DOI: 10.25345/c5kh0fb2s
title: MassIVE MSV000096839 - Structural obstruction to full DNA replication in the chromatin of terminally differentiated skeletal muscle cells
title: Terminal cell differentiation is often associated with permanent withdrawal from proliferation, termed the postmitotic state. Though widespread among vertebrates and determinant for their biology, the molecular underpinnings of this state are poorly understood. Postmitotic skeletal muscle myotubes can be induced to reenter the cell cycle; however, they generally die as a result of their inability to complete DNA replication. Here, we explore the causes of such incompetence. Genomic hybridization of newly synthesized DNA showed that the replicative failure does not concern specific genomic regions, but can stochastically affect any of them. Myoblast and myotube were incubated in replicative Xenopus egg extract, which provides a full DNA replication machinery. While myoblast nuclei attained complete DNA replication, those from myotubes, even in these conditions, duplicated less than half of their genomes, strongly indicating that the very structure of myotube chromatin obstructs DNA replication. Furthermore, disassembling and disorganizing chromatin with a strong salt treatment did not modify the replicative differences between the two nucleus types, suggesting that they are rooted in the core structure of chromatin. Proteomic methods Cells were harvested, centrifuged, then washed once in PBS. To isolate nuclei, pellets were resuspended in Hypotonic Buffer (10mM HEPES pH 7.5, 2 mM KCl, 2 mM MgCl2, 3 mM EDTA, HALT Protease Inhibitors, 1 mM 1,4 DTT) and incubated on ice for 30 or 60 min. Myoblasts were then centrifuged again and resuspended in PBS containing 25 ug/ml trypsin, then incubated for 2 min to release nuclei. Myotubes received a similar treatment, but were resuspended in 37.5 ug/ml trypsin and incubated for 4 minutes. To stop digestion, PMSF was added to a final concentration of 1 mM and the solution was incubated for 5 min on ice. To permeabilize the nuclear membrane, a final concentration of 0.2 percent Triton X-100 was added to the suspension. After 5 min of incubation on ice, isolated nuclei were centrifuged as above and resuspended in Iso Buffer (10 mM HEPES pH 7.5, 25 mM KCl, 2 mM MgCl2, 75 mM sucrose, 0.5 mM PMSF, 1 mM DTT)). For mass spectrometry analyses, freshly isolated nuclei were washed once in Iso Buffer, suspended in 50 mM ammonium bicarbonate, and sonicated. Proteins extracted from nuclei treated or not with 0.5 M NaCl were analyzed by LC-MSMS after cysteine reduction with 1 mM TCEP and alkylation with 12 mM iodoacetamide, and overnight digestion with trypsin (protein ratio 1:50 w:w) at 37 degree. Peptides were then injected into an Ultimate 3000 UHPLC (Dionex, Thermo Fisher Scientific) in line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). After a desalting step in a trap column, analytical separations were performed on a single 45 cm long silica capillary packed in house with a C18, and heated at 45 degree in an oven (Sonation). A 90 min long HPLC gradient was used. The Data Independent Acquisition (DIA) mode was used. MS data were acquired in the 300-1200 m over z range split into 40 contiguous windows with 1 m over z overlap, fragmented by HCD 28 percent, and the MS2 spectra were acquired in the Orbitrap at 30K resolution in the 145 to 1450 m over z range. Protein identification was obtained using the SwissProt mouse database containing the manually curated list of histones (24,945 sequences) obtained from El Kennani. The raw data were analyzed with the DIA-NN software v. 1.8.1. Statistical analyses were performed using the Perseus software (v. 2.0.11). Data analyses regarded only nuclear proteins, based on SwissProt annotations, while contaminating non nuclear proteins were ignored. To evaluate significant changes in protein abundances between NaCl treated and untreated samples, raw intensity values were normalized to total core histone values. After normalization, all values were transformed to log2. Only proteins quantified in at least 5 of 8 samples were considered. Missing values were imputed, replacing them with values from the normal distribution (Width 0.3, Down Shift 1.8). Significance was evaluated using Students t-Test (FDR 0.05, S0 = 0.1, 250 randomization).