scholarly journals An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion

2019 ◽  
Author(s):  
Kizhakke Mattada Sathyan ◽  
Brian D. McKenna ◽  
Warren D. Anderson ◽  
Fabiana M. Duarte ◽  
Leighton Core ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Function ◽  
Specific Protein ◽  
Auxin Signaling ◽  
Auxin Response ◽  
Protein Levels ◽  
Genome Wide ◽  
Pb1 Domain ◽  
Inducible Protein ◽  
Endogenous Loci

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding down-stream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in non-plant systems. However, tagging proteins at their endogenous loci results in chronic, auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the Auxin Response Transcription Factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems, but ARF is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin and we found that expression of the ARF Phox and Bem1 (PB1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transciptional profiling indicates that ZNF143 activates transcription in cis and ZNF143 regulates promoter-proximal paused RNA Polymerase density. Rapidly inducible degradation systems that preserve the target protein’s native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

scholarly journals Genome-wide RNA-seq analysis indicates that the DAG1 transcription factor promotes hypocotyl elongation acting on ABA, ethylene and auxin signaling

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Riccardo Lorrai ◽  
Francesco Gandolfi ◽  
Alessandra Boccaccini ◽  
Veronica Ruta ◽  
Marco Possenti ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Hypocotyl Elongation ◽  
Auxin Signaling ◽  
Rna Seq ◽  
Genome Wide

scholarly journals Protein Levels of Several Arabidopsis Auxin Response Factors Are Regulated by Multiple Factors and ABA Promotes ARF6 Protein Ubiquitination

2020 ◽  
Vol 21 (24) ◽  
pp. 9437
Author(s):  
Keke Li ◽  
Sheng Wang ◽  
Hong Wu ◽  
Hong Wang
Keyword(s):  
Transcript Level ◽  
26S Proteasome ◽  
Auxin Signaling ◽  
Auxin Response ◽  
Response Factors ◽  
Protein Levels ◽  
Multiple Factors ◽  
Protein Ubiquitination ◽  
Regulated Gene Expression ◽  
Arf Protein

The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.

scholarly journals Genome-Wide Identification and Co-Expression Analysis of ARF and IAA Family Genes in Euscaphis konishii: Potential Regulators of Triterpenoids and Anthocyanin Biosynthesis

2022 ◽  
Vol 12 ◽  
Author(s):  
Bobin Liu ◽  
Juanli Zhu ◽  
Lina Lin ◽  
Qixin Yang ◽  
Bangping Hu ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Southern China ◽  
Gene Families ◽  
Auxin Signaling ◽  
Auxin Response ◽  
Chromosomal Distribution ◽  
Element Analysis ◽  
Genome Wide ◽  
Gene Structures

Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.

EIN3-Mediated Ethylene Signaling Attenuates Auxin Response during Hypocotyl Thermomorphogenesis

2021 ◽  
Author(s):  
Jae Young Kim ◽  
Young-Joon Park ◽  
June-Hee Lee ◽  
Zee Hwan Kim ◽  
Chung-Mo Park
Keyword(s):  
Transcription Factor ◽  
Cell Elongation ◽  
Fine Tuning ◽  
Auxin Signaling ◽  
Plant Responses ◽  
Proton Pumps ◽  
Natural Environments ◽  
Auxin Response ◽  
Hypocotyl Growth ◽  
Gene Encoding

Abstract The gaseous phytohormone ethylene plays vital roles in diverse developmental and environmental adaptation processes, such as fruit ripening, seedling establishment, mechanical stress tolerance, and submergence escape. It is also known that in the light, ethylene promotes hypocotyl growth by stimulating the expression of PHYTOCHROME INTERACTING FACTOR3 (PIF3) transcription factor, which triggers microtubule reorganization during hypocotyl cell elongation. In particular, ethylene has been implicated in plant responses to warm temperatures in recent years. However, it is currently unclear how ethylene signals are functionally associated with hypocotyl thermomorphogenesis at the molecular level. Here, we show that ETHYLENE-INSENSITIVE3 (EIN3)-mediated ethylene signals attenuate hypocotyl thermomorphogenesis by suppressing auxin response. At warm temperatures, when the activity of the PIF4 thermomorphogenesis promoter is prominently high, the ethylene-activated EIN3 transcription factor directly induces the transcription of ARABIDOPSIS PP2C CLADE D7 (APD7) gene encoding a protein phosphatase that inactivates the plasma membrane (PM) H+-ATPase proton pumps. In conjunction with the promotive role of the PM H+-ATPases in hypocotyl cell elongation, our observations strongly support that the EIN3-directed induction of APD7 gene is linked with the suppression of auxin-induced cell expansion, leading to the reduction of thermomorphogenic hypocotyl growth. Our data demonstrate that APD7 acts as a molecular hub that integrates ethylene and auxin signals into hypocotyl thermomorphogenesis. We propose that the ethylene-auxin signaling crosstalks via the EIN3-APD7 module facilitate the fine-tuning of hypocotyl thermomorphogenesis under natural environments, which often fluctuate in a complex manner.

scholarly journals High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Pierce ◽  
Jeffrey M. Granja ◽  
William J. Greenleaf
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cancer Cells ◽  
High Throughput ◽  
Regulatory Networks ◽  
Temporal Dynamics ◽  
Chromatin Accessibility ◽  
Regulatory Regions ◽  
Factor Binding ◽  
Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

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