Do differentially regulated regions (DMRs) control imprinting of the maize fie1 and fie2 genes?

Many aspects of early development in plants and animals are controlled by systems of genetic imprinting by which, for a particular gene, either the maternal or the paternal copy (allele) is silenced. Importantly, imprinting in plants affects only the endosperm - one of the two structures (the other being the embryo) formed following double fertilisation. The endosperm can be regarded as analogous to the yolk of an animal egg, providing nutrients to the developing embryo. Importantly the endosperm forms the bulk of the grain of all cereals, and its correct development (and improvement) is thus of great commercial and sociological significance. Recent work on plants, some in the applicant's laboratory, has shown - again as in animals - that expression of imprinted alleles is regulated by chemical modifications to the DNA in the form of methylation. We have now shown that this methylation differs in the maternal and paternal alleles of an important pair of developmental genes in maize (fie1 and fie2), and that, surprisingly, this methylation behaves in different ways in these two genes, suggesting that their imprinting is regulated by differing mechanisms. Interestingly, for one gene (fie1) the paternal copy is methylated in the gametes, while the paternal copy of fie2 is only becomes methylated after fertilisation. The small amount of data from plants suggest that methylation is directlty associated with gene silencing, but this is not the case in animals where it forms only one component of a complex process by which modifications are made both to the DNA and to associated proteins in regions termed DMRs (Differentially Methylated Regions). We now want to dissect this regulatory pathway in plants, first by determining whether it is the methylation itself that silences gene expression, and then by exploring the possibility that the DMRs in plants are marked, prior to DNA methylation, by modified versions of histones, and other proteins which form part of the chromosome matrix (the chromatin). For this work we shall use mutant lines of plants incapable of carrying out methylation, or modifying histones, and a number of well-tried transgenic approaches to investigate changes at the level of individual DMRs. Finally we shall use a recently-developed chromatin-immunoprecipitation system (ChIP) to identify the proteins associated with the DMRs, and to discover whether one group of these proteins, the Polycomb Group, regulate both themselves and imprinting.

Important aspects of endosperm development are regulated by imprinting. Work in the applicant's lab on maize has demonstrated that for a key gene pair (fie1 and fie2; duplicated versions of the Arabidopsis FIE sequence) belonging to the Polycomb Group (Pc-G), imprinting is regulated through 5' differentially methylated regions (DMRs) similar to those found in mammals. Surprisingly the paternal (silenced) copy of fie1 is methylated in the male gamete, while the male version of fie2 only becomes methylated in the primary endosperm cell after fertilisation. The two fie genes thus appear to be differently regulated. In mammals DMRs represent only a single component of a number of complex imprinting pathways, which involve histone modifications, Pc-G complexes, inhibitory proteins and non-coding RNAs. While the picture for plants appears simpler, the fact that the paternal allele of the fie2 sequence is methylated after fertilisation strongly indicates that the DMR is somehow marked in the gamete. In animals DMRs are generally marked by histone modification and also the binding of Pc-G proteins. Such changes also occur in plant chromatin but their significance in imprinting is unknown. Importantly, recent data suggest that Pc-G proteins self-regulate, and thereby control key aspects of imprinting. We shall first investigate the necessity of DMR methylation for imprinting by monitoring allele-specific methylation and expression of 'imprinted' fie reporter constructs in genetic backgrounds defective either in methlation and histone modification, Then, using RNAi to knock down key methylation and histone methylases in the primary endosperm cell, we shall explore the nature and function of any non-methyl marks on the fie2 and fie2 DMRs. Finally, using a downscaled ChIP protocol, we shall (i) identify histone modifications associated with these DMRs, and (ii) explore the putative autoregulatory roles of Pc-G proteins.