Threshold-dependent transcriptional discrimination underlies stem cell homeostasis

Abstract

Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis-regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUSbinding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentrationdependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.WUSCHEL | cis-regulatory module | CLAVATA3 | shoot apical meristem | gene regulation

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning

“…Our work also shows that, at higher WUS levels, HOD1 alone is sufficient to mediate homodimerization (20). The redundant function of HOD1 in promoting homodimerization may explain the inflorescence meristem overproliferation observed in the earlier study (17), despite the use of a WUS form that lacked HOD2.…”

“…S5 B-E) (n = 4), lacking the coding sequences of the WUS protein, revealed the stable nuclear accumulation of the protein upon Dex treatment, showing that the protein destabilization is specific to WUS. As seen with the 35S::WUS-GR-expressing seedlings, the 35S::eGFP-WUS-GR seedlings also failed to develop on Dex-containing plates and activated CLV3 (20), upon Dex application, confirming its functionality (Fig. S5A).…”

“…The analysis presented in the accompanying manuscript (20) shows that HOD1 is also required for DNA binding. Consistent with the compromised DNA binding ability of HOD1, wus-7 homozygous mutants failed to activate pCLV3::H2B-mYFP expression at levels comparable with the WT (Fig.…”

“…Nevertheless, the variations in WUS protein levels may also be due to a reduction of protein accumulation caused perhaps by the targeted destabilization in cells located outside the rib meristem. To test this hypothesis, we first expressed eGFP-WUS from a central zone-specific promoter, which led to lower levels of the protein than the eGFP-WUS expressed from the WUS promoter (20). This result shows that ectopic overexpression of WUS in the central zone does not result in higher protein levels, suggesting a destabilization of WUS.…”

“…The destabilization of WUS protein observed upon misexpression of eGFP-WUS in the central zone could also be due to an indirect consequence of meristem overproliferation (10,11,20). To exclude such a possibility, we transiently overexpressed WUS, using a dexamethasone (Dex)-inducible form of WUS protein where eGFP-WUS was fused with the hormone binding domain of the rat glucocorticoid receptor (GR).…”