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Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA

Nature Structural & Molecular Biology volume 31, pages 568–577 (2024)Cite this article

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Abstract

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.

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Fig. 1: Most human mitochondrial nucleoids are inaccessible.
Fig. 2: Accessibility patterns reveal mtDNA architecture.
Fig. 3: Altered TFAM levels shift the population of accessible nucleoids.
Fig. 4: In vitro reconstituted nucleoids reveal preferential TFAM binding and nucleation sites throughout the genome.

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Data availability

Raw and processed sequencing data are available from the Gene Expression Omnibus (GEO) at accession number GSE212611. PolG and TWINKLE ChIP–seq data were provided by X. Zhu. All microscopy data are available on Open Microscopy Environment (OMERO) at https://omero.hms.harvard.edu/webclient/?show=project-10105. Custom genome fasta files used for alignment based on the Hg38 reference genome (https://ncbi.nlm.nih.gov/grc/human) are available at https://www.github.com/churchmanlab/mtFiberseq. Source data are provided with this paper.

Code availability

Code for analysis of PacBio sequencing data available on GitHub: https://github.com/churchmanlab/mtFiberseq.

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Acknowledgements

We thank H. Merens and additional members of the Churchman laboratory for helpful discussions and assistance; K. Choquet, N. Kramer and B. Smalec for critical reading of the manuscript; L. Tallon, L. Sadzewicz and the University of Maryland School of Medicine Genomics Resource Center for PacBio sequencing; X. Zhu (University of Gothenburg) for Polɣ and TWINKLE ChIP–seq data from HeLa S3 cells; B. Battersby (Institute of Biotechnology, University of Helsinki) for human myoblasts from anonymous healthy control samples; P. Montero Llopis and P.V. Anekal of the MicRon facility (Harvard Medical School) for providing the microscope and consultation for imaging and analysis; A. Bergen and M. Burdyniuk (Arivis) for help with analysis; the Harvard University Bauer Core Facility for Illumina sequencing services and the IDDRC Molecular Genetics Core at Boston Children’s Hospital for NanoString services. This work was supported by the National Institutes of Health (R01-GM123002 to L.S.C., DP5-OD029630 to A.B.S. and F32-GM130028 to R.S.I.) and a Helen Hay Whitney Foundation Fellowship (F-1240 to K.G.H.). A.B.S. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is a Pew Biomedical Scholar.

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  1. These authors contributed equally: Thomas W. Tullius, Katja G. Hansen.

Authors and Affiliations

  1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    R. Stefan Isaac, Thomas W. Tullius, Katja G. Hansen, Mary Couvillion & L. Stirling Churchman

  2. Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA, USA

    Danilo Dubocanin & Andrew B. Stergachis

  3. Department of Genome Sciences, University of Washington, Seattle, WA, USA

    Andrew B. Stergachis

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Contributions

R.S.I. and L.S.C. conceptualized the study. R.S.I. developed the methodology, with input from K.G.H, T.W.T., A.B.S. and L.S.C. R.S.I. led the investigation, with assistance from K.G.H. R.S.I., K.G.H., T.W.T., M.C. and D.D. performed formal analyses. R.S.I. wrote the original draft of the manuscript. R.S.I., K.G.H., T.W.T., M.C., A.B.S. and L.S.C. reviewed and edited the manuscript. R.S.I., K.G.H., A.B.S. and L.S.C. acquired funding. A.B.S. and L.S.C. supervised the study.

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Correspondence to Andrew B. Stergachis or L. Stirling Churchman.

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Extended data

Extended Data Fig. 1 Resolution of adenine methyltransferases with mtDNA.

(a) Histogram showing the distance to the next neighboring A/T nucleotide from the 9,218 A/T nucleotides present in the human mitochondrial genome. (b) Histogram showing the distance to the next GC dinucleotide from the 711 present in the human mitochondrial genome. (c) Bar plot showing the percent of reads binned by the number of m6A modifications per read comparing untreated samples and those treated with 500 U of the m6A-MTase Hia5. Individual dots represent three and six biological replicates for 0 U Hia5 and 500 U Hia5, respectively. Error bars represent s.d. of the mean. (d) Bar plot showing the percent of reads binned by the number of m6A modifications per read for samples treated with 200, 500, 750, and 1000 U of the m6A-MTase Hia5. Individual dots represent six biological replicates for 500 U Hia5. Error bars represent s.d. of the mean. (e) Bar plot showing the percent of reads binned by the number of m6A modifications per read for samples treated with 500 U of the m6A-MTase Hia5 for 10, 30, 45, 60, and 120 minutes. The 120 minute sample received an additional SAM spike-in after 60 minutes. Individual dots represent 6 biological replicates for 10 minutes and 2 biological replicates for 60 minutes. Error bars represent s.d. of the mean.

Source data

Extended Data Fig. 2 Reproducibility of mtFiber-seq and HMM footprint calling.

(a) Correlation scatter plots comparing six mtFiber-seq samples from HeLa S3 cells for (bottom left) fraction of reads methylated at each adenine and (top right) fraction of reads with a footprint at each genomic position. PacBio chemistry version is indicated for each replicate. Pearson’s correlation coefficient is shown for each correlation. (b) Correlation scatter plots comparing two mtFiber-seq samples from U2-OS cells for (bottom left) fraction of reads methylated at each adenine and (top right) fraction of reads with a footprint at each genomic position. PacBio chemistry version is indicated for each replicate. Pearson’s correlation coefficient is shown for each correlation. (c) Correlation scatter plots comparing three mtFiber-seq samples from undifferentiated human skeletal muscle myoblasts for (bottom left) fraction of reads methylated at each adenine and (top right) fraction of reads with a footprint at each genomic position. PacBio chemistry version is indicated for each replicate. Pearson’s correlation coefficient is shown for each correlation.

Extended Data Fig. 3 ATAC-see reveals intracellular nucleoid heterogeneity.

(a) Schematic depicting experimental design. Tn5 was loaded with ATTO488-labeled oligo nucleotides to form active transposomes. U2-OS cells were treated with transposome and imaged by confocal fluorescence microscopy (b) Schematic depicting the ATAC-see segmentation and analysis pipeline. Background corrected images were masked and segmented in Arivis. Features of assigned objects were extracted and analyzed. (c) Representative image of a U2-OS cell showing ATAC-see and DNA signals. DNA was labeled with an anti-ss/dsDNA antibody that shows preferential labeling of mtDNA (Scale bars, 5 µm for single cell, 1 µm for zoom) (d) Histogram and violin plot showing the distribution of ATAC-see and DNA signal. Shown are the min-max normalized mean intensities from 27,079 segmented objects. Box-plot elements are as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points represent outliers. N = 54,459 segmented objects from 57 cells across two biological replicates. (e) Distribution of ATAC-see and DNA signal from 6 individual U2-OS cells. Shown are the min-max normalized mean intensities from each segmented object. Box-plot elements are as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points represent outliers. N = 1,394, 792, 1,890, 1,558, 3,440, and 1,254 segmented objects from Cells 1, 2, 3, 4, 5, and 6, respectively.

Extended Data Fig. 4 Comparison of ATAC-see reaction conditions.

(a) Tn5 in vitro activity in four different reaction buffers measured by DNA fragment analysis. Assembled transposome was mixed with 50 ng plasmid DNA for 30 minutes at 37 °C and DNA fragments were assessed by Agilent TapeStation D1000. Tn5 activity fragments the plasmid DNA, resulting in the appearance of smaller ( < 500 bp) bands. Four buffers were tested: B1 (50 mM Tris, pH 7.4, 10 mM potassium chloride, 75 µM disodium phosphate, 274 mM sodium chloride), B2 (33 mM Tris, pH 7.8, 66 mM potassium acetate, 11 mM magnesium acetate, 16% N,N-dimethylformamide), B3 (20 mM Tris, pH 7.6, 10 mM magnesium chloride, 20% N,N-dimethylformamide), and B4 (50 mM TAPS, pH 8.5, 25 mM magnesium chloride, 40% PEG8000). Buffer B2 was used for ATAC-see reactions performed in Extended Data Figs. 3 and 4. (b) Z-projection of the max intensities of a background corrected field-of-view of U2-OS cells treated with Tn5 transposomes in the presence and absence of EDTA. DNA was labeled with an α-ss/dsDNA antibody. (Scale bar, 10 µm) Representative image of four independently repeated experiments. (c) U2-OS cells sowing ATAC-see signal after treatment with Tn5 over a 60 minute time course. Two intensity ranges are shown to highlight the nuclear and mitochondrial signals.(Scale bar, 10 µm). Experiment performed once to determine optimal labeling time for subsequent experiments. (d) Confocal fluorescence microscopy showing mtDNA labeling throughout the mitochondrial network. A single Z-plane (0.3 µm) is shown. The mitochondrial network is labeled with an α-TOM20 antibody, mtDNA with an α-ss/dsDNA antibody, and chromatin with DAPI (Scale bars, 10 µm, 2 µm for zoom). Experiment performed once to confirm mtDNA signal within the mitochondrial network.

Extended Data Fig. 5 mtFiber-seq exposes unique D-loop features.

(a) UpSet plot showing the co-occurence of footprints at the TAS, CSBI, and MTERF1 binding site. Maximum footprint sizes were set based on the footprint size distributions: 60 bp (TAS), 140 bp (CSBI), and 35 bp (MTERF1). Reads with larger footprints were omitted. N = 6 biologically independent samples. Paired two-sided Student t-tests were used to compare the frequency of footprint co-occurrence: n.s. P > 0.05; ***P < 0.001. From left to right, specific p-values are 0.0008 (Category 2 vs Category 3) and <0.0001 (Category 3 vs Category 4). The categories in the plot represent 1.74%, 1.94%, 3.75%, 1.66%, 4.05%, and 2.08% of the total molecule population for replicates 1-6. (b) Hia5 activity on ssDNA and dsDNA. The Km for dsDNA is 0.233 µM. A lower limit of 3.48 µM was set for the Km for ssDNA as the reaction never saturated. N = 3 replicates. (c) Methylation strand bias from positions 1,000 to 3,000 in untreated HeLa cells and cells treated with 2CMA. Methylation bias is calculated as the number of methylations on the light and heavy strands, averaged using a 150 nt window and normalized against the region’s AT content. (d) Same as (c) but for the NCR for three biological replicates. (e) Log2 fold change in the methylation strand bias score between 2CMA treated and control samples at four loci. N = 4 biological replicates. Samples were compared with a two-sided Student’s t-test: *P < 0.05; n.s.P > 0.05. Exactly p-value is 0.015 for D-loop region 2CMA vs DMSO. (f) Heatmap showing RNA levels in HeLa cells after treatment with 2CMA as measured by NanoString. Counts were internally normalized to GAPDH and calculated relative to the DMSO control. All RNAs shown are mitochondrially encoded except for NDUFA7. N = 3 biological replicates. (g) Bar plot showing the percent of total nucleoids containing D-loops. A nucleoid was defined as having a D-loop if it had at ≥7 methylations within the region and a strand methylation ratio ≥3.01. N = 6 biological replicates for HeLa S3, 2 biological replicates for U2-OS, and 3 biological replicates for HSMM. For all plots, error bars represent s.d. of the mean.

Source data

Extended Data Fig. 6 TAS/CSBI footprints are associated with D-loop containing nucleoids.

(a) Histogram of the natural log of the ratio of light strand methylation to heavy strand methylation for reads with a minimum of 7 methylations in the D-loop. A Gaussian mixture model was applied and a threshold was identified based on a GMM posterior probability of 0.99. Red and green lines indicate each Gaussian fit. The blue and orange lines indicate the posterior probability of each population. A threshold of 3.01 was determined and used to identify reads with D-loop as shown in main Fig. 2h,i and Extended Data Figure 6b, c (b, c) Heatmap of the footprint size enrichment at the D-loop region in (b) reads containing a D-loop and in (c) reads lacking a D-loop in HeLa cells. Each row represents a footprint size, and each column shows a position in the genome. Presence of a D-loop was calculated using a GMM with a threshold of 3.01 from the log distribution of the ratio of Light Strand and Heavy Strand methylation.

Extended Data Fig. 7 Changes in OXPHOS levels and TFAM:mtDNA ratio during myoblast differentiation.

(a) Volcano plot showing differential expression analysis of human skeletal muscle myoblasts in differentiation media for 3 days compared to 0 days (left) and 6 days compared to 0 days (right). Red dotted lines are shown for a padj value of 0.01 and a fold-change value of 1.5. OXPHOS genes are shown in yellow and key nucleoid-associated proteins are labeled in light blue. P-values were obtained using the Wald test and corrected for multiple testing using the Benjamini and Hochberg method. (b) Western blot of nuclear-encoded (NDUFS1) and mitochondrial-encoded (ATP6) OXPHOS subunits from human skeletal muscle myoblasts in differentiation media for 0, 3, and 6 days. GAPDH was used as a loading control. Shown are two biological replicates. (c) mtFiber-seq methylation strand bias at the NCR from three biological replicates of human skeletal muscle myoblasts after 0, 3, and 6 days in differentiation media. Methylation bias is calculated as the number of methylations on the light and heavy strands, averaged over a 150 nt sliding window and normalized against the region’s AT content. Each window was required to have at least 2,250 methylations across all reads combined. (d) Western blot of TFAM levels from human skeletal muscle myoblasts in differentiation media for 0, 3, and 6 days. GAPDH was used as a loading control. Shown are three biological replicates. (e) Quantification of relative mtDNA levels by qPCR from human skeletal muscle myoblasts in differentiation media for 0, 3, and 6 days. Shown are mtDNA levels relative to day 0 from three biological replicates. Separate replicates are indicated by circle, triangle, and square shapes. Error bars represent s.d. of the mean. (f) Quantification of TFAM levels by western blot. TFAM bands were quantified and normalized against GAPDH. Shown are the TFAM levels relative to day 0. Samples were compared with a one-sided Student’s t-test. Exact p-value 0.031 for 6d vs 0d. Shown are three biological replicates. Separate replicates are indicated by circle, triangle, and square shapes. Error bars represent s.d. of the mean.

Source data

Extended Data Fig. 8 Increased TFAM levels decrease the accessible population without changing mtDNA levels in HeLa cells.

(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.

Source data

Extended Data Fig. 9 In vitro reconstituted nucleoids reveal TFAM protection patterns.

(a) TFAM binding to a 28mer corresponding to the HSP binding site measured by fluorescence polarization. The KD was determined to be 6.2 nM. Results shown are the mean with s.d. from three replicates. (b) (Left) 0.5% agarose gel showing mtDNA LR-PCR product before and after column cleanup. (Right) Genomic DNA ScreenTape analysis showing LR-PCR product. Representative gel of five independently repeated experiments (c) Two replicates of a dot blot assessing methylation of mtDNA with increasing concentrations of TFAM. A dilution series of DNA amounts were adsorbed onto a nitrocellulose membrane and detected with an anti-m6A antibody. (d, e) Tables of Pearson correlation coefficients between replicates and TFAM concentrations for (d) the fraction of reads methylated at each adenine and (e) the fraction of reads with a footprint at each genomic position from two replicates with each TFAM concentration. (f) Hill coefficients calculated from genomic position 2,500 to 14,000 from a four parameter logistics regression based on the fraction of reads protected with a footprint at least 20 bp long. Results are from the average of two replicates. (g) Meta density plots of footprint sizes from (top) 26 high affinity sites and (bottom) 64 low affinity sites at each TFAM concentration. The center line represents the kernel density estimate with the 95% confidence intervals shaded. Distributions between high and low affinity sites are significantly different at 5 µM TFAM (two-sample KS test, P < 2.2*10−16, D = 0.32) and 10 µM TFAM (two-sample KS test, P < 2.2*10−16, D = 0.29). (h) Heatmaps of footprint size enrichment at the light strand promoter (LSP) (top) and heavy strand promoter (HSP) (bottom) from HeLa cells subsampled to each TFAM concentration. Each heatmap row represents a footprint size, and each column shows a position in the genome.

Source data

Extended Data Fig. 10 TFAM nucleates from preferred binding sites in vitro and in cells.

(a, b) Heatmaps of the footprint size enrichment from two high affinity sites for each concentration of TFAM and from HeLa cells subsampled to each concentration: (a) from position 5,040 to 5,540 and (b) from position 11,900 to 12,400. Each heatmap row represents a footprint size, and each column shows a position in the genome. Line plots indicating the 1/K1/2 across these loci are shown above the heatmaps. (c, d) Density plots showing the footprint size distribution between 20 and 180 bp at (c) position 5,290 and (d) position 12,150 as a function of TFAM concentration. (e, f) Heatmaps of the footprint size enrich from two low affinity sites for each concentration of TFAM and from HeLa cells subsampled to each concentration: (a) from position 5,991 to 6,491 and (b) from position 10,332 to 10,802. Each heatmap row represents a footprint size, and each column shows a position in the genome. Line plots indicating the 1/K1/2 across these loci are shown above the heatmaps. (g, h) Density plots showing the footprint size distribution between 20 and 180 bp at (g) position 6,241 and (h) position 10,552 as a function of TFAM concentration.

Supplementary information

Reporting Summary

Supplementary Table

mtFiber-seq dataset summaries; TFAM fraction bound and K1/2 values genome-wide; List of primers and oligonucleotides; List of antibodies; List of NanoString probes; Read counts for footprint co-occurrence.

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Isaac, R.S., Tullius, T.W., Hansen, K.G. et al. Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA. Nat Struct Mol Biol 31, 568–577 (2024). https://doi.org/10.1038/s41594-024-01225-6

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