Marker gene tethering by nucleoporins affects gene expression in plants

Abstract

In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localize at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants.

Light and temperature shape nuclear architecture and gene expression

“…It is worth mentioning that several light-induced genes such as CHLOROPHYLL A/B-BINDING ( CAB ), RUBISCO SMALL SUBUNIT ( RBCS ), PLASTOCYANIN ( PC ) and GENOMES UNCOUPLED 5 ( GUN5 ) have been observed to re-position to the nuclear periphery in response to light [ 35 •• ]. Tethering experiments of reporter genes to a plant nucleoporin showed an increase in gene expression [ 36 ], whereas NUP1-associated sequences were enriched in repressed genes [ 31 ].…”

“…These studies indicate that the plant NE potentially holds a dual role in regulating gene expression depending on the chromatin context and precise topology on the nuclear periphery (NPC versus lamin-like protein association) [ 31 , 35 •• , 36 ]. Although yeast and mammalian systems have provided key examples demonstrating a direct association between gene repositioning to the nuclear periphery with gene activation or repression, there is very limited information correlating nuclear positioning and gene expression in plants [ 14 , 35 •• ].…”

Dynamic Changes in Plant Nuclear Organization in Response to Environmental and Developmental Signals

“…In Drosophila melanogaster, chromatin mobility is important for interacting with distinct nuclear compartments during differentiation (Thakar and Csink, 2005). Furthermore, altering nuclear morphology changes chromatin organization and disrupts genome function (Lanctôt et al, 2007;Ramdas and Shivashankar, 2015;Smith et al, 2015).…”

“…Feng et al (2014) visualized lightmediated chlorophyll a/b-binding protein (CAB) loci repositioning from the nuclear interior to the periphery, which was associated with increased CAB gene expression. Smith et al (2015) showed that artificially tethering a locus to a nucleoporin enhances its expression. Another group performed an in vivo study that identified and characterized Arabidopsis chromatin regions that preferentially associated with the nuclear periphery.…”

Chromatin Tagging Systems Contribute to Live Imaging Analyses for Chromatin Dynamics

“…The chromatin tagging system revealed that the arrangement of chromatin positioning was closely related to the regulation of gene activity. For example, the nuclear lamina regulates the transcriptional activity through repositioning of specific chromatins in animal cultured cells and A. thaliana (Reddy et al 2008, Kumaran and Spector 2008, Finlan et al 2008, Smith et al 2015. The chromatin tagging system also contributes to the analyses of the high-order chromatin structure.…”