Positive Regulation of Minichromosome Maintenance Gene Expression, DNA Replication, and Cell Transformation by a Plant Retinoblastoma Gene

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Positive regulation of minichromosome maintenance gene expression, DNA replication, and cell transformation by a plant retinoblastoma gene

Paolo A. Sabellia,1, George Hoersterb, Lucina E. Lizarragaa,2, Sara W. Browna, William J. Gordon-Kammb, and Brian A. Larkinsa,1

aDepartment of Plant Sciences, University of Arizona, 303 Forbes Hall, Tucson, AZ 85721; and bPioneer Hi-Bred International, Inc., Johnston, IA 50131

Contributed by Brian A. Larkins, January 6, 2009 (sent for review October 29, 2008) Retinoblastoma-related (RBR) genes inhibit the cell cycle primarily papillomavirus E7, and certain plant viral proteins, such as wheat by repressing adenovirus E2 promoter binding factor (E2F) tran- dwarf virus RepA (16–19), interact with the A/B pocket domain scription factors, which drive the expression of numerous genes through a conserved LxCxE motif (20); thus, disrupting the required for DNA synthesis and cell cycle progression. The RBR-E2F ability of RBRs to bind and repress E2F transcription factors. pathway is conserved in plants, but cereals such as maize are Also, mutation of key residues essential for pocket conformation characterized by having a complex RBR gene family with at least 2 can disrupt RBR activity (21). One such mutation involves the functionally distinct members, RBR1 and RBR3. Although RBR1 has cysteine at position 706 in human RB (22–25). In maize, the a clear cell cycle inhibitory function, it is not known whether RBR3 equivalent cysteine residue is required for interaction of RBR1 has a positive or negative role. By uncoupling RBR3 from the and RBR3 with viral oncoproteins and RepA (18, 19). negative regulation of RBR1 in cultured maize embryos through a The plant RBR-E2F pathway appears similar to that in combination of approaches, we demonstrate that RBR3 has a animals (4, 5, 26); however, although many plants appear to positive and critical role in the expression of E2F targets required possess only 1 RBR gene, maize and related cereals have at least for the initiation of DNA synthesis, DNA replication, and the 2 distinct types, RBR1 and RBR3 (19, 27, 28). Maize RBR1

efficiency with which transformed plants can be obtained. Titra- represses expression of RBR3 through a mechanism that most PLANT BIOLOGY tion of endogenous RBR3 activity through expression of a domi- likely involves inhibition of E2F transcription factors (19), and nant-negative allele with a compromised pocket domain suggests can be circumvented by expression of RepA (19, 29), which that these RBR3 functions require an activity distinct from its inhibits RBR1 through a pocket-dependent mechanism (17, 18, pocket domain. Our results indicate a cell cycle pathway in maize, 30). Ectopic expression of RepA provides an effective means to in which 2 RBR genes have specific and opposing functions. Thus, down-regulate RBR1 and release E2F activity in an inducible the paradigm that RBR genes are negative cell cycle regulators manner, and considerably stimulates cell transformation and cannot be considered universal. callus growth in maize embryos (29). Inhibition of RBR3 expression by RBR1 suggests 2 possible alternative functions for cell cycle ͉ DNA synthesis ͉ E2F ͉ RepA RBR3 in cell cycle control: RBR3 could have a novel positive role or, similar to p107 in mammals, it could provide a fail-safe etinoblastoma-related (RBR) proteins are negative cell cy- mechanism by restraining the cell cycle after loss or inactivation Rcle regulators conserved in animals and plants (1–5). They of RBR1 (15, 19, 27, 31). are known as pocket proteins by virtue of a conserved region The ability of plant cells to undergo transformation and located in their C-terminal half (i.e., the pocket domain), which regeneration is associated with cell cycle activity (29, 32, 33), and is largely responsible for protein–protein interactions and bio- increasing evidence supports the idea of a critical role played by logical function. It comprises 2 highly conserved A and B the RBR/E2F pathway (29, 33, 34). A compensatory mechanism domains separated by a nonconserved spacer of varying length, between RBR1 and RBR3 could explain, at least in part, the as well as a less-conserved C-terminal region. In mammals, these well-known recalcitrance of maize and related grasses to genetic proteins are well characterized and comprise RB, p107 and p130. transformation (19, 27, 35). However, the role of RBR3 in RBR proteins repress cell cycle progression primarily through E2F-dependent gene expression, cell cycle progression, and cell the inhibition of E2 promoter binding factor (E2F)-dependent transformation is unknown. A negative cell cycle role for RBR3 transcription, which is required to express many genes involved would be consistent with other RBR genes, but formal evidence in S-phase and cell cycle progression, including the minichro- is lacking. Also, the existence of an RBR1/RBR3 compensatory mosome maintenance 2–7(MCM2–7) family of DNA replication mechanism has not been demonstrated in vivo. factors (4, 6–11). Thus, repression or expression of E2F targets, Using a combination of approaches, involving the up- or such as MCM2–7 genes, is a good indicator of the activity of down-regulation of RBR3 (either alone or in combination with RBR and E2F proteins. RBR1 down-regulation) in cultured maize embryos, we demon- In overexpression studies, all 3 mammalian pocket proteins inhibit E2F-dependent gene expression, recruit chromatin remod- eling complexes, actively repress transcription, and arrest cell Author contributions: P.A.S., G.H., W.J.G.-K., and B.A.L. designed research; P.A.S., G.H., L.E.L., S.W.B., and W.J.G.-K. performed research; P.A.S., G.H., and W.J.G.-K. analyzed data; growth (3, 12, 13). However, gene knockouts in mice have shown and P.A.S. and B.A.L. wrote the paper. that, in addition to having overlapping functions, individual pocket The authors declare no conflict of interest. proteins have unique properties (14). In particular, although only Freely available online through the PNAS open access option. RB is considered a bona fide tumor suppressor, both p107 and p130 1To whom correspondence may be addressed. E-mail: [email protected] or larkins@ can functionally compensate for RB inactivation or loss in certain ag.arizona.edu. contexts and restrain cell proliferation (15). 2Present address: College of Pharmacy, University of Arizona, 1295 North Martin Avenue, Inactivation of RBRs can occur by various mechanisms. For Tucson, AZ 85721. example, it is well documented that several oncoviral proteins, This article contains supporting information online at www.pnas.org/cgi/content/full/ such as SV40 large T antigen (Tag), adenovirus E1A, human 0813329106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813329106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Table 1. Constructs used in transformation experiments Plasmid no. Plasmid name Promoter Coding sequence 3Ј sequence Reference or source

PHP9058 UBI::PATϳGFP UBI moPAT fused to moGFP pinII ref. 29 PHP21829 UBI::PATϳYFP UBI moPAT fused to ZsYFP-N1 pinII Clontech Laboratories PHP3953 UBI::GUS UBI ␤-glucuronidase from E. coli pinII ref. 54

PHP17008 Nos::RepA Nos WDV RepAm pinII ref. 29 PHP25473 UBI::RBR3NASx2 UBI N/A None This work PHP25475 UBI::RBRDSH UBI N/A None This work PHP30001 UBI::RBR3HA UBI RBR3HA pinII This work PHP30003 UBI::RBR3HA-C788G UBI RBR3HA-C788G pinII This work PHP30004 35S::RBR3HA-C788G 35S RBR3HA-C788G pinII This work

Maize-optimized moPAT and moGFP indicate maize codon-optimized PAT and GFP sequences. RepAm indicates an ЉintronlessЉ version of RepA. N/A, not applicable.

strate that RBR3 has a necessary and positive role in the pression of RBR3 RNAi (RBR3DSH) or antisense expression of the MCM gene family, DNA replication, and cell (RBR3NASx2) constructs, each driven by the ubiquitin 1 (UBI) transformation. Also, evidence is presented indicating that an promoter, resulted in only 3.2 and 6.1% transformation effi- activity attributable to one or more domains distinct from the ciency, respectively. This result indicates that RBR3 has a pocket domain is required for RBR3 function. These results rule positive role in maize transformation, and that its down- out a compensatory mechanism between RBR1 and RBR3 in regulation negatively impacts this process. Coexpression of maize, and shed light on important differences concerning the RepA and RBR3DSH or RBR3NASx2 constructs resulted in function of the RBR-E2F pathway between cereals and other transformation efficiencies close to control values (8.3 and 9.9%, dicotyledonous plants. respectively), consistent with both RepA and RBR3 having positive roles in the pathway controlling cell transformation. Results Gene expression analysis by RT-PCR showed that on expres- To understand the function of RBR3, we uncoupled its expression of RepA, RBR3 was up-regulated 3.8-fold relative to sion from the inhibitory control of RBR1, and analyzed how this control calli (Fig. 1), which confirmed previous findings indi- condition impacts maize cell transformation, DNA replication, cating that RBR3 is negatively controlled by RBR1, most likely and the expression of selected E2F targets. We first investigated through inhibition of E2F activity (19). In fact, we demonstrated the consequences of RBR3 down-regulation on cell transforma- earlier that up-regulation of RBR3 depends on the presence of tion. Ectopic expression of RepA is an effective means to inhibit the LxCxE motif in RepA that mediates interaction with and RBR function in plants by a mechanism that specifically involves inhibition of RBR1, and that E2F and their partner DP proteins the release of E2F transcription factors, which increases cell transformation in maize (29, 36). However, RepA-dependent inhibition of RBR1 in maize resulted in increased expression of RBR3 (19), making it difficult to dissect their individual roles. Therefore, we uncoupled RBR3 from RBR1 by specifically down-regulating RBR3 using RNAi and RNA antisense approaches. A description of the constructs used in these experiments is given in Table 1. On expression of RepA with the nopaline synthase (Nos) promoter, transformation efficiency of embryos increased from 10.9% in control experiments to 27.5% (Table 2), consistent with previous results (29). However, ex-

Table 2. RBR3 is required for maize cell transformation Embryos, transformed/ Transformation Construct bombarded rate, %

RBR3 down-regulation Control 38/350 10.9 Nos::RepA 103/375 27.5** UBI::RBR3DSH 13/400 3.2** Nos::RepA ϩ UBI::RBR3DSH 31/375 8.3 UBI::RBR3NASx2 26/425 6.1* ϩ Nos::RepA UBI::RBR3NASx2 42/425 9.9 Fig. 1. Down-regulation of RBR3 expression reduces levels of all 6 MCM2–7 RBR3 overexpression transcripts. Maize embryos were transformed with different constructs by UBI::GUS, control 27/260 10.4 particle bombardment, the resulting calli genotyped, and RNA was extracted, UBI::RBR3HA 28/270 10.4 pooled, and assayed for gene expression by RT-PCR (19). See Table 1 for UBI::RBR3HA-C788G 23/283 8.1 construct description. Calli transformed with constructs for RBR3 down- 35S::RBR3HA-C788G 16/281 5.7* regulation by RNAi (RBR3DSH) and antisense (RBR3NASx2) displayed reduced expression of RBR3 RNA as well as transcripts of the MCM2–7 family. Expres- * and **, signiﬁcance levels (*, 0.05 probability; **, 0.01 probability) sion of 3 non-E2F target RNAs (i.e., RBR1, RBR2, and actin) was not affected by relative to control in each of the 2 independent experiments (RBR3 down- down-regulation of RBR3. A representative experiment is shown. See Fig. S2 regulation and RBR3 overexpression). for comprehensive quantitative analysis.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813329106 Sabelli et al. Downloaded by guest on September 25, 2021 can interact with the RBR3 promoter (19). Accumulation of RBR3 transcript was reduced Ϸ50% in calli transformed with either the RBR3DSH or RBR3NASx2 construct, and coexpression of RepA and RBR3DSH or RBR3NASx2 resulted in RBR3 RNA expression levels that were 2.1- and 1.8-fold higher, respectively, than the control (Fig. 1). No major change in RBR1 or RBR2 RNA expression was detected, indicating that the transgenic experiments involving RepA expression and RBR3 down-regulation impinged specifically on the expression of RBR3 (Fig. 1). We next examined the effect of down-regulating RBR3 on the expression of MCM2–7 genes. Three MCM genes from maize were previously characterized, MCM3 (37), MCM6 (38), and MCM7 (39). Through database mining, we identified the 3 remaining members of the MCM2–7 family [supporting information (SI) Fig. S1]. On expression of RepA, all 6 MCM genes were up-regulated 2- (i.e., MCM3) to 7.4-fold (i.e., MCM5) (Fig. 1 and Fig. S2), indicating maize MCM2–7 genes are negatively regulated by RBR1 and are most likely targets of E2F activity, consistent with what is known about MCM gene expression in higher eukaryotes (4, 9–11). However, down-regulation of RBR3 resulted in decreased expression of all members of the MCM2–7 gene family, which ranged from 10 to 80% of the control (Fig. 1 and Fig. S2). Thus, expression of RBR3 is required for transcription of at least a subset of known E2F targets (i.e., MCM2–7 genes), although individual MCM genes appeared to differ in their sensitivity to RBR3 down-regulation.

These data support the transformation results, and indicate the PLANT BIOLOGY positive role of RBR3 in cell transformation could be mediated by its regulation of DNA synthesis through controlling the expression of at least some essential DNA replication factors. Fig. 2. Overexpression of RBR3 induced increased expression of MCM3–7 We next asked whether RBR3 is sufficient to increase cell genes, whereas expression of the C788G mutant resulted in the down- transformation and MCM gene expression. RBR3 was engi- regulation of all 6 MCM2–7 genes. RBR3 was overexpressed under the control neered with a hemagglutinin (HA) epitope tag (RBR3HA) and of the maize ubiquitin1 (UBI) promoter (RBR3HA). Also, an RBR3HA-C788G mutant was expressed with the UBI and CaMV 35S promoters. Expression overexpressed under the control of the UBI promoter (29). As analysis was carried out as in Fig. 1. Expression of RBR1 and actin RNAs was expected, RBR3 RNA was expressed at high levels (21.7-fold largely unaffected. A representative experiment is shown. See Fig. S3 for over control values), and, with the exception of MCM2, all of the comprehensive quantitative analysis. MCM genes were up-regulated 1.6- to 2.2-fold (Fig. 2 and Fig. S3). Expression of RBR3HA had no noticeable effect on actin and RBR1 gene expression. However, no significant change in control. However, it not only failed to stimulate expression of MCM transformation efficiency was observed (Table 2). One expla- genes, but also resulted in their collective down-regulation (Fig. 2 nation for these results relates to the inability of RBR3 to and Fig. S3). Thus, high expression of the dominant-negative stimulate expression of the full complement of genes required to RBRHA-C788G allele interfered with endogenous RBR3 activity. achieve cell proliferation (see below). Because the spectrum of Essentially similar results were obtained on expression of the genes regulated by RBR3 is unknown, understanding of the C788G mutant under control of the weaker cauliflower mosaic reasons why RBR3 is not a limiting factor in maize transforma- virus (CaMV) 35S promoter (Fig. 2 and Fig. S3), indicating that tion will probably require genome-wide profiling of gene ex- high expression levels of RBR3HA-C788G are not necessary for pression. Thus, we conclude that RBR3, although required, is the inhibition of endogenous RBR3, and that expression of not limiting for transformation. UBI::RBR3HA-C788G probably saturated the system. Consistent Having established that RBR3 has a positive role in both with the inhibition of MCM gene expression, a decrease in trans- regulating MCM gene expression and cell transformation, we formation efficiency was also observed on expression of the RBR3- asked whether these effects solely depend on its pocket region C788G mutant allele (Table 2). This result indicates that expression or involved a pocket-independent mechanism. Although de- of the C788G allele competed with endogenous RBR3 for some tailed information is available regarding the role of the C- essential protein–protein interaction, which appears to be mediated terminal pocket domain of RBR proteins, relatively little is by a domain(s) distinct from the pocket region (Fig. S4 and known about activity that is pocket-independent. We previously Discussion). Thus, we conclude that RBR3 controls MCM gene showed that RBR3 has a conventional pocket protein activity expression and cell transformation through both the pocket domain that requires a conserved cysteine in the B domain at position and a pocket-independent activity. 788, similar to other RBR proteins (19). In vitro interaction Because RBR3 controls expression of key genes involved in studies indicated that mutating this residue to glycine (C788G) the regulation of DNA synthesis, we investigated whether its greatly reduced the ability of RBR3 to interact with RepA, overexpression or down-regulation would be sufficient to stim- adenovirus E1A, and human papillomavirus E7 proteins (19). ulate or repress DNA replication, respectively. Control and Therefore, we reasoned that ectopically expressing the C788G- transgenic calli were pulse-labeled with the thymidine analogue mutagenized form of RBR3 (RBR3HA-C788G) in callus would BrdU and analyzed for their rate of DNA synthesis by using a create the means, in a dominant-negative manner, to establish Southwestern method with anti-BrdU antibodies (Fig. 3). Ex- whether or not RBR3 also functions through a pocket-independent pression of UBI::RBR3HA in 2 independent experiments re- mechanism (Fig. S4). Expression of RBR3HA-C788G driven by the sulted in 1.6- and 2-fold greater rates of DNA synthesis, com- strong UBI promoter increased RBR3 transcript 16.7-fold over the pared with control, approaching the stimulation obtained by

Sabelli et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 nuclei in G2 phase on expression of RBR3HA. This analysis revealed that cells tend to stabilize in the G2 phase on overexpression of RBR3, as shown by the Ϸ50% reduction in the G1/G2 nuclei ratio in UBI::RBR3HA-transformed nuclei, compared with control (0.56 versus 1.0) (Fig. 4B). This result suggests that although RBR3 overexpression stimulates DNA replication, it is not equally effective at stimulating the transition of G2 cells to M-phase. This apparent block in G2 would compensate for the stimulatory role of RBR3 in DNA replication, and curtail a potential net gain in the rate of cell proliferation. This interpretation is in agreement with the observation that RBR3 fails to stimulate cell transformation when overexpressed (Table 2). Therefore, we conclude that RBR3 does not promote the G2/M-phase transition as effectively as it does the G1/S-phase transition, which would explain the failure of RBR3 overexpression to stimulate cell transformation. Discussion We demonstrated that a maize member of the RB gene family, RBR3, has an essential and positive role in regulating MCM gene expression, DNA replication, and cell transformation. These Fig. 3. RBR3 is necessary and sufficient for DNA replication. Two indepen- properties appear to be unique among pocket proteins. In fact, dent experiments involving in vivo incorporation of BrdU into newly synthe- sized DNA in different transgenic maize calli are shown. Calli were pulse- it is well established that all RB genes in animals have negative labeled with BrdU, and their DNA samples were pooled and probed with an roles in DNA replication and cell cycle progression (3, 40). anti-BrdU antibody. Pools ranged from 5 to 30 independent samples, as Similar findings were reported for plant RBRs, and a number of indicated. DNA loadings were confirmed by hybridization to 32P-labeled total studies have shown that cell proliferation is stimulated on maize DNA. Expression of either Nos::RepA and UBI::RBR3HA increases callus down-regulation or loss of RBR activity in tobacco, Arabidopsis, DNA replication, whereas expression of the dominant negative allele and maize (29, 36, 41–44). Interaction with and inhibition of UBI::RBR3HA-C788G impairs it. Pairwise differences in BrdU incorporation plant RBR by geminivirus RepA proteins has been established levels for each treatment relative to the control were significant as deter- Ͻ (17–19, 45), and shown to cause up-regulation of E2F-dependent mined by t test (P 0.05). gene expression (19, 36), which is in agreement with the current model in which RBR inhibits the G1/S-phase transition during RepA expression (2.6-fold). In contrast, DNA replication was the cell cycle by repressing E2F transcription factors (6, 7, 46, severely inhibited on expression of the RBR3HA-C788G dom- 47). The requirement for RBR3 for the expression of MCM2–7 inant negative mutant allele (i.e., 0.2-fold relative to control). genes is novel and appears unique within the RBR family. For example, several previous studies profiled gene expression in These results indicate that overexpression of RBR3 is necessary animal systems on down-regulation or loss of pocket protein and sufficient to stimulate DNA replication in vivo, and are function (9, 10, 48–51); however, in no instance were MCM consistent with the positive roles of RBR3 in regulating MCM genes down-regulated. Also, our data indicate that RBR3 is gene expression and cell transformation. limiting for the expression of all but one of the MCM2–7 genes. To understand whether RBR3 also promotes the G2/M-phase Thus, we have identified a previously undescribed function of a transition, we compared the relative frequencies of G1 and G2 plant RBR gene: maize RBR3 appears to have a key, positive nuclei in calli expressing RBR3HA (UBI::RBR3HA) with con- regulatory role between RBR1 and MCM2–7 genes in the trol calli (Fig. 4). Representative flow-cytometric profiles are RBR-E2F pathway leading to DNA replication (Figs. 1–3 and 5). shown in Fig. 4A, which indicate an increase in the fraction of Because MCM2–7 are conserved E2F targets in all systems studied so far, it is likely that RBR3 impinges directly or indirectly on at least a subset of E2F transcription factors. A detailed interaction analysis between RBR3 and prospective E2F protein binding partners will be required to define the specificities of RBR3/E2F binding. The outcomes of expressing the C788G allele of RBR3 suggest that the function(s) of RBR3 in cell cycle regulation and transformation requires an activity that is distinct from the canonical role of the pocket domain. The C788G mutation strongly diminishes the pocket binding activity of RBR3 (19). Ectopic expression of this allele would be expected to have no consequence if RBR3 function was exerted solely through its pocket domain, because an intact complement of RBR3 pocket activity would be retained due to the presence of endogenous RBR3. However, a marked reduction in MCM2–7 gene expression, DNA synthesis, and cell transformation was observed on expressing the C788G mutant allele. We interpret these out- Fig. 4. Overexpression of RBR3 results in a block in the G2 phase of the cell comes as an indication of a requirement of RBR3 for an activity cycle. Control and RBR3-overexpressing calli (UBI::RBR3HA) were analyzed by flow cytometry. (A) Representative flow-cytometric profiles. (B) Histogram that is independent from its pocket domain. In the model shown showing an Ϸ50% decrease in the G1/G2 nuclei ratio in the calli transformed in Fig. S4, we represent this unspecified activity as a protein or with UBI::RBR3HA compared with control, suggesting an inhibition of the protein complex binding to the N-terminal region of RBR3. G2/M-phase transition in the cell cycle. Differences in G1/G2 ratios were Overexpression of RBR3 with a functional pocket domain would significant as determined by t test (P Ͻ 0.01). increase the pool of RBR3 bound to the unknown factor,

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813329106 Sabelli et al. Downloaded by guest on September 25, 2021 pression is not, indicates that this scenario is not the case. Clearly, understanding the precise mechanism of action of RBR3 represents a challenge for future research. Profiling global gene expression changes on up- or down-regulation of RBR3, and investigating its interactions with members of the E2F family could help address this question. Because RepA expression results in up-regulation of MCM genes, increased DNA replication, and transformation, all of which require RBR3 function, our findings appear to rule out a role for RepA in inhibiting RBR3 in vivo, although the 2 proteins clearly interact in vitro in a pocket- and LxCxE-motif-dependent fashion (Fig. 5) (19). At least 2 alternative explanations fit with these results. RepA and RBR3 may not interact in vivo, or, even if such an interaction does occur, it does not influence RBR3 activity. Alternately, because binding of RepA (or other oncoviral protein partners) to pocket proteins through their LxCxE- motifs results in a conformational change of the pocket domain Fig. 5. Model showing RBR3 role in the RBR/E2F pathway controlling the resulting in the displacement of E2F, and because RBR3 pocket expression of MCM2–7 genes, DNA replication, and cell transformation. RepA seems necessary for its activity, we favor the view that RepA and inhibits RBR1; thus, stimulating the pathway leading to S-phase gene expres- RBR3 do not interact in vivo in this context. Thus, our results sion, DNA synthesis, and cell transformation through up-regulation of RBR3. rule out a compensatory mechanism between RBR1 and RBR3 The transgenic approaches to down- or up-regulate RBR3 are indicated in in maize embryogenic callus that results in inhibition of the cell italics. The dotted line illustrates a potential inhibitory effect of RepA on RBR3 cycle and transformation (19, 27). ruled out by this work. The organization of the RBR gene family in maize appears to be shared by and unique to cereals, but the biological implica- effectively enhancing the normal role of RBR3 in controlling tions are unknown. Because RBR genes have been universally MCM gene expression and DNA replication (Fig. S4B). Accord- viewed as repressors of cell cycle activity, the 3 maize RBR genes PLANT BIOLOGY ing to this interpretation, the RBR3 expression level, rather than were expected to play largely redundant roles in controlling the the concentration of the unknown binding factor, is limiting for cell cycle. Consequently, it was thought that stimulation of the RBR3 activity. Expression of the C788G allele would out- pathways they control could be achieved by their simultaneous compete endogenous RBR3 for binding to the unknown fac- down-regulation. Our findings indicate that at least 1 type of tor(s), but, because of its compromised pocket domain, it could RBR gene in maize (and possibly its orthologs in related grass not function, causing the negative effects described above (Fig. species as well) has a positive cell cycle role. Therefore, strategies S4C). Thus, both the unspecified activity independent of the aimed at the manipulation of the RBR/E2F pathway to stimulate RBR3 pocket and its pocket domain would be required for cell proliferation and increase crop yield in cereals will need to RBR3 function. More research will be needed to elucidate the be revised. Although certain RBR genes may be down-regulated, identity of the unknown factor required for RBR3 activity. others may be up-regulated, possibly simultaneously, to stimu- Investigation in various systems has primarily focused on de- late the cell cycle. Clearly, understanding the RBR/E2F pathway scribing the function of the pocket domain of RBRs, and there in Arabidopsis and other dicots provides only limited insight is little information on pocket-independent activities. For ex- relative to cereals. Improvement of cereals through the manip- ample, the N-terminal domains of RBRs show a good degree of ulation of the RBR/E2F pathway will have to consider the sequence conservation, at least within RBR subfamilies; how- different and opposing roles of distinct RBR genes. ever, they have not been thoroughly investigated (52). We previously identified an amino acid sequence in proximity of the Materials and Methods RBR3 N terminus spanning residues 78–124, which is similar to For details, see SI Materials and Methods. a conserved domain in p107 and p130 that is required for growth suppression and inhibition of CDK/CycA/E complexes (19, 53). Plasmids. The promoters and terminators used in this study were described However, it is unknown whether this domain has a role in the earlier (29), and include maize UBI, Nos, and CaMV 35S promoters; the potato proteinase inhibitor (pin) II terminator was used as the flanking 3Ј end RBR3 functions we have described. ϳ Several nonmutually exclusive pathways downstream of RBR3 sequence in most constructs (Table 1). The UBI::PAT GFP and Nos::RepA constructs were previously described (29). In some cases, YFP (Clontech Lab- could be envisioned that eventually stimulate E2F-dependent oratories) replaced GFP in the UBI::PATϳGFP plasmid, or the Escherichia coli expression of MCM genes and subsequent DNA replication and uidA gene (encoding ␤-glucuronidase) replaced the PATϳGFP fusion to give transformation. They include the stimulation of activator E2Fs, the UBI::GUS plasmid (54). the inhibition of repressor E2Fs, and an inhibition of RBR1 in a dominant-negative fashion. All 3 scenarios, in their most direct Cell Transformation Experiments. Immature maize (Zea mays L.) high-type II interpretation, would involve RBR3 binding to E2Fs, which embryos were transformed by particle bombardment essentially as described appears likely as mentioned above. Although our data do not earlier (29). Each transformation experiment included the selectable-visible rule out the existence of partial overlap between RBR1 down- marker UBI::moPATϳmoGFP (or YFP)::PinII, which is a fusion between maize- regulation and RBR3 overexpression, they suggest that the optimized (mo) phosphinothricin acetyl transferase (PAT) that confers resis- pathways controlled by RBR1 and RBR3 are not identical, and tance to the herbicide BASTA, and GFP or YFP. Transgenic calli were selected that RBR3 does not stimulate downstream processes simply by and scored as described (29). Transformation efficiency is defined as the percentage of embryos resulting in transgenic calli relative to the total num- inhibiting RBR1 activity. If RBR3 solely down-regulated RBR1 ber of embryos targeted for transformation by particle bombardment. Sig- activity, then RBR3 overexpression would essentially recapitu- nificance was determined by using a 2 ϫ 2 (pairwise fashion) contingency table late the full spectrum of phenotypes resulting from inhibition of to determine Chi2 values for each treatment relative to control with a 0.05 RBR1, such as those obtained by expressing RepA. However, the probability (*) or a 0.01 probability (**). Significance was determined sepa- fact that ectopic expression of RepA is sufficient to trigger a rately for the 2 different experiments (RBR3 down-regulation and RBR3 substantial increase in transformation, whereas RBR3 overex- overexpression).

Sabelli et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 RT-PCR Analyses. Individual calli originating from independent transforma- different times and averaged. X-ray films were scanned and digitally quantified. tion events were screened for the presence/absence of the transgene(s) by PCR Blots were then probed with 32P-labeled total genomic B73 DNA, exposed to and, where appropriate, the expression of transgenic RNA by RT-PCR. RNA Phosphoimager screen, and digitally quantified. BrdU incorporation was quan- samples were pooled before RT-PCR analysis. Description of RT-PCR targets, tified relative to the amount of DNA in each sample. primers used and RNA pools are given in Tables S1–S3. Each RT-PCR was repeated at least 3 times, and average expression and SD values are given in Flow Cytometry. Ten control calli, transformed with the UBI::PATϳGFP::pinII Fig. S2 and Fig. S3. construct and 10 calli transformed in addition with the UBI::RBR3HA::pinII construct were analyzed by flow-cytometry to investigate the effect of over- BrdU Incorporation Analysis. Calli were incubated in an Eppendorf tube with expressing RBR3 on the relative frequency of unreplicated (G1 phase) and Murashige and Skoog Basal Salt Mixture containing 1 mM BrdU for5hat28°C replicated (G2 phase) nuclei. Nuclei were released from callus tissue and analyzed with a Partec CAA-II flow analyzer as previously described (55, 56). on a rotating incubator in the dark. Callus DNA was extracted from a number of independent transformants ranging from 5 to 30, depending on the treatment, ACKNOWLEDGMENTS. We thank Drs. Ricardo A. Dante (Alellyx Applied and genotyped according to standard procedures (19). Pooled DNA samples (250 Genomics, Campinas, Brazil) and Ramin Yadegari (University of Arizona) for ng) were denatured, neutralized, and blotted onto a nitrocellulose membrane. comments on the manuscript. This work was supported in part by grants from BrdU was detected by using a primary anti-BrdU antibody and a secondary Pioneer Hi-Bred International Inc., Johnston, Iowa, and the U.S. Department antibody conjugated to horse radish peroxidase. Three exposures were taken at of Energy Grant DE-FG02-96ER20242.

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