-myc intron I were also identified and designated MIF-2 and MIF-3 (33), and somatic mutations found in Burkitt's lymphoma samples were shown to be frequently clustered within discrete domains that define these recognition sequences (33). These findings suggested that the mutations clustering in this region of the c-myc gene in Burkitt's lymphoma may be targeting specific regulatory elements for c-myc regulation, although direct evidence for this is lacking.It was recently demonstrated that the RFX1 protein, which binds to the X box of the major histocompatibility complex (MHC) class II genes, the methylated DNA-binding protein (MDBP), and the EP protein, which binds to the enhancer of hepatitis B virus (HBV), all represent the same DNA binding activity (29,36). The X-box sequence has been shown to play a role in regula...

The Epstein-Barr Virus Promoter Initiating B-Cell Transformation Is Activated by RFX Proteins and the B-Cell-Specific Activator Protein BSAP/Pax5

“…2B demonstrate that the formation of site C complexes in the presence of both DG75 (B-cell) and Jurkat (T-cell) nuclear extracts was inhibited by the addition of the HBV enh-I, MIF-1, and HLA DRA X competitor oligonucleotides, as well as by addition of a site C competitor itself. In contrast, the mutant sequences HBV enh-I m and MIF-1 m, previously reported to have lost RFX/MIBP1 binding (45), and the mutant site C sequence m1 (6) were unable to compete. These findings provide the first evidence that site C is indeed a functional RFX binding site.…”

“…2C). Interestingly, similar complexes have been observed in in vitro binding assays with several other viral enhancer sequences, notably from cytomegalovirus, polyomavirus, and HBV, and in the latter two cases there is direct functional evidence that such binding has a regulatory role (7,10,45,46,51). However, in contrast to the polyomavirus and HBV enhancers, where activation requires RFX binding to the full inverted repeat sequence (10), it appears that only the 5Ј half of Wp site C needs to be conserved both for RFX complexes to form and for optimal Wp activity to be maintained (Fig.…”

“…Of these, RFX1, RFX2, and RFX3 have been implicated in the regulation of viral and cellular genes and shown to bind target sites as both homodimers and heterodimers (22,31,45,46,48,50,51), while RFX5 specifically associates with the promoters of HLA class II genes as a complex with two other proteins, RFX-AP and RFX-ANK/ RFX-B (12,34,37,55). In addition, another cellular factor, MIBP1 (myc intron binding factor 1), first identified as a regulator of c-myc gene expression (65), has been implicated in the regulation of RFX-dependent promoters (45) and can form heterodimers with at least one member of the RFX family, RFX1 (7).…”

A complete screening of the IL4 gene

“…17 The regulatory factor X1 complex was shown to bind to the promoter region of MHC class II genes and to be required for distinct promoter and enhancer-repressor activities in several viral genes. 18 Furthermore, binding of Myb proteins to the promoter of the Ig switch region might play an important role in the regulation of IL4-dependant germline transcription. 19 Thus all 3 transcription factors putatively interacting with the conserved sequence of the variable element found in intron 3 of the IL4 gene have thus far been associated with regulatory mechanisms in which IL-4 might also play a role.…”

The kinase activity of c-Abl but not v-Abl is potentiated by direct interaction with RFXI, a protein that binds the enhancers of several viruses and cell-cycle regulated genes

“…Obviously, the most immediate aim is to de®ne the role of the kinase domain in the transcription machinery. In contrast to the positive role of EP in viral enhancers, the EP-DNA element, as a multimer and an isolation, does not activate transcription (Ostapchuk et al, 1989;Dikstein et al, 1990;Reinhold et al, 1995;Labrie et al, 1995;Safrany and Perry, 1995;Katan et al, 1997). RFX1 itself contains several independent functional domains.…”