Plant genomes are extremely sensitive to, and can be developmentally reprogrammed by environmental light cues. Here using rolling-circle amplification of gene-specific circularizable oligonucleotides coupled with fluorescence in situ hybridization, we demonstrate that light triggers a rapid repositioning of the Arabidopsis light-inducible chlorophyll a/b-binding proteins (CAB) locus from the nuclear interior to the nuclear periphery during its transcriptional activation. CAB repositioning is mediated by the red/far-red photoreceptors phytochromes (PHYs) and is inhibited by repressors of PHY signalling, including COP1, DET1 and PIFs. CAB repositioning appears to be a separate regulatory step occurring before its full transcriptional activation. Moreover, the light-inducible loci RBCS, PC and GUN5 undergo similar repositioning behaviour during their transcriptional activation. Our study supports a light-dependent gene regulatory mechanism in which PHYs activate light-inducible loci by relocating them to the nuclear periphery; it also provides evidence for the biological importance of gene positioning in the plant kingdom.
Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes ( PhAPG s). PhAPG s are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.
An improved understanding of the factors that regulate the migration of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) would provide new insights into human heart development and suggest novel strategies to improve their electromechanical integration after intracardiac transplantation. Since nothing has been reported as to the factors controlling hESC-CM migration, we hypothesized that hESC-CMs would migrate in response to the extracellular matrix and soluble signaling molecules previously implicated in heart morphogenesis. To test this, we screened candidate factors by transwell assay for effects on hESC-CM motility, followed by validation via live-cell imaging and/or gap-closure assays. Fibronectin (FN) elicited a haptotactic response from hESC-CMs, with cells seeded on a steep FN gradient showing nearly a fivefold greater migratory activity than cells on uniform FN. Studies with neutralizing antibodies indicated that adhesion and migration on FN are mediated by integrins a-5 and a-V. Next, we screened 10 soluble candidate factors by transwell assay and found that the noncanonical Wnt, Wnt5a, elicited an approximately twofold increase in migration over controls. This effect was confirmed using the gap-closure assay, in which Wnt5a-treated hESC-CMs showed approximately twofold greater closure than untreated cells. Studies with microfluidic-generated Wnt5a gradients showed that this factor was chemoattractive as well as chemokinetic, and Wnt5a-mediated responses were inhibited by the Frizzled-1/2 receptor antagonist, UM206. In summary, hESC-CMs show robust promigratory responses to FN and Wnt5a, findings that have implications on both cardiac development and cell-based therapies.
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