Acetylation and phosphorylation of human TFAM regulate TFAM–DNA interactions via contrasting mechanisms

Abstract

Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression and transmission of mitochondrial DNA (mtDNA). However, mechanisms for the post-translational regulation of TFAM are poorly understood. Here, we show that TFAM is lysine acetylated within its high-mobility-group box 1, a domain that can also be serine phosphorylated. Using bulk and single-molecule methods, we demonstrate that site-specific phosphoserine and acetyl-lysine mimics of human TFAM regulate its interaction with non-specific DNA through distinct kinetic pathways. We show that higher protein concentrations of both TFAM mimics are required to compact DNA to a similar extent as the wild-type. Compaction is thought to be crucial for regulating mtDNA segregation and expression. Moreover, we reveal that the reduced DNA binding affinity of the acetyl-lysine mimic arises from a lower on-rate, whereas the phosphoserine mimic displays both a decreased on-rate and an increased off-rate. Strikingly, the increased off-rate of the phosphoserine mimic is coupled to a significantly faster diffusion of TFAM on DNA. These findings indicate that acetylation and phosphorylation of TFAM can fine-tune TFAM–DNA binding affinity, to permit the discrete regulation of mtDNA dynamics. Furthermore, our results suggest that phosphorylation could additionally regulate transcription by altering the ability of TFAM to locate promoter sites.

Mitochondrial DNA: Epigenetics and environment

“…mtDNA transcription is still an active area of research as several unknown variables related to initiation, elongation, and termination remain to be elucidated. Of the mitochondrial transcription components, TFAM appears to be regulated via post-translational modifications, where the precise implications of these modifications on epigenetic regulation of mitochondrial transcription are unknown (Lu et al, 2013;King et al, 2018). Studies involving in vitro reconstitution of mtDNA transcription minimally requires TFAM, mitochondrial transcription factor B2 (TFB2M), and mitochondrial RNA polymerase where the precise role of TFAM has been debated.…”

“…1; reviewed in Shokolenko and Alexeyev, 2017). Of the mitochondrial transcription components, TFAM appears to be regulated via post-translational modifications, where the precise implications of these modifications on epigenetic regulation of mitochondrial transcription are unknown (Lu et al, 2013;King et al, 2018).…”

“…TFAM, the main structural constituent of mitochondrial nucleoids is one example of a mitochondrial protein that gets post-translationally modified via acetylation, O-linked glycosylation, and phosphorylation (Suarez et al, 2008;Lu et al, 2013;King et al, 2018). Two serine residues (S55 and S56) within the high-mobility group 1 (HMG1) domain are primary sites for phosphorylation, whereas four lysine residues (K62, K76, K111, and K118) also within HMG1 appear to be sites for acetylation ( Fig.…”

Supercoiling DNA optically

“…S9), it is still lower than the maximum limit for rotationcoupled diffusion along the DNA backbone. 28 This implies that the protein is slowed down by more stable transient interactions with the DNA. The current work suggests that 9/25 these interactions are either stronger and / or less transient when the DNA is underwound.…”

“…eGFP-RPA and alexa-555-TFAM were prepared as detailed previously. 20,28 Generation of negatively supercoiled DNA. The method employs the following protocol to generate negative supercoiling in DNA (see also Fig.…”