Signal transducer and activator of transcription 5a (STAT5a) represses mitochondrial gene expression through direct binding to mitochondrial DNA

https://doi.org/10.1016/j.bbrc.2020.04.152Get rights and content

Highlights

  • IL-3 stimulation represses mitochondrial DNA expression from all three promoters.

  • Repressed mitochondrial DNA expression involves STAT5a binding to D-loop in vivo.

  • Mitochondrial targeting of exogenous STAT5a reduces mitochondrial DNA expression.

  • STAT5a DNA-binding activity is involved in reducing mitochondrial DNA expression.

  • Mitochondrial STAT5a association with PDC-E2 correlates with this repression.

Abstract

Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors essential for cytokine signaling. Our previous study showed that interleukin-3 (IL-3) induced STAT5 translocation to mitochondria and binding to mitochondrial DNA (mtDNA) in vitro. In this report, we further demonstrated in vivo binding of endogenous STAT5a to mtDNA transcriptional control region and reduced gene expression from all three mtDNA promoters after IL-3 stimulation. To specifically define the function of mitochondrial STAT5a, we generated mitochondrial-targeting wild-type and mutant STAT5a proteins. Compared with non-targeting STAT5a, mitochondrial-targeting wild-type STAT5a significantly reduced mitochondrial gene expression in transfected HEK293 cells. The level of attenuation was amplified in cells expressing constitutively active STAT5a, but abrogated in cells expressing DNA-binding-defective STAT5a. STAT5a-mediated repression of mtDNA expression also positively correlated with STAT5a binding to the E2 subunit of pyruvate dehydrogenase complex (PDC-E2), both a gate-keeping metabolic enzyme and a component of mtDNA nucleoid in mitochondrial matrix. Metabolic shift away from mitochondrial respiration is known in many cytokine-stimulated cells and cancer cells. STAT5a-mediated repression of mitochondrial gene expression and its interaction with PDC-E2 may provide important insights into its underlying mechanisms.

Introduction

One of the cancer hallmarks is metabolic shift away from oxidative phosphorylation in mitochondria and toward aerobic glycolysis in cytoplasm, widely known as the “Warburg effect” [1,2]. This characteristic metabolic reprogramming also occurs in highly proliferating cells, such as in cytokine-stimulated cells [3]. Mitochondrial oxidative phosphorylation is carried out by the electron transport chain (ETC) complexes embedded in the inner membrane to generate ATP. While the majority of ETC components are encoded by the nuclear genome, 13 ETC components are encoded by the mitochondrial genome [4]. In addition to the 13 ETC polypeptides, the circular mitochondrial DNA (mtDNA) also encodes 2 ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs) as components of the mitochondrial translational machinery. Consistent with reduced oxidative phosphorylation, lower levels of mitochondrial gene expression have been reported in both cancer cells [5] and cytokine-stimulated cells [6]. However, the mechanisms underlying altered mtDNA regulation remain largely unknown.

Expression of mtDNA is controlled by the D-loop region that drives transcription from 2 heavy-strand promoters (HSP) and 1 light-strand promoter (LSP) [4]. Each promoter drives the expression of a polycistronic precursor RNA that gives rise to different mRNAs, rRNAs and tRNAs. There is extensive crosstalk between mtDNA and the nuclear genome. Mitochondrial transcriptional components, including RNA polymerase and transcription factors, are all encoded by nuclear DNA. Increasing evidence further demonstrates that distinct nuclear transcription factors exhibit additional functions in the mitochondrion [7]. In neurons, binding of cAMP response element-binding protein (CREB) to mtDNA D-loop is linked to elevated mitochondrial gene expression and ETC activity [8]. On the other hand, NF-κB (RelA) binding to mtDNA D-loop inhibits mitochondrial gene expression [9]. These findings point to the existence of a complex nuclear-mitochondrial network through dual functions of transcription factors in regulating both genomes.

Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors essential for cellular response to cytokines [10]. Upon stimulation, tyrosine-phosphorylated STAT proteins dimerize, translocate to the nucleus, and regulate specific gene expression to modulate cellular functions. We reported previously that tyrosine-phosphorylated STAT5 also translocated to mitochondria in cytokine-stimulated cells [11]. Consistent with constitutive STAT5 activation in human cancers, mitochondrial STAT5 was detected in leukemic T cells [11]. Other than STAT5, STAT3 has also been detected in the mitochondrion and contributes to cellular respiration and transformation [12,13]. Initial reports of mitochondrial STAT3 showed STAT3 interaction with the ETC component without binding to mtDNA. On the contrary, we showed that mitochondrial STAT5 recognized a potential STAT5 binding site within mtDNA D-loop region [11]. These findings suggest that STAT5 may regulate mitochondrial gene expression through direct binding to mtDNA. STAT5a and STAT5b are two closely related STAT proteins that exhibit overlapping and distinct functions [14]. In this report, we specifically examined the role of mitochondrial STAT5a in regulating mitochondrial gene expression.

Section snippets

Cell lines and culture conditions

Maintenance of the mouse pro-B-cell line BaF3 and the human HEK293 cell line has been described previously [15]. For cytokine stimulation experiments, BaF3 cells were deprived of interleukin-3 (IL-3) for 16 h and then either left untreated or stimulated with 10 ng/ml of recombinant mouse IL-3 (Millipore, Billerica, MA) for 30 min.

Construction and transfection of plasmids

Mitochondrial-targeting STAT5a expression constructs were generated in the pEF/myc/mito cloning vector (Invitrogen). pEF6/V5-His plasmids expressing wild-type mouse

Results and discussion

We showed previously that IL-3 stimulation of BaF3 cells led to mitochondrial translocation of tyrosine-phosphorylated STAT5 [11]. In our in vitro assay, mitochondrial STAT5 binds to a potential STAT5 site between HSP and LSP that drive transcription of circular mtDNA from two opposite directions. To confirm whether endogenous STAT5a binds to mtDNA in vivo, we performed ChIP using primers flanking the mtDNA STAT5 site. As shown in Fig. 1A, significant STAT5a binding to mtDNA was detected in

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported in part by research grants from Chang Gung University (EMRPD1G0191 and EMRPD1H0341), the Ministry of Science and Technology, Taiwan (MOST 105-2320-B-182-041 and MOST 106-2320-B-182-042) and Chang Gung Medical Research Foundation (CMRPD1F0361, CMRPD1F0362 and CMRPD1F0363), Taiwan (to C.L.Yu).

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    These authors contributed equally to this work.

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