The zinc finger transcription factor, MOK2, negatively modulates expression of the interphotoreceptor retinoid-binding protein gene, IRBP.

The human and murine MOK2 orthologue genes encode Krüppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain as MOK2, we have suggested that PAX3 and IRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention on IRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3'-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.

A gene that encodes a protein consisting solely of zinc finger domains is preferentially expressed in transformed mouse cells.

We describe the cloning and characterization of the mouse MOK-2 gene, a new member of the Krüppel family of zinc finger proteins. Sequencing of both cDNA and genomic clones showed that the predicted MOK-2 protein consists of seven zinc finger domains with only five additional amino acids. The finger domains of MOK-2 are highly homologous to one another but not to those of other zinc finger proteins. MOK-2 is preferentially expressed in transformed cell lines, brain tissue, and testis tissue. Its possible role in cellular transformation is discussed.

Contribution of different GC-motifs to the control of simian virus 40 late promoter activity.

During the course of lytic infection the 21-bp repeat region regulates differentially the late gene expression; a mutant deleted for this region expresses late genes either to a higher level in the absence of T antigen or to a lower level in the late phase of infection as compared to wild type (23). By analysing a series of clustered point mutations generated within the GC-motifs we show that i) mutations within motifs I and II stimulate late transcription two to three-fold, suggesting that competition for transcription machinery between early-early and late promoters is mediated by these two motifs, ii) after viral replication, simultaneous mutations within motifs IV, V and VI decrease to 23% the efficiency of late transcription, indicating that these motifs are elements of the late promoter. Moreover comparison of results presented in this paper with results published by Barrera-Saldana et al. strongly suggest that late-early and late promoters are regulated in a similar manner.

The sequence motifs that are involved in SV40 enhancer function also control SV40 late promoter activity.

The simian virus 40 (SV40) enhancer element is constituted of two domains which contain sequences important for late transcription (M. Ernoult-Lange, F. Omilli, D. O'Reilly and E. May, J. Virol. 61, 167-176, 1987). By analysing a series of clustered point mutations generated throughout the enhancer region we mapped domain I from nt 232 to 272 and domain II from nt 184 to 216. These two domains which are required for late promoter activity both in the presence and in the absence of T antigen correspond closely to the domains B and A respectively, identified for enhancer function (M. Zenke, T. Grundström, H. Matthes, M. Wintzerith, C. Schatz, A. Wildeman and P. Chambon, EMBO J., 5, 387-397, 1986). Similarly to the enhancer function the late promoter elements defined by these two domains contain multiple sequence motifs. Moreover there is a striking overlap between the sequence motifs within domain A, active for early enhancer function and those within domain II involved in efficient late transcription.

SV40 late promoter: contribution of the initiation site sequences to basal late promoter activity.

We have previously shown that the +7 to -53 element of the SV40 late promoter (nt 273 to nt 332) does not have any promoter activity, but is able to stimulate the late promoter activity of the enhancer element. The +7 to -53 element contains several late transcriptional initiation sites and we have shown that its removal results in an increase in the heterogeneity of initiation sites. Furthermore we found that inversion of the +7 to -53 element does not adversely affect the efficiency of late transcription. However, when the +7 to -53 element was inverted, transcription initiated from a single site at nt 302. In fact, we noticed that there is a consensus TATA box signal 26 nt upstream of this single site in the inverted +7 to -53 element. These results may indicate that the ability of +7 to -53 element to function in both orientations is due to the fact that, in both orientations, it possesses sequences capable of fixing the initiation sites of transcription.

Characterization of the simian virus 40 late promoter: relative importance of sequences within the 72-base-pair repeats differs before and after viral DNA replication.

We examined sequences involved in the simian virus 40 (SV40) late promoter in vivo, by using quantitative S1 nuclease analysis of a series of deletion mutants within the SV40 regulatory region. These mutants were constructed so as to place the altered promoter region in its normal position relative to the SV40 late genes. The effects of the deletions on late transcriptional activity were analyzed before and after viral DNA replication, by omitting or including SV40 large T antigen. The data show that (i) in the absence of large T antigen, the deletion of the 21-base-pair (bp) repeats results in a fourfold increase in late transcription, and (ii) the sequences within the 72-bp repeats are a component of the SV40 late promoter, acting not only before, but also after viral DNA replication. We identified two domains which contain sequences important for efficient late transcription. Domain I, at the late proximal end of each 72-bp repeat, was found to function before replication and was possibly also involved after replication. The contribution of domain II, at the late distal end of each 72-bp repeat, was much more significant after replication but only of minor importance before replication.

Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen.

We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G-C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication.

Simian virus 40 late promoter region able to initiate simian virus 40 early gene transcription in the absence of the simian virus 40 origin sequence.

To improve our knowledge of the simian virus 40 (SV40) late promoter control region, we took advantage of the fact that T antigen can be expressed with a heterologous promoter. We constructed three chimeric plasmids (pEMP-273, pEMP-LCAP-162, and pEMP-LCAP-113) each with a putative late promoter sequence positioned immediately upstream from the SV40 early gene coding region but in an orientation opposite to its natural orientation in the SV40 genome. After transfection of the recombinant DNA into HeLa or CV1 cells, T antigen accumulation, as scored by indirect immunofluorescence, was used as a functional test for promoter activity. We found that the sequence mapping from nucleotides 332 to 273 is not sufficient for promoting transcription of SV40 early gene but does potentiate the promoter activity of the 72-base-pair repeats in initiating the transcription at natural late cap sites. Considering that both plasmids pEMP-LCAP-162 and pEMP-LCAP-113 lack all of the sequence of the SV40 replication origin, we conclude that SV40 transcription can be mediated through a putative late promoter in the absence of the sequence for the SV40 replication origin.

Evidence of transcription from the late region of the integrated simian virus 40 genome in transformed cells: location of the 5' ends of late transcripts in cells abortively infected and in cells transformed by simian virus 40.

By means of S1 mapping, we observed that spliced 16S and 19S viral late mRNAs--in addition to early mRNAs--were present in cytoplasmic polyadenylated RNA preparations from simian virus 40-transformed cell lines of rat or mouse origin containing no detectable amount of free viral DNA. The amounts of early and late virus-specific mRNAs in these lines were quantified by hybridization of radioactive cytoplasmic polyadenylated RNA with cloned region-specific restriction fragments. The relative amount of late viral mRNA produced in these transformed cells was found to be of the same order as that produced in simian virus 40-infected, nonpermissive baby mouse kidney cells. Moreover, by using the S1 nuclease protection method, we compared the 5' ends of late mRNAs produced (i) in transformed cells, (ii) in abortively infected mouse cells, and (iii) in the late phase of the lytic cycle. The 5' ends of late mRNAs both in abortively infected and in transformed cells were less heterogeneous than the 5' ends of late mRNAs produced during the lytic cycle; however, they were a subset of the 5' ends of late transcripts produced in the lytic cycle.