We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

There are two hypotheses explaining a fulminant outcome after hepatitis B virus (HBV) infection, both of which may be applicable at the same time: (i) basal core promoter (BCP) mutations increase viral replication, allowing rapid spread of the virus through the liver, and (ii) pre-core (pre-C) mutations abrogating hepatitis B e antigen (HBeAg) synthesis remove its tolerogenic effect, leading to a vigorous immune response. This study investigated the effect of these mutations on virus replication efficiency and HBeAg production. Substitutions A1762T/G1764A and T1753C, C1766T and T1768A in the BCP region, and G1896A and G1899A in the pre-C region, were examined either alone or in combination, using a common genetic background. Huh7 cells were transfected with these constructs and real-time PCR was used to quantify released virion-associated and intracellular HBV DNA, pregenomic RNA and pre-C mRNA. In addition, culture supernatants were tested for hepatitis B surface antigen (HBsAg) and HBeAg. The double BCP mutation (A1762T/G1764A) and the pre-C mutations (G1896A, G1899A), either alone or in combination, had no appreciable effect on the replication capacity of the virus. In contrast, clones with mutations at positions

Previous studies have shown that the cell cycle-regulated B-myb promoter contains a conserved E2F binding site that is critical for repressing transcription in quiescent cells. To investigate its significance for permanent promoter silencing, we have inactivated this binding site in the mouse genome. Mice homozygous for the mutant B-myb mE2F allele were fully viable, however, B-myb transcription was derepressed during quiescence in mouse embryo fibroblasts (MEFs) derived from mutant animals. Moreover, it was found that mutation of the E2F site resulted in abnormal maintenance of B-myb expression in senescent MEFs and in differentiated brain tissue. These findings therefore reveal a direct and primary role for repressive E2F complexes in silencing gene expression in post-mitotic cells. Analysis of histone modifications at the promoter showed that histone H3 lysine 9 was constitutively acetylated throughout the cell cycle in homozygous mutant MEFs. This mouse system is the first description of an E2F site mutation in situ and will facilitate the study of E2F function in vivo.

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Transcription from the B-myb (MybL2 gene) promoter is strictly cell cycle-regulated by repression mediated through an E2F site during G 0 /early G 1 . We report here the characterization of a corepressor site (downstream repression site (DRS)) required for this activity that is closely linked to the E2F site. Systematic mutagenesis of the DRS enabled a consensus to be derived, and it is notable that this sequence is compatible with cell cycle gene homology region sequences associated with cell cycle-dependent elements in the cyclin A, cdc2, and CDC25C promoters. The B-myb promoter is inappropriately active during G 0 in mouse embryo fibroblasts lacking the p107 and p130 pocket proteins, and we show that the ability of transfected p107 and p130 to re-impose repression on the promoter is dependent on the DRS. In contrast, transfected Rb was unable to repress the B-myb promoter. Consistent with the notion that Rb⅐E2F complexes are unable to bind the B-myb promoter E2F site in vivo, footprinting showed that this site is unoccupied in cells lacking p107 and p130. Chromatin immunoprecipitation assays showed a requirement for the DRS in recruiting p107 and p130 complexes to the B-myb promoter, indicating that in vivo the DRS governs the occupancy of the adjacent E2F site by transcriptional repressors.

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The A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound eects on the growth and/or dierentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the ®rst exon, part of the ®rst intron as well as upstream regulatory sequences. Multiple transcription start sites have been identi®ed which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (7183714), of a positive upstream (75387183) and a negative downstream regulatory region (NRE) (+83+374) have been de®ned. The NRE is promoter-and orientation-independent but position speci®c. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box eciently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Sp1 and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction. Oncogene (2000) 19, 3931 ± 3940.

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