Comparing the epigenetic landscape in myonuclei purified with a PCM1 antibody from a fast/glycolytic and a slow/oxidative muscle

Abstract

Muscle cells have different phenotypes adapted to different usage, and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of the epigenetic landscape by ChIP-Seq in two muscle extremes, the fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where up to 60% of the nuclei can be of a different origin. Since cellular homogeneity is critical in epigenome-wide association studies we developed a new method for purifying skeletal muscle nuclei from whole tissue, based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labelling and a magnetic-assisted sorting approach, we were able to sort out myonuclei with 95% purity in muscles from mice, rats and humans. The sorting eliminated influence from the other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the differences in the functional properties of the two muscles, and revealed distinct regulatory programs involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles were also regulated by different sets of transcription factors; e.g. in soleus, binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SIX1 binding sites were found to be overrepresented. In addition, more novel transcription factors for muscle regulation such as members of the MAF family, ZFX and ZBTB14 were identified.

Exercise‐induced histone H3 trimethylation at lysine 27 facilitates the adaptation of skeletal muscle to exercise in mice

“…myonucleus-specific marker (Bengtsen et al, 2021;Winje et al, 2018), confirming that the histones extracted from myonuclei were enriched in this fragment (Figs 5D and S3). Furthermore, the blots show that exercise training drastically increased the H3K27me3 level in the muscle fibres of the GSK343-treated group (3.4-fold vs. GSK343-treated control).…”

“…The myonuclear H3K27me3 levels were also analysed by western blotting in plucked single fibres. Immunohistochemical labelling of the single fibres showed that >95% of observed nuclei were positive for PCM1, a myonucleus‐specific marker (Bengtsen et al., 2021; Winje et al., 2018), confirming that the histones extracted from myonuclei were enriched in this fragment (Figs 5 and S3). Furthermore, the blots show that exercise training drastically increased the H3K27me3 level in the muscle fibres of the GSK343‐treated group (3.4‐fold vs .…”

“…In contrast, these modifications were prevalent at the loci specific to the fast-twitch plantaris muscle (Kawano et al, 2015). Bengtsen et al (2021) compared the epigenetic landscape in purified myonuclei between slow-twitch soleus and fast-twitch extensor digitorum longus muscles in adult mice and reported that the peaks of H3K4me3 and histone H3 acetylation at lysine 27 (H3K27ac) were commonly found at the loci specific for soleus or extensor digitorum longus muscle. Although the whole aspect of exercise-induced epigenetics in skeletal muscle has recently been uncovered, the dynamics of histone modifications and their roles in the adaptation of skeletal muscle to exercise are still largely unknown.…”

Transplantation of PSC-derived myogenic progenitors counteracts disease phenotypes in FSHD mice

“…1b ). We validated the position of the RFP + nuclei inside the myofiber and the subsequent quantifications using a specific marker for myonuclei, PCM1 56 , 57 (Fig. 1c, d ).…”

Cross Talk rebuttal: Schwartz and Gundersen

“…Based on lineage tracing studies in mouse, they suggest that ∼4% of nuclei are newly acquired in homeostatic muscle (Masschelein et al., 2020). However, as the authors of that paper warn, this represents a baseline‐value with methodological problems associated with: promoter leakiness, imperfect sorting (see Bengtsen et al., 2021), and diffusible reporters. On the presumption that myonuclear numbers do increase over time, Kirby and Dupont‐Versteegden (2022a) reason that some mechanism for nuclear elimination must maintain steady‐state numbers.…”