Extended Data Fig. 8: Increased TFAM levels decrease the accessible population without changing mtDNA levels in HeLa cells.
From: Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA
(a) Proteinase K protection assay to validate TFAM-HA localization to mitochondria. TFAM-HA appears as a third upper band. Representative blot of two independently repeated experiments (b, c) Pearson correlation coefficients from three mtFiber-seq replicates from HeLa S3 TetOn-TFAM-HA cells treated with (b) DMSO or (c) doxycycline comparing (purple) fraction of reads methylated at each adenine and (grey) fraction of reads with a footprint at each position. PacBio chemistry version is indicated for each. Pearson’s correlation coefficients are shown. (d) Relative mtDNA levels from HeLa S3 TetOn-TFAM-HA cells treated with DMSO or doxycycline. N = 3 biological replicates. (e) UpSet plot showing the co-occurrence of footprints at TAS, CSBI, and MTERF1 from HeLa S3 TetOn-TFAM-HA cells treated with DMSO or doxycycline. Maximum footprint sizes based on the footprint size distributions: 60 bp (TAS), 140 bp (CSBI), and 35 bp (MTERF1). A maximum footprint size was set at 170 bp. Reads with larger footprints at these loci were omitted. N = 3 biological replicates. The categories represent 0.59%, 0.98%, and 1.67% of the total molecule population for DMSO replicates 1-3 and 0.44%, 0.54% and 1.31% of the total molecule population for doxycycline replicates 1-3. (f) UpSet plot showing the co-occurrence of footprints at the TAS and CSBI with containing a D-loop in HeLa S3 TetOn-TFAM-HA cells treated with DMSO or doxycycline. Maximum footprint sizes: 60 bp (TAS) and 140 bp (CSBI). N = 3 biological replicates. The categories represent 1.23%, 1.85%, and 3.37% of the total molecule population for DMSO replicates 1-3 and 1.00%, 1.14%, and 2.61% of the total molecule population for doxycycline replicates 1-3. (g) Log2 fold change in the fraction of reads containing a D-loop from total and accessible nucleoids ( > 1% m6A) in HeLa S3 TetOn-TFAM-HA cells treated with doxycycline relative to DMSO. Data were subsampled to match methylation distributions. Samples were compared with two-sided chi-squared tests for each replicate: *P < 0.05. Exact p-values were 0.011, 5.321*10−6, and 2.037*10−7 for doxycycline vs DMSO for total reads, and 0.026, 9.487*10−6, and 5.823*10−9 for doxycycline vs DMSO for accessible reads. N = 3 biological replicates for each condition. For all plots, error bars represent s.d. of the mean.