The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures

Abstract

Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein-DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation.

Figure 1.

Electrophoretic mobility shift assay and atomic force microscopy analyses of TFAM association with pUC19 DNA. (A) Gel shift analysis of TFAM binding to linearized pUC19 DNA. TFAM concentration (micrograms per milliliter) is indicated for each lane, and it correlates with samples analyzed in B–F. Image contrast is inverted. (B–E) AFM height scans of TFAM-DNA complexes at varying concentrations of TFAM (3-nm height scale). (F) AFM height scan of TFAM–DNA complexes at 100 μg/ml TFAM (5-nm height scale). Bar, 1 μm. TFAM concentrations are indicated in the bottom left corner of each panel in micrograms per milliliter. All AFM data are raw flattened images.

Figure 2.

Quantitation of protein occupancy and compaction derived from AFM images. (A) Relative protein occupancy per structure with SE for each TFAM concentration. (B) Hill plot of adjusted TFAM occupancy. Transformed data points and quadratic best fit are shown with a Hill coefficient (n = 2.01). (C) Coefficient of 2-dimensional compaction per TFAM concentration with SE. (D) Coefficient of 3-dimensional compaction per DNA molecule for each TFAM concentration with SE. (E) Representative AFM image from 7 μg/ml TFAM (see Figure 1C). Bar, is 1 μm. (F) Colorized example of DNA (red) and DNA bound by TFAM (yellow) derived from AFM data shown in E with inset zoom of uncompacted DNA molecule. (G) Colorized individual DNA molecules from E as determined by computational analysis. Examples of ellipses used for calculations used to generate panel C and D are indicated in white.

Figure 3.

Analysis of TFAM-DNA association and TFAM oligomerization state by surface plasmon resonance and size exclusion chromatography. (A and B) SPR sensorgrams for TFAM association and dissociation with immobilized 99-base pair COXI sequence and control region sequence, respectively. (C and D) SPR sensorgrams for the 45-base pair LSP-Half and HSP-Half of control region DNA, respectively. Insets, expanded view of the first 30 s of association phase. The concentration of TFAM is indicated in the legend of each graph. (E) Size exclusion analysis of recombinant TFAM preparations and mitochondrial extracts. UV absorption chromatographs of isolated TFAM and TFAM-HS are shown as the percentage of maximum absorbance (abs) and milliliters retention (mls). Inset, Western blot analysis of even fractions of a mitochondrial extract isolated from HEK293 cells. (F) Schematic representation of mouse TFAM protein. Shown are the major domains, including mitochondrial targeting sequence (MTS), MTS cleavage site, DNA binding domains (HMG box 1 and 2), linker region between HMG boxes, predicted coiled-coil domain, and the C-terminal extension. Also shown are predicted α-helices (Chou and Fasman, 1974) and the HMG box helices inferred from sequence alignment with the crystal structure of HMG-D (Murphy et al., 1999; Matsushima et al., 2003).

Figure 4.

Predicted kinetic, cooperative, and stoichiometric characteristics of TFAM by SPR. (A) Kinetic analysis of TFAM binding to DNA. DNA ligands are indicated to the left of each box. Data were fitted locally using 1:1 binding model with mass transport correction. (B) Hill plot derived from kinetic parameters of TFAM binding to COXI sequence, yielding a positive Hill constant of 2.48 for TFAM. (C) TFAM–DNA stoichiometry, binding site size estimate and binding sites per genome calculations.

Figure 5.

Bioscope atomic force microscopy analysis of TFAM–DNA with decreasing protein occupancy. (A–D) An example titration of TFAM bound on DNA reveals loop structures at low protein occupancy. All images are 250-nm scans. Bar, 100 nm.

Figure 6.

Ultrahigh vacuum noncontact atomic force microscopy of DNA compaction intermediates. (A) NC-AFM scan of naked DNA. (B) NC-AFM scans show TFAM at low occupancy induces the formation of loop structures on DNA. Arrows indicate TFAM protein at the base of two omega (Ω) structures. Steps of compaction are numbered for each row. (C) Outline of protein–DNA complexes observed in B. (D) Model for DNA compaction. The process of compaction progresses from initial binding to fully compacted DNA with numerous stages of intermediate organization between. The arrowhead indicates a possible cleft in a TFAM dimer. Bars (A and B), 100 nm. The raw data are contrast inverted.

Figure 7.

Analysis of atomic force microscopy images of higher-order nucleoprotein structures. The left two panels are height images of TFAM–DNA structures at 100 μg/ml protein. The center two panels are 3-D projections of AFM images. Each band in the Z-plane is 2 nm. The right three panels are cross-section traces of the 3-D projections as indicated by color.