Investigating the roles of arabidopsis polycomb-group genes in regulating flowering time and during plant development by (I) challenging silencing and (II) developing approaches to dissect Pc-G action
Abstract
Polycomb-group (Pc-G) proteins regulate homeotic gene silencing associated with the
repressive covalent histone modification, trimethylation of histone H3 lysine 27 (H3K27me3).
Pc-G mediated silencing is believed to remodel chromatin, rendering target genes
inaccessible to transcription factors. Pc-G mediated silencing might result in irreversible
changes in chromatin structure, however, there has been little analysis addressing whether
Pc-G mediated silencing is reversible. In this work we focused on CURLY LEAF (CLF), the
first Pc-G homologue discovered in Arabidopsis. CLF mediated repression of the floral
homeotic gene AGAMOUS (AG) was challenged during early and late leaf development. AG
was activated by the late leaf promoter, revealing that Pc-G mediated silencing can be
overcome in old leaves in the presence of CLF. AG was also activated in young leaf
primordia, yet did not persist in older leaves, revealing that transient activation of a Pc-G
target is not epigenetically stable. To address the mechanism of Pc-G action within an
endogenous environment, the histone dynamics at the APETALA1 (AP1) locus were
characterized by Chromatin Immunoprecipitation. Unexpectedly, we found that the activation
of AP1 in leaves did not require the removal of H3K27me3, questioning whether H3K27me3
is sufficient to silence. The roles of CLF in leaf and flower development are masked due to partial redundancy with
SWINGER (SWN). clf- swn- mutants form a callus-like mass on sterile-tissue culture with no
distinguishable plant organs. The role of CLF in regulating flowering time in natural
populations of A. thaliana was investigated by complementing clf- mutants with CLF alleles
from two accessions. We found that natural variation in CLF did not affect flowering time. To
dissect the roles of CLF and SWN in late leaf and flower development, two approaches were
developed for targeted expression. Firstly, CLF was introduced into the LhG4/ pOp
transactivation system to provide CLF during early plant development. For mosaic analysis,
CLF was introduced into the CRE lox recombination system in order to create clf- sectors
surrounded by CLF+ SWN+ and CLF+ swn- cells.