Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

Figure 32.2. Deletion analysis of the b-PDE promoter suggests a regulatory DNA element in the -45 to -23 region. Serial 5¢-end deletion constructs of the -45 to +53 b-PDE promoter were transfected in Y79 retinoblastoma cells (light bars) and in ex vivo dissected Xenopus embryonic heads (dark bars). Luciferase activity was measured in cell lysates and normalized to the corresponding b-galactosidase activity for each sample. The results are expressed as percent of the mean activity produced by the -72 to +53 b-PDE construct ± standard deviation. Each transfection was done in triplicate and repeated several times. (Reprinted with permission from Lerner et al., J Biol Chem, 2002).

previously for studying the regulation of photoreceptor-specific gene expression, including that of b-PDE (Lerner et al., 2001). Luciferase activities were measured and normalized to the b-galactosidase activities obtained with a control plasmid in Y79 cells, or expressed per embryo and averaged statistically as described previously for Xenopus transfections (Batni et al., 2000). When the -45 to -23 region was deleted, further reduction in promoter activity was observed in both transfection systems (Figure 32.2). The activity level of the -23 to +53 promoter was not significantly different from that observed with the promoter-less control vector when tested in Y79 cells or Xenopus embryos. Luciferase activity remained low when the +4 to +53 promoter construct carrying further 5¢-end deletion past the major transcription start site was tested. High evolutionary conservation of the -45 to -23 region (Di Polo et al., 1996) that comprises the consensus Crx response element (CRE, -41/-36) and the T/A-rich sequence (b/TA) located at a consensus position for the TATA box is evident between mouse and human also suggesting its functional importance.

2.3. Functional Testing of Nucleotide Substitution Mutants in the Proximal Promoter and the 5¢-Untranslated Region of the b-PDE Gene in Neuroretina-Related Transfection Systems

The initial proximal promoter deletion analysis described above prompted us to investigate whether the putative CRE (-41/-36) and its flanking sequences in the b-PDE proximal promoter were functionally relevant to the transcriptional regulation of the b-PDE gene in vivo in the context of a retina-relevant environment. A series of b-PDE promoter mutants carrying small oligonucleotide substitutions within the -45 to -23 region was transiently transfected in Y79 retinoblastoma cells and also in Xenopus embryos maintained ex vivo

(summarized in Figure 32.3, top). Interestingly, the -41/-38 m mutation that completely disrupted the consensus CRE motif, had little effect on the b-PDE promoter activity in both transfection systems (Figure 32.3A). In addition, no significant changes were seen with mutations in the CRE flanking sequences (i.e. -37/-36 m, -35/-34 m and -33/32 m). The -30/-27 m mutant containing nucleotide substitutions in positions 2 through 5 of the T/A- rich b/TA sequence TAAGAAA to TCCTCAA) also showed no significant effect on promoter activity in transient transfections.

Although neither the mutations in consensus CRE or b/TA affected promoter activity, a cooperative interaction of transcription factors at both sites located in close proximity of each other could not be ruled out. Therefore, a double-mutant was constructed that contained both -30/-27 m and -41/-38 m. However, transient transfections of Y79 cells using the double-mutant showed no significant alterations in promoter activity compared to the wild type b-PDE promoter (Figure 32.3B). To search for other regulatory sequences in this TATA- and Inr-less gene, additional b-PDE promoter mutants (n = 14) containing nucleotide substitutions spanning the proximal 5¢-flanking and the 5¢-untranslated regions (-23 to +53; Figure 32.3, top) were tested in transient transfections of Y79 retinoblastoma cells. Promoter activity determined in these mutants ranged between approximately 0.5- and 1.5-fold that of the wild type control (Figure 32.3B). A 3¢-end deletion mutant (-72 to +4) lacking most of the 5¢-UTR showed approximately 3-fold reduction of promoter activity, which may be attributed to the deletion of certain translational control elements such as the Kozak sequence.

In summary, our results suggest that the b-PDE promoter may not have well-defined core elements responsible for basal transcription in Y-79 cells or Xenopus embryo heads. Rather, it appears that the transcription factors that interact with this region and mediate low-level b-PDE gene expression may not require a rigid sequence, but can accommodate a range of nucleotides.

2.4. TBP and TFIIB Bind the b-PDE Promoter

The possibility of an additional regulatory sequence(s) in the b-PDE basal promoter region or the 5¢-UTR is suggested by the tight regulation of the transcriptional initiation site selection in this gene. There are only one major and one minor transcription start sites in both human and murine b-PDE genes (Di Polo et al., 1996). This indicates the assembly of the basal transcription machinery at a specific core promoter element rather than random binding to a variety of sequences. Sequence analysis of the b-PDE promoter showed that there are no known consensus core promoter elements present in this gene. However, there is the b/TA sequence that has a high T/A content and is located in the close proximity of the transcription start site (-31TAAGAAA-25, which is the consensus location for the TATAbox element) of the b-PDE promoter. However, this b/TA sequence is quite different from the known functional TATA-box elements (TATAAA, consensus).

Thus, we tested whether the b/TA sequence was able to bind purified TBP separately or in complex with TFIIB in GMSAs. As a control, we compared the binding of TBP, TFIIB and the TFIIB-TBP combination to the AdML promoter. Shifted bands were observed with the addition of either TBP alone or TFIIB alone to the b/TA probe (Figure 32.4A). Addition of the combination of TBP and TFIIB resulted in a slower migrating band with about three-fold increase in band intensity compared to TBP alone, producing a characteristic supershifted pattern described previously for the AdML promoter (Wolner and Gralla, 2000).

Figure 32.4. TBP and TFIIB bind the b-PDE promoter as single proteins with cooperative enhancement of binding by TFIIB-TBP complex. The -45b/TA probe sequence comprising the -45/-16 region of the b-PDE promoter is shown at the bottom. In mutant competitors, nucleotide transversions were introduced into the -45/16 sequence. Electrophoretic mobility shift assays: A: In the control experiment (lane 1), protein was not included in the binding reaction. Purified TFIIB (lane 2), TBP (lane 3), or a combination of TFIIB-TBP (lane 4) was added to the binding reactions. In lane 5, the 200-fold molar excess of unlabeled -45b/TA oligonucleotide was added to the reaction mixture identical to that resolved in lane 4. B: Combination of TFIIB-TBP (lane 2) produces a typical supershifted complex with a 3-fold increase in intensity compared to TBP alone (lane 1). In lanes 3 and 4, a 20-fold and 200fold molar excess of the -31/-27 m mutant unlabeled competitor, and in lanes 5 and 6, a 20-fold and 200-fold molar excess of the -35/-27 m competitor were included in the same binding reaction as in lane 2. Retarded proteinDNA complexes are labeled with arrows. (Reprinted with permission from Lerner et al., J Biol Chem, 2002.)

These results suggest an enhanced cooperative binding by the TFIIB-TBP complex to the b-PDE promoter compared to TBP alone.

In contrast, when comparable protein concentrations were used, the AdML promoter interacted with TBP and TFIIB-TBP, but did not form a stable TFIIB-DNA complex in GMSA (data not shown), as previously demonstrated (Wolner and Gralla, 2000). Although, the addition of a 200-fold molar excess of the wild type -45/-16 competitor to the binding reaction prevented the shifted complex formation (Figure 32.4A, lane 5), the mutant -30/ -27 m and -35/-27 m competitors also showed substantial competition with the wild type sequence for TFIIB-TBP binding (Figure 32.4B). These results further corroborate our functional transfection data that a well-defined core promoter sequence could not be found in the b-PDE 5¢-flanking region.

2.5. The b-PDE Promoter is a Specific Target for Activation by the Sp4

Transcriptional Regulator

The most significant finding of the present investigation was the demonstration of the functional involvement of members of the Sp family in transcriptional regulation of the b-

Figure 32.5. Sp4 is a potent activator of the b-PDE gene promoter. The fold induction of the b-PDE or SV40 reporter constructs was determined relative to the uninduced reporter activity. A: The -93 to +53 minimal rodspecific b-PDE promoter (2 mg) was cotransfected with increasing amounts of Sp1, Sp3 and Sp4 plasmids and compared to the uninduced promoter contransfected with an empty plasmid. B: The SV40 promoter/luciferase vector (2 mg, pGL2-Control, Promega®) was cotransfected with 2 mg of the plasmid containing either Sp1, Sp3 or Sp4, and compared to an empty plasmid. Luciferase activity was measured in cell lysates and normalized to the corresponding b-galactosidase activity for each sample. The results are expressed as the fold induction of the mean activity of the uninduced -93 to +53 b-PDE reporter construct ± SD. (Reprinted with permission from Lerner et al., J Biol Chem, 2002.)

PDE promoter. Our previous investigations of promoter activity using transient transfections of multiple b-PDE promoter mutants, as well as protein-DNA binding studies using the b-PDE promoter sequences, have revealed the b/GC element (-55/-46) as an important enhancer of the b-PDE promoter that binds different transcription factors of the Sp family (Lerner et al., 2001). Members of the Sp family bind to GC-rich DNA sequences through three zinc finger motifs. The residues involved in the determination of the target site specificity and binding affinity are highly conserved between different family members. Recently, we compared different Sp proteins (Sp1, Sp3 and Sp4) that share similar DNA-binding characteristics (Hagen et al., 1992) for their effects on transcription from the b-PDE promoter. Interestingly, Sp1, Sp3 and Sp4 transcription factors showed differential effects on the b- PDE promoter activity. Wild-type minimal rod-specific b-PDE promoter (-93/+53, 2 mg) was transiently co-transfected with increasing amounts of expression plasmids (0.08 mg, 0.4 mg and 2 mg) each carrying a full-length cDNA for either Sp1, Sp3 or Sp4. Compared to other members of the Sp family, Sp4 was the only transcription factor that showed significant dose-dependent effect on the b-PDE promoter (Figure 32.5A). Promoter-specificity of the Sp4-mediated transactivation was confirmed by comparing its effect on transcription

from the b promoter (approximately 21-fold enhancement) to that on the SV40 promoter (no significant change) relative to the uninduced transcription, respectively (Figure 32.5B). The maximum activation was observed using 2 mg of pRC/CMV-Sp4 without any further increase in promoter activity using 5 mg of pRC/CMV-Sp4, indicating a saturation effect. In contrast, neither Sp1 nor Sp3 showed any significant effect on transcription from the b-PDE promoter.

3. POST-TRANSCRIPTIONAL STUDIES

Regulation of expression of PDE subunits is also controlled at the post-transcriptional level. We examined the retinal steady-state mRNA and protein levels, protein biosynthesis rate, as well as the translational efficiency of rod-specific cGMP-phosphodiesterase. Our findings indicated that in mouse retina the number of mRNA molecules for b-PDE is approximately 5 times higher than that for a-PDE and that the levels of a-PDE and b-PDE transcripts in 10-day-old mice are approximately 85% of those in 30-day-old animals. The relative concentrations of two endogenously expressed b-PDE transcripts differing by the length of their 5¢-UTR are similar in the developing and adult retina. At the protein level, a-PDE and b-PDE show equimolar expression in retinas of 10and 30-day-old mice. Furthermore, we observed similar turnover rates for both subunits through pulse-chase experiments. The discordance between the mRNA and protein levels suggested that PDE expression is regulated post-transcriptionally and most likely at the translational level that is generally accepted to modulate the global synthetic activity of the cell.

To investigate whether the production of equal amounts of a-PDE and b-PDE from different amounts of the corresponding mRNAs was controlled at the level of translation, we determined the translational efficiency of a-PDE and b-PDE mRNAs and examined the role of their 5¢ and 3¢ UTRs as well as coding regions in the regulation of protein synthesis. Using constructs containing the full-length cDNA for a-PDE or b-PDE we were able to conclusively demonstrate that a-PDE mRNA is translated approximately 5 times more efficiently than its b-PDE counterpart (Figure 32.6).

Thus, our results indicated that the low level of a-PDE mRNA found in retina is counterbalanced by its efficient translation. These data also point at possible regulation of PDE expression in photoreceptor cells by the feedback mechanism: protein synthesis efficiency dictates the level of mRNA transcription.

After determining the translational efficiency of a-PDE and b-PDE mRNAs, we investigated which factors contribute to their differential translation. Since protein synthesis is controlled primarily at the initiation step and is generally dependent on the structural properties of individual mRNAs, we evaluated the role of the 5¢ and 3¢ UTRs, known to be involved in the regulation of protein synthesis (Day and Tuite, 1998; Kozak, 1987; Kozak, 1997; Geballe and Morris, 1994), as well as the coding sequences of a-PDE and b-PDE mRNAs on this process. Sequence analysis revealed the presence of an upstream AUG and the absence of a “strong” initiation sequence in the 5¢ UTR of b-PDE mRNA (Piri et al., 2003). In contrast, the a-PDE mRNA has a consensus translation initiation sequence and no upstream AUG. Furthermore, neither of the 5¢ UTRs of a-PDE and b-PDE mRNAs contains stable secondary structures that can reduce the rate of protein synthesis. On the basis of these analyses we hypothesized that these differences could account for the lower protein synthesis efficiency of b-PDE. Indeed, when we mutated the upstream AUG and restored

Figure 32.6. In vitro translation of full-length a-PDE and b-PDE cDNAs. Ia-IVa: The different regions of a-PDE and b-PDE mRNAs are shown in the constructs as: hatched boxes, a-PDE 5¢ and 3¢ UTRs; open box, a-PDE coding region; filled boxes, b-PDE 5¢ and 3¢ UTRs; grey box, b-PDE coding region. Mutations in the translation initiation sequence and upstream AUG are depicted. Ib-IVb: Autoradiographs and Ic-IVc: quantitative analysis of the in vitro synthesized a-PDE and b-PDE proteins.

the consensus translation initiation sequence in the b-PDE 5¢ UTR, the protein production was increased. However, using several chimeric constructs we demonstrated that, in fact, the 5¢ UTR of a-PDE leads to lower protein synthesis than the 5¢ UTR of b-PDE mRNA (Figure 32.6). Therefore, the differential translation of the PDE subunits cannot be solely explained based upon the primary or secondary structures of their 5¢ UTRs. Multiple examples describe the involvement of cis-elements within the 3¢ UTR in translational regulation and the mechanisms by which these elements influence protein synthesis (Jackson and Standart, 1990; Conne et al., 2000; Stuart et al., 2000). We demonstrated that both a-PDE and b-PDE 3¢ UTRs have a stimulatory effect on translation. The results of our studies undoubtedly implicate the involvement of the coding regions in the differential translation of a-PDE and b-PDE mRNAs. All eight constructs containing the b-PDE coding region resulted in lower protein expression than the four constructs containing the a-PDE coding region, regardless of the flanking 5¢ or 3¢ UTR.

In summary, our results indicate that the low level of a-PDE mRNA found in retina can be compensated by its more efficient translation to achieve equimolar expression with b-PDE. Moreover, the a-PDE and b-PDE coding regions are involved in the differential expression of these subunits, with the former producing more protein than the latter.

Figure 32.7. Schematic model of the molecular events required for rod-specific transcription from the minimal -93 to +53 b-PDE promoter. Functionally relevant DNA response elements in the b-PDE promoter are represented by tall graphic figures, whereas consensus binding sequences for known transcriptional regulators that do not affect transcription from this promoter are shown as narrow rectangles. Nucleotides are numbered relatively to the major transcription start site at +1. Potential protein-DNA interactions are shown as vertical arrows, whereas their functional effects on promoter activity are represented by semicircular arrows. Basal transcription factors TBP and TFIIB may interact with the b-PDE promoter and their higher affinity cooperative binding is indicated as a hatched double-arrow. (Reprinted with permission from Lerner et al., J Biol Chem, 2002.)

4. CONCLUSIONS

Sp4 is the least characterized member of the Sp family of transcriptional regulators, possibly because of its restricted pattern of expression that is predominant in the central nervous system. Here, we demonstrate that the rod-specific b-PDE gene represents the first natural target gene for Sp4. In addition, the b-PDE promoter seems to lack transcriptional regulation by the other related members of the Sp family, Sp1 and Sp3. The fact that Sp4 is able to specifically transactivate the b-PDE promoter supports our finding of this relatively restricted protein, compared to the ubiquitous Sp1 or Sp3, being abundantly expressed in the mammalian retina. The lack of other known specific Sp4 targets, combined with its regulation of a rod-restricted b-PDE gene, implies that this transcription factor functions in a very narrow cell type-specific manner.

In addition, Sp4 could have a more universal role in cell-specific expression of certain genes in rods and possibly other retinal cell populations by interacting with different arrays of transcription factors. We have previously shown that another nuclear factor, Nrl, regulates transcription from the b-PDE promoter (Lerner et al., 2001). Considering the additional b-PDE transcriptional mechanism described in this chapter and those from our previous studies, we can suggest that a unique combination of molecular interactions may be required for rod-specific transcription from this TATAand Inr-less promoter (Figure 32.7). This model is consistent with the combinatorial principles of transcriptional regulation of cell-specific gene expression.

Finally, the expression of PDE subunits is also regulated at the post-transcriptional level, with a-PDE mRNA translated approximately 4.1- and 5.5-fold more efficiently than b-PDE short and long transcripts, respectively. This indicates that the low level of a-PDE mRNA

found in retina can be compensated by its more efficient translation to achieve equimolar expression with b-PDE.

5. ACKNOWLEDGEMENTS

We wish to thank Ms. Lisa Mohan and Dr. Silvia Reid for their invaluable assistance in the preparation of this manuscript. This work was supported by National Institutes of Health grants EY02651 (DBF) and EY00367 (LEL), and grants from The Foundation Fighting Blindness (DBF). DBF is the recipient of a Research to Prevent Blindness Senior Scientific Investigators Award.

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