With the completion of the sequencing of some model genomes, the attention of molecular biologists has been diverted to understanding gene functions and the role of regulatory genes such as transcription factors in signal transduction cascades. This is because it has now been realized that the natural variation between species is due to transcription factors, which play a key role in regulating gene families at different functional levels.
In a review article in the June, 03 issue of Trends in Plant Science (vol 8:279-285), John W. Chandler and Wolfgang Werr at the University of Cologne, Germany, discuss the advantages of a dominant negative functions approach over classical techniques in determining individual gene functions, particularly those of transcription factors gene families.
Gene disruption approaches at the DNA level: The authors draw attention to the fact that although gene disruption through T-DNA disruption is a powerful method to study gene function, it has a number of limitations. First, the method is applicable to a few model species such as Arabidopsis. Secondly, it is highly labor intensive in view of the necessity to screen large population of plants. Thirdly, many phenotypes remain undetected or only noticeable under extreme environmental conditions
Gene disruption approaches at the RNA level: The authors discuss the advantages of using RNA interference (RNAi) for studying gene expression in preference to the antisense method or the one in which a gene is disrupted through T-DNA insertion. Unlike the last-mentioned method, RNAi inactivates a gene without changing the genome structure. In the RNAi method, the mRNAs of the targeted gene is degraded by double-stranded RNAs (dsRNAs). The latter is formed from the complementary strand of the targeted mRNA by the activity of an RNA-dependent RNA polymerase (RdRP). The newly formed dsRNA form of mRNA is cleaved by the activity of an enzyme called Dicer into a number of fragments, leading to the inactivation of the targeted gene. In the past five years it has proved to be one of the most powerful molecular tools to determine the function of a gene in plants and other phyla.
Protein domain conservation: By means of suitable examples, the authors have described the evolution of transcription factors, accounting for their multidomain nature. According to them, reproductive developments in angiosperms have taken place as a result of structural changes in the MADS box genes. The authors cite a recent publication in which homology between the cloned 26 AP3 (Apetala) and PI (Pistillata) genes from distantly related species was reported. Such a close homology indicates the evolutionary tendency of strong conservation of the PI-specific motif and the AP3 motif between diverse dicot species at various evolutionary levels.
Such a high degree of protein domain conservation also suggests the possibility of restoration of the normal phenotype of a mutant by orthologous genes from a widely divergent species. The authors have cited a number of examples in which such an assumption was substantiated. For instance, the NEEDLY gene from Pinus radiatus was found to complement the lfy (leafy) mutant gene of Arabidopsis; the normal allele ofDEFICIENS from Antirrhinum was found to partly complement the ap3 mutant of Arabidopsis. The normal allele of pi and ap3 control different flowering traits in Arabidopsis and yet the normal functioning of these mutant genes can be partly restored by a class B homeotic gene, GGM2 from a unrelated gymnosperm group, Gnetum gnemon. It has also been shown that LMADS1, a MADS-box protein from Lilium is the functional orthologue of AP3 and that AGL2-lke ovule-specific MADS-box proteins from Petunia and rice bear close homology. These findings suggest that dominant-negative effects can be utilized to transfer genes from Arabidopsis to important crops such as enhancing their nutritional status by suppressing genes that cause toxicity or those that make the phenotype tall thus minimizing chances of its lodging in adverse climate.
The authors draw attention to certain highly conserved domains between plants and animals such as the homeodomain, myb motif, leucine zippers and helix-loop-helix domains. Such close gene regulatory domains between plants and animals suggest that domains in animals that function as repressor may also act as plant transcriptional activators.
Conversion of transcriptional repressors into activators or enhancement of transcriptional activator function:
The LEAFY (LFY) gene has been shown to have dual role: (a) the gene is essential for the conversion of the inflorescence meristem to a floral meristem and (b) it is also involved in the development of petals and stamens. On the other hand, the homeotic gene AGAMOUS (AG) is essential for the development of stamens and carpels as deduced from the mutant phenotype ag. The mutants are characterized by replacements of stamens with petals and carpels with another ag flower in floral whorl 3 and 4,respectively. In order to explain the function of the above two genes LEAFY and AG, the authors give the example of gain-of-function of the LFY mutant. When lfy was fused with a viral protein transcription factor gene VP16, the LFY phenotype was partially rescued. This finding indicates that tissue specificity for the expression of a particular gene can be altered as demonstrated by the AG gene which is activated by the action of the LFY gene. LFY:VP16 fusion was also shown to act as a dominant-negative LFY allele by reconverting abnormal flowers to the normal floral type. This restoration of the flower morphology from abnormality to the normal type indicates that LFY can act independently of AGAMOUS in suppressing the expression of genes involved in shoot development.
Conservation of protein domains and transcriptional machinery: The authors describe the importance of using ENGRAILED (En) dominant-negative approaches in redundant gene families. En encodes a homeodomain-containing DNA binding protein. To elaborate their point, they mention that in Arabidopsis there are 21 members of HSF (heat shock factor 1) gene family and their individual functions cannot be detectable because phenotypes of the knockout mutants are indistinguishable. The authors describe the mode of action of two floral genes APETALA3 (AP3) and PISTILLATA (PI). There is hardly any redundancy in their pathways and that the two genes act somewhat directly to regulate the floral organ formation
The divergence in functional domains of different gene families is small.: With an illustrated account, the authors have explained the different steps involved in the dominant-negative reaction.Transcription factors that participate in the reaction are constituted of (a) activation or repression unit; (b) a protein interaction surface; and (c) a DNA binding domain. These units function in the form of dimers which could be homodimers of two transcription factors within the family or heterodimers of two different gene families.
Dominant-negative versus loss-of-function approaches: The authors describe the effects of a gene when it is inactivated due to loss of its functional part. Such inactivated genes are designated loss-of-function genes. The function of such defective genes can be restored by inserting related fragments into it. In the dominant-negative method, researchers study protein-protein interactions – the interactions between a truncated protein produced by a transgene and the related native protein inside the cell. The truncated protein forms a chimera with an integral part of a larger protein molecule. The chimeric protein blocks the reaction that brings the two integral parts of a protein together to make it functional. According to the authors, the chimeric protein interacts with a whole range of native compatible gene products. In the process several native cognate proteins and their native partners belonging to several gene family interact, creating phenotypes not revealed by conventional methods.
Implications and perspectives:
The authors underpin the importance of using high-throughput screens to identify heterologous protein interactions between species. Use of such automated device will allow researches assemble dominant-negative constructs without prior knowledge of sequences of particular gene homologues.
The authors discuss a recently reported powerful technique called CHRIS (the chimeric repressor interference system). The technique provides information on heterologous genes or protein domains, and has therefore a great potential for determining gene functions in species where genomic sequencing is difficult or has not yet been done. Recently the technique has been successfully used in Arabidopsis to identify novel phenotypes attributed to hitherto unknown genes. With the help of more refined techniques, it is expected that future researches would shed more light on (a) ectopic expression of transcription factors under tissue-specific promoter control, (b) the sequential expression of a transcript factor gene at different time frame, and (c) modus operandi of transcription factors to target a specific developmental aspect.
Finally, the authors hope that with characterization of more plant repressors, it may be possible to construct more and more dominant-negative tools to decipher the functions of hitherto unknown genes.