transcription at multiple loci throughout many different stages of development (Mozgova and Hennig, 2015; Comet et al., 2016). In A. thaliana, two lncRNAs have been identified that bind to PRC2. COLDAIR is located within the first intron of FLC and recruits PRC2 to this loci (Heo and Sung, 2011). COLDAIR recruits PRC2 to the first intron of FLC, where PRC2 deposits the repressive mark H3K27me3 (Wood et al., 2006; De Lucia et al., 2008; Heo and Sung, 2011). Subsequently, COLDWRAP is transcribed from the FLC promoter, binds to PRC2 and H3K27me3 is then deposited at the FLC promoter (Kim and Sung, 2017). By recruiting PRC2, COLDAIR and COLDWRAP facilitate long term epigenetic silencing of FLC to enable flowering. Studies in humans have shown that approximately 20% of lncRNAs bind to PRC2 and additional lncRNAs are bound by other chromatin-modifying complexes (Khalil et al., 2009). First identified genetically in Drosophila as the bithorax gene complex (Lewis, 1978), molecular studies have shown PRC2 is composed of four subunits; Enhancer of zeste E(z), Suppressor of zeste Su(z), Extra sex combs (Esc) and p55. The Drosophila subunits have multiple conserved counterparts within plants and at least three forms of PRC2 are functionally active during different life stages of A. thaliana (Mozgova and Hennig, 2015). In Drosophila, E(z) possesses the repressive H3K27 methyltransferase activity (Czermin et al., 2002; Muller et al., 2002). In plants, the three different E(z) methyltransferase homologs are CURLY LEAF (CLF), SWINGER (SWN) and MEDEA (MEA) (Goodrich et al., 1997; Grossniklaus et al., 1998; Luo et al., 1999; Chanvivattana et al., 2004). CLF is expressed in vegetative tissues and it has been demonstrated that COLDAIR and COLDWRAP bind to this subunit during vernalisation (Heo and Sung, 2011; Kim and Sung, 2017). PRC2 exists in a dimeric state, containing at least two RNA-binding subunits, however in mammals, each PRC2 dimer only binds to one RNA molecule (Davidovich et al., 2014). Mammalian lncRNAs can have strong binding specificity to PRC2 in vitro, however, binding specificity in vivo remains to be
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demonstrated (Davidovich et al., 2015). Many identified PRC2-bound lncRNAs are predicted to act in cis. This could be the result of the PRC2 being recruited by nascent RNA that is tethered to Pol II (Lee, 2012; Kaneko et al., 2013).
By analogy with studies in humans, it is likely that many plant lncRNAs bind to PRC2. Furthermore, lncRNAs potentially recruit PRC2 to the locus of lncRNA transcription, facilitating repression in cis. In this chapter, lncRNAs will be identified in PRC2 mutants that have demonstrated roles in seed development. FERTILISATION INDEPENDENT SEEDS (FIS) PRC2 is only active during A. thaliana reproductive development, being expressed in the central cell and endosperm nuclei for a short time (Mozgova and Hennig, 2015). FIS PRC2 is distinct from other forms of PRC2 that are active during vegetative development and vernalisation, as not all subunits are shared between different forms of PRC2. The four subunits of FIS PRC2 are; E(z) homolog MEA, Su(z) homolog FIS2, Esc homolog FERTILISATION INDEPENDENT ENDOSPERM (FIE) and p55 homolog MULTICOPY SUPPRESSOR OF IRA1 (MSI1) (Mozgova and Hennig, 2015). Both MEA and FIS2 are specific to FIS PRC2; FIE and MSI1 are common to other forms of PRC2 (Mozgova and Hennig, 2015). Both MEA and FIS2 have reproductive-specific expression in the central cell and endosperm, whereas other components are expressed in many tissues (Luo et al., 2000; Qiu et al., 2017). In A. thaliana, certain mutations in MEA, FIS2, FIE and MSI1 enable autonomous endosperm development in the absence of fertilisation (Ohad et al., 1996; Chaudhury et al., 1997; Luo et al., 1999; Kohler et al., 2003). Therefore, FIS PRC2 controls key aspects of endosperm and seed development. In this chapter, lncRNAs will be identified in homozygous MEA and FIS2 mutants; mea1-1/mea1-1 and fis2-3/fis2-3 (originally reported as fis1/fis1 and fis2/fis2) (Chaudhury et al., 1997; Luo et al., 1999). These homozygotes are rare as mea1-1 and fis2-3 are maternal gametophytic lethal, being transmitted at very low levels (Chaudhury et al., 1997; Luo et al., 1999). Accordingly, the heterozygous plants MEA/mea1-1 and FIS2/fis2-3 can be identified by a 1:1 ratio of viable and embryo-arrested seeds. However, when the progeny of these heterozygotes was examined, Chaudhury et al. (1997) identified that 1 plant in 450 and 2 in 1,621 of the F1 were homozygous; mea1-1/mea1-1 and fis2-3/fis2-3
respectively. These plants give rise to relatively few (if any) viable seeds, but enough to maintain a homozygous line. Predominantly the embryo of these mutants arrests which is followed by seed atrophy. It is considered that a functional FIS PRC2 is not present in these plants. As the heterozygotes contain wild type alleles with a functional FIS PRC2, the homozygous lines will be used. 1 DAP siliques will be harvested from mea1-1/mea1-1 and
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fis2-3/fis2-3 plants, where seed development is occurring despite eventual arrest and atrophy. RNA-seq of these siliques will provide insights into novel lncRNAs regulated by FIS PRC2 during early silique and seed development. It is anticipated that lncRNAs and protein coding genes located in the endosperm will also be identified. It is estimated that the endosperm constitutes 0.1 – 1% of 1 DAP siliques, as each endosperm contains 44 – 48 nuclei during this stage (Boisnard-Lorig et al., 2001). However, as high-throughput next- generation sequencing technology can generate 4x108 reads (Illumina® HiSeq 2000), transcripts from the endosperm will be sampled (Goodwin et al., 2016).
3.1.2 Aims
In this chapter, A. thaliana mutations mea1-1/mea1-1 and fis2-3/fis2-3 will be utilised to identify lncRNAs. MEA and FIS2 expression is primarily in the central cell and endosperm; it is predicted that RNA-seq of mea1-1/mea1-1 and fis2-3/fis2-3. Altered lncRNA populations may potentially provide insights into lncRNAs that are regulated by or associated with FIS PRC2. Aims are as follows:
1. Identify lncRNAs in A. thaliana mea1-1/mea1-1 and fis2-3/fis2-3 1 DAP siliques.
2. Determine if lncRNAs undergo differential expression in mea1-1/mea1-1 and fis2-
3/fis2-3 mutants compared to wild type.
3. Identify novel lncRNA candidates for further investigation.
RNA-seq will be performed on homozygous A. thaliana mea1-1/mea1-1 and fis2- 3/fis2-3 1 DAP siliques. LncRNAs will be identified, annotated and their expression will be compared to wild type. LncRNA candidates exclusively identified in mea1-1/mea1-1 and fis2- 3/fis2-3 mutants will be further investigated bioinformatically. These will provide a foundation for further research into lncRNAs potentially regulated by FIS PRC2 or associated with FIS PRC2. Furthermore, these may have roles in silique and seed development.
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