The mammalian pRb/E2F complex has been reported to recruit chromatin remodeling enzymes, including Rpd3-type HDACs, to actively repress transcription of genes involved in G1/S transition during cell cycle progression (Harbour, JW and Dean, DC, Genes Dev 14:2393-2409, 2000). To investigate whether the maize Rpd3-type histone deacetylase, ZmRpd3, (Rossi, V, et al., Mol Gen Genet 258:288-296, 1998) and the maize retinoblastoma-related protein, ZmRBR, can repress transcription, we prepared a set of effector plasmids, in which either the complete ZmRpd3I coding sequence or the ZmRBR1 large pocket region were fused to the DNA-binding domain of the tetracycline repressor (TetR). The expression of these chimeric genes was driven by the 35S constitutive promoter of the cauliflower mosaic virus (CaMV). The TET system is based on the targeting of TetR-fused proteins to a synthetic promoter containing four tet operators upstream of a minimal promoter (TAX promoter, Bohner, S, et al., Plant J 14:87-95, 1999). Our experimental strategy was based on the following considerations. First, the reporter plasmid, containing the b-glucuronidase gene (GUS) under the control of the TAX promoter was stably integrated into the tobacco genome (tobacco TAX transgenic lines) allowing formation of a natural chromatin structure. Second, analysis of the repression activity was carried out by means of competition assays, in which tobacco protoplasts from TAX lines were co-transformed with a TetR-fusion of the strong viral activator VP16 together with one of the TetR-fusion plasmids expected to repress transcription. To avoid heterodimerization of the TetR domains, which might interfere with VP16 transactivation efficiency, we employed different TetR DNA-binding domains unable to heterodimerize (TetR-B for VP16 fusions and TetR-B/E for the other TetR-fusions, respectively (Forster, K, et al., Nucl Acids Res 27:708-710, 1999). Third, the effect on transcriptional activity of the GUS gene, due solely to competition for binding the TET operators, was assessed by co-transforming TetR-B-VP16 (TET-VP16) and a TetR-B/E-fused maize zein (TET-zein), a structural protein unable to control transcriptional activity (Spena, A, et al., J Mol Biol 169:799-811, 1983). Therefore, evaluation of transcription repression activities of ZmRpd3I/ZmRBR1 TetR-fusion proteins was achieved by comparison with results obtained from TetR-B/E-zein transformed protoplasts.

The results of these experiments showed that VP16 strongly activated expression of the GUS gene, which was only slightly reduced by the presence of competing TetR-zein. Conversely, a plasmid carrying the ZmRpd3I TetR-fusion strongly reduced GUS activity. Furthermore, both a mutation in histidine 153, believed to be critical for HDAC activity, as well as treatment with the HDAC inhibitor TSA, were able to relieve the repression activity of ZmRpd3I. These results indicate that ZmRpd3I is likely to use its HDAC activity to repress transcription when artificially targeted to a promoter within a chromatin context. A negative effect on gene transcription was also observed for the ZmRBR1 TetR-fusion, although it was lower with respect to ZmRpd3I and almost insensitive to TSA treatment. Significantly, GUS activity was reduced to background levels by the simultaneous presence of TET-RBR and CaMV-Rpd3, a plasmid expressing ZmRpd3I not fused to the TetR DNA-binding domain. In this case, TSA relieved the effect of ZmRBR1 on GUS activity repression to a level observed with ZmRBR1 alone. The introduction of the C653F mutation, which affects A/B pocket integrity, resulted in a complete loss of transcription repression activity regardless of whether CaMV-Rpd3 was present or not. These findings suggest that ZmRBR1 possesses an HDAC-independent capacity to repress gene transcription, which relies on the integrity of the A/B pocket. However, our results also indicate that the ability of ZmRBR1 to recruit a ZmRpd3I-related HDAC enhances its effect in repressing gene transcription. The integrity of the A/B pocket domain is also a prerequisite for this repression mechanism. Finally, we observed that similar results were obtained when the reporter plasmid (pUC-TAX-GUS/INT) was co-transformed into protoplasts prepared from tobacco wild type plants together with the above described effector plasmids. In this last experiment the reporter plasmid may not adopt a complete chromatin structure suggesting that non-histone proteins may also be ZmRBR1/ZmRpd3I substrates.
 
 

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