Distinct isoforms of Nrf1 diversely regulate different subsets of its cognate target genes

Meng Wang,Lu Qiu,Yiguo Zhang,Xufang Ru,Yijiang Song

doi:10.1038/s41598-019-39536-0

Abstract

The single Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1α, Nrf1β and Nrf1γ, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have herein established three cell lines on the base of the Flp-In T-REx system, which are allowed for the tetracycline-inducibly stable expression of Nrf1α, Nrf1β and Nrf1γ. Consequently, the RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1α and/or Nrf1β following induction by tetracycline. By contrast, the other DEGs regulated by Nrf1γ were far less than those regulated by Nrf1α/β (i.e. ~11% of Nrf1α and ~7% of Nrf1β). However, further transcriptomic analysis revealed that the tetracycline-induced expression of Nrf1γ significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1γ acts as a major dominant-negative inhibitor competitively against Nrf1α/β activity, such that a number of DEGs regulated by Nrf1α/β are counteracted by Nrf1γ.

Highlights

Nuclear factor-erythroid 2-related factor 1 (Nrf[1], called Nfe2l1) acts as a transcription factor belonging to the cap’n’collar (CNC) basic-region leucine zipper family, which is indispensable for maintaining both cellular homoeostasis and organ integrity during normal development and growth, as well as the adaptative responses to other pathophysiological processes[1,2,3]
This demonstrates that Nrf1γ exerts a distinguishable effect from the other two isoforms Nrf1α/β, on the transcriptional expression of Nrf1-target genes, because its inducible expression by Tet significantly increased the percentage of down-regulated genes among the differentially expressed genes (DEGs) detected
Nrf1α, Nrf1β and Nrf1γ) to the precision regulation of different subsets of its target genes, each of these isoform-expressing cell lines was established by Flp recombinase-mediated integration on the base of the Flp-In T-REx-293 host cells

Summary

Introduction

Nuclear factor-erythroid 2-related factor 1 (Nrf[1], called Nfe2l1) acts as a transcription factor belonging to the cap’n’collar (CNC) basic-region leucine zipper (bZIP) family, which is indispensable for maintaining both cellular homoeostasis and organ integrity during normal development and growth, as well as the adaptative responses to other pathophysiological processes[1,2,3]. On the contrary, when Nrf1γ is forcedly expressed, the consequence enables it to make a possible interference with the functional assembly of each of the active transcription factors (i.e. Nrf1α or Nrf2) with its heterodimeric partner (i.e. sMaf and other bZIP proteins), in order to down-regulate expression of AP1-like ARE-driven target genes[12,13]. To date, it is, unknown how each isoform of Nrf[1] contributes to its unique role in regulating expression of ARE-driven cytoprotective genes against various physiopathological stresses. This work provides a further better understanding of distinctions in the transcriptional regulation of cognate Nrf1-target genes by its different isoforms

Methods

Results

Conclusion