Study on the Imprinting Mechanism of the UPWARD CURLY LEAF1 (UCL1) Gene in Arabidopsis

Abstract

Genomic imprinting, an epigenetic process in mammals and flowering plants, refers to the differential expression of alleles of the same genes in a parent-of-origin-specific manner. In flowering plants like Arabidopsis, genomic imprinting has been detected only in the endosperm and it is regulated by Polycomb Repressive Complex 2 (PRC2) through trimethylation of lysine 27 on histone H3 (H3K27me3). Recent high-throughput sequencing analyses revealed that more than 200 loci are imprinted in Arabidopsis

however, only a few of these imprinted genes and their imprinting mechanisms have been examined in detail. In a previous study, it was reported that UPWARD CURLY LEAF1 (UCL1), a gene encoding an E3 ligase that degrades the CURLY LEAF (CLF) polycomb protein, is a paternally expressed imprinted gene (PEG). After fertilization, paternally inherited UCL1 is expressed in the endosperm, but not in the embryo. FERTILIZATION INDEPENDENT SEED2-PRC2 (FIS2-PRC2) silences the maternal UCL1 allele in the central cell before fertilization and in the endosperm after fertilization. In this study, the expression pattern of the UCL1::GUS genes suggests that the polycomb response element (PRE) of UCL1 is located between -2.5 and –2.4 kb upstream of the UCL1 translation start codon. To investigate exact PRE sequences of UCL1, I generated UCL1_2.7k::GUS constructs with 10 bp-scanning transversion mutagenesis between -2.5 and –2.4 kb. Their GUS expressions need to be checked in the Col-0 background transformants after floral dip. The PRE cooperated with the endosperm-specific factor binding element (ESFE), -1.0 kb upstream of UCL1, to drive the paternal imprinting and endosperm-specific expression of the UCL1::GUS gene. However, the imprinting pattern of the UCL1_PRE+ESFE::GUS was relatively unstable. To identify additional element which is essential for repression of maternal UCL1 allele with PRE, new construct containing -1814 to –1478 bp upstream of UCL1, the putative differentially methylated region (DMR), was generated. UCL1_PRE+DMR+ESFE::GUS showed complete suppression of maternal UCL1 allele, that indicates DMR is essential for the paternal imprinting of the UCL1 gene. On the other hand, the expression pattern of the UCL1::GUS genes those contain sequential deleted ESFE suggests that ESFE of UCL1 is located between –271 bp and –171 bp. Specific transcription factor may bind to this ESFE sequence for endosperm-specific expression of UCL1.