Figure 1.
Characterization of TFAM monoclonal antibodies.
(A) Immunoprecipitation of TFAM from cell lysates. HeLa cell lysate was applied to sheep anti-mouse Dynabeads conjugated to anti-Myc, 20G2C12 TFAM antibody, 20F8A9 TFAM antibody, or a 50/50 mixture of 20G2C12 and 20F8A9 TFAM antibodies. The labeled bands are: 1) Antibody heavy chain; 2) antibody light chain; 3) TFAM. (B) Western blot using the 20G2C12 antibody detects a ~23kDa band. (C and D) Immunocytochemistry showing TFAM localization. Mitochondria were identified by PPIF staining; mtDNA was identified by anti-DNA staining. There was no evidence for nuclear localization of TFAM using either antibody.
Figure 2.
ChIP-seq analysis of genome-wide TFAM binding.
(A) Overview of computational processing of data. Reads were trimmed to 36 bp and then either mapped against the mitochondrial genome (ChrM), or the complete hg19 version of the genome. After removing multireads and alignments to the mitochondrial genome, peaks in the nuclear genome were called using MACS2. (B) The proportion of sequencing reads mapping to chrM in ChIP and input datasets. All replicates of the ChIP-seq resulted in at least 30% of reads mapping to the mitochondrial genome, much greater than the 0.4-1.9% of reads mapping to mtDNA in the input datasets. Replicates 1-3 were performed using the 20G2C12 antibody, while Replicate 4 was performed using the 20F8A9 antibody.
Figure 3.
Coating of the mitochondrial genome by TFAM in HeLa cells.
Circos plot of plus strand and minus strand TFAM ChIP-seq and input read density signal over chrM. (A, E) Annotation of protein coding (green on forward/heavy strand, red on reverse/light strand), ribosomal RNA (blue) and tRNA (blue on forward/heavy strand, grey on reverse/light strand) transcripts. (B) D-loop (black), LSP promoter (large red tile), known LSP TFAM binding site (small red tile), HSP promoter (large blue tile), known HSP1 TFAM binding site (small blue tile), and origins of heavy strand replication (Ori-b, orange tile; OH, yellow tile). (C) TFAM ChIP-seq signal on forward (red) and reverse (blue) strands. (D) Input signal on forward (red) and reverse (blue) strands. (F) Origin of light strand replication (yellow tile). Note that the input signal is exaggerated 60-fold relative to the ChIP-seq signal in order to visualize coverage irregularities. The signal from the TFAM ChIP-seq largely follows that of the input, indicating generalized binding across the mitochondrial genome.
Figure 4.
Absence of TFAM binding to the nuclear genome.
(A) Cross-correlation plot of input DNA computed over the nuclear genome. (B) Cross-correlation plot of TFAM ChIP-seq computed over the nuclear genome. (C) Distribution of ChIP-seq reads mapping to the plus and minus strand around called binding sites in a ChIP-seq dataset for the NRSF transcription factor [51] in HeLa cells, generated by the ENCODE consortium [52]. (D) Distribution of TFAM ChIP-seq reads mapping to the plus and minus strand around called binding sites indicates lack of real binding sites. (E) No ChIP-seq enrichment around the promoter of the SERCA2/ATP2A2 gene, previously suggested to be a TFAM target.