AMPK promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors DNMT1, RBBP7, and HAT1 - PubMed (original) (raw)

AMPK promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors DNMT1, RBBP7, and HAT1

Traci L Marin et al. Sci Signal. 2017.

Abstract

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) acts as a master regulator of cellular energy homeostasis by directly phosphorylating metabolic enzymes and nutrient transporters and by indirectly promoting the transactivation of nuclear genes involved in mitochondrial biogenesis and function. We explored the mechanism of AMPK-mediated induction of gene expression. We identified AMPK consensus phosphorylation sequences in three proteins involved in nucleosome remodeling: DNA methyltransferase 1 (DNMT1), retinoblastoma binding protein 7 (RBBP7), and histone acetyltransferase 1 (HAT1). DNMT1 mediates DNA methylation that limits transcription factor access to promoters and is inhibited by RBBP7. Acetylation of histones by HAT1 creates a more relaxed chromatin-DNA structure that favors transcription. AMPK-mediated phosphorylation resulted in the activation of HAT1 and inhibition of DNMT1. For DNMT1, this inhibition was both a direct effect of phosphorylation and the result of increased interaction with RBBP7. In human umbilical vein cells, pharmacological AMPK activation or pulsatile shear stress triggered nucleosome remodeling and decreased cytosine methylation, leading to increased expression of nuclear genes encoding factors involved in mitochondrial biogenesis and function, such as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), transcription factor A (Tfam), and uncoupling proteins 2 and 3 (UCP2 and UCP3). Similar effects were seen in the aortas of mice given pharmacological AMPK activators, and these effects required AMPK2α. These results enhance our understanding of AMPK-mediated mitochondrial gene expression through nucleosome remodeling.

Copyright © 2017, American Association for the Advancement of Science.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1

Fig. 1. AMPK regulated a nucleosome remodeling network by phosphorylating DNMT1, RBBP7, and HAT1

(A) Protein domains and putative AMPK-mediated phosphorylation sequence of DNMT1, RBBP7, and HAT1. DMAP1, DNA methyltransferase 1–associated protein 1 binding domain; BAH, bromo-adjacent homology domain. (B) Illustrated hypothesis of AMPK-mediated phosphorylation of DNMT1, RBBP7, and HAT1 and effects on mitochondrial biogenesis and function. (C) Kinase assays using recombinant target protein in the presence or absence of activated recombinant AMPKα2β1γ1. Top: Kinase assays conducted with peptides. Bottom: Kinase assays with full-length proteins. n = 4 independent experiments. CPM, counts per minute. (D) Kinase assays using immunoprecipitated wild-type (WT) and mutated DNMT1, RBBP7, and HAT1 proteins from HUVECs. Top: Autoradiograph. Lower: Total protein and immunoglobulin G (IgG) immunoblots for loading. n = 3 independent experiments. (E and F) Coimmunoprecipitation (IP) immunoblots in HUVECs transfected with WT DNMT1, RBBP7, or HAT1 or their corresponding Ser-to-Ala mutants (DNMT1-S730A, HAT1-S190A, and RBBP7-S314A) and treated with AICAR or left untreated for 30 min (top) or 10 min (bottom). P-ACC (phospho–acetyl-CoA carboxylase), T-ACC (total acetyl-CoA carboxylase), T-RBBP7, T-DNMT1, and T-HAT1 immunoblotting was conducted with the input IP crude cell lysate. n = 4 independent experiments. Comp C, compound C. (G and H) Densitometry analysis of coimmunoprecipitation immunuoblots comparing coimmunoprecipitated protein to total protein. *P < 0.05. AU, arbitrary units.

Fig. 2

Fig. 2. AMPK decreased DNMT1 activity

(A and B) DNMT1 activity in HUVECs (A) and AMPK+/+ or _AMPK_−/− MEFs (B) treated with AICAR or metformin or left untreated (CTRL). n = 4 independent experiments for (A) and (B). (C) DNMT1 activity in _AMPK_−/− MEFs infected with Ad-AMPK-CA or Ad-AMPK-DN [50 multiplicity of infection (MOI)]. n = 3 independent experiments. (D) DNMT1 activity in HUVECs transfected with control, AMPK, PARP-1, or RBBP7 small interfering RNA (siRNA) and treated as indicated. n = 3 independent experiments. (E) DNMT1 activity in cells transfected with the indicated forms of DNMT1 or RBBP7 and treated as indicated. n = 4 independent experiments. (F) DNMT1 activity in HUVECs transfected with control, AMPK, PARP-1, or RBBP7 siRNA and subjected to pulsatile shear stress (PSS). n = 3 independent experiments. (G) DNMT1 activity in HUVECs transfected with the indicated forms of DNMT1 or RBBP7 and subjected to PSS. n = 3 independent experiments. *P < 0.05.

Fig. 3

Fig. 3. AMPK activation decreased promoter methylation of _PGC-1_α, NRF1, NRF2, Tfam, UCP2, and UCP3 genes

(A to C) Methylation-specific quantitative polymerase chain reaction (qPCR) analysis of promoter methylation in AICAR- or metformin-treated AMPK+/+ and _AMPK_−/− MEFs compared to nontreated AMPK+/+ group (A); HUVECs transfected with DNMT1-WT, DNMT1-S730A, or DNMT1-S730D (B); or HUVECs transfected with RBBP7-WT, RBBP7-S314A, or RBBP7-S314D (C). Cells were treated as indicated. n = 4 independent experiments for (A) to (C). (D) Promoter methylation status in HUVECs transfected with DNMT1-WT, DNMT1-S730A, or DNMT1-S730D and subjected to PSS. n = 3 independent experiments. *P < 0.05. FC, fold change.

Fig. 4

Fig. 4. AMPK increased HAT1 activity

HAT1 activity was assessed in cells treated with AICAR or metformin or left untreated. (A) AMPK+/+ and _AMPK_−/− MEFs. (B) _AMPK_−/− MEFs were infected with Ad-AMPK-CA or Ad-AMPK-DN AMPK (50 MOI) before treatment. n = 3 independent experiments for (A) and (B). (C) HAT1 activity in HUVECs transfected with control, AMPK, HAT1, or RBBP7 siRNA before the indicated treatment. n = 4 independent experiments. (D) HAT1 activity in cells transfected with control, AMPK, HAT1, or RBBP7 siRNA and then subjected to PSS. n = 3 independent experiments. (E and F) HUVECs were transfected with the indicated forms of HAT1 or RBBP7 and treated as indicated. n = 4 independent experiments for (E) and n = 3 independent experiments for (F). *P < 0.05.

Fig. 5

Fig. 5. AMPK activation decreased nucleosomal compaction of _PGC-1_α, NRF1, NRF2, Tfam, UCP2, and UCP3 genes

(A) H4K5 acetylation in AMPK+/+ or _AMPK_−/− MEFs treated with or without AICAR or metformin. n = 4 independent experiments. (B) H4K5 acetylation in HUVECs expressing indicated forms of HAT1 and treated as indicated. n = 4 independent experiments. (C) H4K5 acetylation in HUVECs expressing the indicated forms of RBBP7 and treated as indicated. n = 4 independent experiments. (D) H4K5 acetylation in cells expressing the indicated forms of HAT1 and then subjected to PSS. n = 3 independent experiments. (E to G) FAIRE analysis in HUVECs transfected with the indicated forms of DNMT1, RBBP7, or HAT1 and treated as indicated. n = 4 independent experiments. *P < 0.05.

Fig. 6

Fig. 6. AMPK activation increased mitochondrial biogenesis and function

HUVECs expressing the indicated forms of DNMT1, RBBP7, or HAT1 were treated with AICAR or left untreated. (A) JC-1 staining. n = 6 independent experiments. (B) MitoTracker staining. n = 6 independent experiments. (C) Quantification of MitoTracker staining of six fields. n = 6 independent experiments. (D) Mitochondrial DNA (mtDNA) abundance. (E to H) Activity of citrate synthase (E), complex I (F), complex IV (G), and complex V (H). (I) ATP abundance. n = 3 independent experiments. RFU, relative fluorescence unit. (J) ROS abundance. n = 3 independent experiments for (D) to (J). *P < 0.05.

Fig. 7

Fig. 7. AICAR regulates nucleosome remodeling and gene expression through AMPKα2 in vivo

Aortas were isolated from AMPK_α_2+/+ and _AMPK_α_2_−/− mice administered AICAR or metformin. (A) DNMT1 binding to promoters. (B) HAT1 binding to promoters. (C) Euchromatin abundance. (D) Methylation status. (E) Abundance of the indicated mRNAs. (F) Complex IV activity. n = 12 mice per genotype and treatment. *P < 0.05.

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