Fase I Análisis estratégico ambiental 59

The study of the functional components of the RdDM pathway and its operations has been fa- cilitated by mutations perturbing the pathway. In bothArabidopsis and maize they have proved invaluable in demonstrating direction of DNA methylation and through this the dissection of para- mutation (McGinnis et al. 2006; Sidorenko et al. 2009; Hollick and Chandler 2001), small RNA movement (Molnar et al. 2010) and TGS (Madzima et al. 2011; McGinnis et al. 2006) to name a few. Table.11 provides a summary of maize RdDM mutants (all used in analysis later in the chapter), those that affect the accumulation of siRNA and those that have shown to interfere with TGS and hypothesisied to be involved in mediating silencing.

Genetic screening by theb1 andpl1 systems have allowed for the identification of mutants involved in paramutation, these include the characterisation ofmop1 andmop2 mutants (using the b1 al- lele) (Dorweiler et al. 2000; Sidorenko et al. 2009) andrmr1,rmr2 andrmr6 mutants (using the

pl1 allele) (Hale et al. 2007; Erhard et al. 2009; Hollick and Chandler 2001). Furthermore,rmr1,

mop1 and rmr2 have also shown to be directly involved in the epigenetic silencing of transcrip- tionally silent transgenes in maize (McGinnis et al. 2006). mop1,mop2 andrmr6 are mutations of known elements within the RdDM pathway (see 1.4.2), knocking out RDR2, NRPD1-like (Pol IV subunit) and NRDP2/E2-like (Pol IV and V subunit) respectively (Arteaga-Vazquez and Chan- dler 2010). All three affect the accumulation of 24 nt siRNAs in vivo and are associated with

loss of DNA methylation. However, genome-wide analysis conducted onmop1 mutants has shown retrotransposons and DNA TEs to be differentially regulated (Jia et al. 2009) and has shown the removal of DNA methylation following the reversal of silencing (for example of, Mutator (Mu1) elements) (Lisch et al. 2002), demonstrating a role in regulating TEs and genes. Bothrmr6 and

mop2 (semi-dominant mutation available,Mop2-1) are close tomop1 in RdDM , encoding the Pol IV and Pol V subunits required for the initial steps of the pathway. The study of Pol IV mutants however, has mostly been conducted inArabidopsis,withnrpd1aandnrpd2 mutants (Pol IV and V subunits) showing great loss in DNA methylation in heterochromatin (Onodera et al. 2005).

mop2 is also referred to asrmr7, an allele ofmop2 that was under investigation at the same time by different research teams (Stonaker et al. 2009).

Thermr1 andrmr2 mutants, identified through genetic screens with thepl1 allele, however, have less defined roles. Thermr1 gene is required for the efficient accumulation of 24 nt siRNAs, just as the genes previously described, but encodes an SNF2 protein thought to interact with chromatin marks (Hale et al. 2007). A more recent study also indicates thatrmr1 operates upstream and independently of Pol IV in the RdDM pathway and the loss of which induces hypomethylation of

Mutator elements (Hale et al. 2009). The role ofrmr2 is less clear but it is known to influence the accumulation of 24 nt siRNAs and the maintenance of DNA methylation at distinct loci, such as the 3’ end ofPl1-Rh (Barbour et al. 2012). Greater understanding of this element may only arise through the discovery of directly interacting proteins.

In addition, other mutants demonstrate potential roles in epigenetic TGS, with less well understood functions. Firstly, Unstable factor for orange 1 (Ufo1), a trans-acting modifier of P1-wr alleles which controls phlobaphene pigmentation in vegetative and reproductive tissues, was shown to release epigenetic repression and induce hypomethylation (Chopra et al. 2003). Moreover, recently the role ofUfo1 in paramutation was probed, observing its requirement for the paramutation ofb1

and p1 loci (Sekhon et al. 2012). Current evidence also suggests thatUfo1 has roles in multiple epigenetic pathways, due to the loss of H3K9me2 from the P1-wr allele following reactivation by this mutation.

morpheus’ molecule 1-like (mom1-like) is the homologue of MOM1, which, is required for the maintanence of TGS in Arabidopsis. Work conducted has shown that the mutation of mom1

results in loss of methylation from previously silenced genes and heterochromatic repeat regions (Amedeo et al. 2000). Interestingly, changes in expression of genes were also observed, without alterations in DNA methylation, indicating thatmom1 acts independently of the RdDM pathway or downstream (Amedeo et al. 2000; Saze et al. 2012).

Finally,Rpd3-like encodes a histone deaceytlase in maize required for gene silencing, similar to a mammalian protein which is recruited by the human retinoblastoma tumour suppressor gene (Rossi et al. 2003). The focus of this study found maize genes that can recruit the plant equivalent,Rpd3- like, for the same result. Earlier studies have also found roles for similar genes in Drosophila

associated with PEV, altering gene expression (De Rubertis et al. 1996). However, little else is known of the role of this protein in plant gene silencing networks.

The RdDM and TGS mutants described have been employed to reactivate NYR-v expression in maize, releasing reporter repression to identify the mechanisms involved.

Table 11: Epigenetic maize mutants affecting siRNA biogenesis and/or TGS

Name Abrev. Pathway Function Reference

required to maintain

repression 1 rmr1 RdDM Sucrose nonfermenting 2 (SNF2)-like adeno-sine triphosphatase chromatin remodeller Hale et al., 2007

required to maintain

repression 2 rmr2 unknown unknown Barbour et al., 2012

required to maintain

repression 6 rmr6 RdDM NRPD1-like (largest subunit of DNA-dependent RNA polymerase IV and V) Erhard et al., 2009

mediator of

paramutation 1 mop1 RdDM RNA-dependent RNA polymerase 2 (RDR2) Dorweiler et al., 2000

mediator of

paramutation 2 mop2 RdDM NRPD2/E2-like (second largest sububit ofDNA-dependent RNA polymerase IV) Sideorenko et al., 2009

unstable factor for

orange 1 ufo1 TGS unknown Chopra et al., 2003

morpheus’ molecule

1-like mom1-like TGS unknown pers. comm. Biogemma

Rpd-3 like Rpd-3

like TGS(PEV) unknown De Rubertis et al., 1996

Rossi et al., 2003

4.2 Results