Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 103 (52), 19689-94

Sequence-specific Modification of Mitochondrial DNA Using a Chimeric Zinc Finger Methylase

Affiliations

Sequence-specific Modification of Mitochondrial DNA Using a Chimeric Zinc Finger Methylase

Michal Minczuk et al. Proc Natl Acad Sci U S A.

Abstract

We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Efficient mitochondrial targeting of multidomain ZFP proteins requires a combination of a specific MTS and NES. (A) ZFPs containing four fingers (F1–F4) were selected from two libraries (L1 and L2) to bind targets in mtDNA (SI Fig. 5). All ZFPs (with or without a GGG linker) were closely related. Sequences of recognition helices (positions −1 to 6) are shown with DNA-contacting amino acids in boldface. ZFPs were fused to N-terminal MTSs of the F1β subunit of the human mitochondrial ATP synthase (F-ZFP) or subunit VIII of human cytochrome c oxidase (C8-ZFP) in the presence or absence of C-terminal GFP (C8-ZFP-GFP), and their localization was assessed as exclusively nuclear (N), exclusively mitochondrial (M), or mixed with either predominantly mitochondrial (M/N) or predominantly nuclear (N/M). (B) The localization of a particular ZFP, ZFP30, was analyzed further in the context of different MTSs with or without additional sequences such as GFP (image 2) or 3′ UTR of the mRNA for F1β subunit of human ATP synthase (32) (image 8). In addition to C8 (images 1 and 2) and F (images 7 and 8), we tested the MTS from the subunit 6 of ATP synthase from Chlamydomonas reinhardtii (R) (image 6) and MTSs from the following zinc finger proteins: MP42 from Trypanosoma brucei (T1) (image 3), MP63 from T. brucei (T2) (image 4), and 7b from Leishmania tarentolae (T3) (image 5). In the merged immunofluorescence images mitochondria are stained in red and ZFPs are labeled in green, and partially mitochondrial localization of F-ZFP30 is marked by arrows (image 7). (C and D) A number of F-ZFPs were fused with the C-terminal NES (F-ZFP–NES) and tested for their ability to enter mitochondria when attached to additional domains including GFP (F-GFP–ZFP–NES, 48 kDa), a catalytic domain of the hDNMT3a methylase (F-ZFP–meth–NES, 61 kDa), or both (F-GFP-ZFP–meth–NES, 86 kDa). Intracellular localization of individual proteins was assessed and presented (C) and additionally, for clone ZFP30, illustrated by images in D (abbreviations as in A and B).
Fig. 2.
Fig. 2.
Design and DNA binding of the NARP-specific mitochondrial F-ZFPNARP. (A) DNA recognition by a three-finger protein, F-ZFPNARP. A Zif268-based F-ZFPNARP has been selected to bind a sequence containing the T8993G mutation in the L-strand of mtDNA (the 8993G is marked as a black box in a target site). The amino acid sequences of the α-helices of zinc fingers F1, F2, and F3 are listed below with a single-letter code. The fingers F1, F2, and F3 are represented by α-helix and two β-strands stabilized by a zinc ion depicted as a gray sphere. Predicted contacts by residues in positions −1, 3, and 6 with the L-strand of mtDNA are shown as solid black arrows. The curved gray arrows indicate possible cross-strand interactions (33) between the amino acid in position 2 and the complementary H-strand at the interface between adjacent 3-bp binding sites for each finger. (B) F-ZFPNARP discriminates between closely related sequences. In vitro-synthesized F-ZFPNARP and a control ZFP F-ZFPcont were tested in the gel retardation assays for their binding to the target DNA, which contained the mutant T8993G (NARP-G) or T8993C (NARP-C) or the WT sequence 8993T (WT). All of the peptides were used in successive 5-fold dilutions (marked as gradient symbols), and DNA probes were used at a concentration of 0.3 nM. The letter “f” denotes free DNA, and “b” denotes protein-bound complexes. Two mobility forms of protein bound complexes “b” can be attributed to two different degrees of compaction of F-ZFPNARP-DNA, occurring in the presence or absence of a “cross-strand interaction.” Note that F-ZFPNARP has not been optimized for these interactions. (C) F-ZFPNARP retains its binding ability upon import to mitochondria. Gel retardation assay on the DNA target containing the T8993G mutation (NARP-G) was performed on the mitochondrial extract from the cells transiently expressing mitochondrially targeted F-ZFPNARP or F-ZFPcont. The cytosolic fraction was used as a control. Sequential dilutions of the proteins and concentration of the probe were as in B.
Fig. 3.
Fig. 3.
Chimeric zinc finger methylase F-ZFPNARP–meth–NES is targeted to the mitochondrial matrix and colocalizes with mtDNA. (A) Schematic structure of mitochondrially targeted methylases. To construct NARP-specific (F-ZFPNARP–meth–NES) or control (F-ZFPcont–meth–NES) chimeric methylases F-ZFPNARP or F-ZFPcont was linked by using a 17-aa flexible linker of (SGGGG)3SS to a catalytic domain (residues 592–909) of the human DNMT3a DNA methylase (hDNMT3a CD). The NES was added to the C terminus. As an additional control the mitochondrially targeted methylase lacking the DNA binding domain was constructed (F–meth–NES) by deleting ZFP from the F-ZFPNARP–meth–NES construct. Both constructs use the HA epitope tag to facilitate further detection. (B) F-ZFPNARP–meth–NES zinc finger methylase localizes inside mitochondria. The NARP cells transiently overexpressing F-ZFPNARP–meth–NES were fractionated, and the protein fractions were analyzed by Western blotting using anti-HA mAb. The localization of the F-ZFPNARP–meth–NES precursor (p) and its mature (m) form in total cell lysate (T), cytosolic (C), and a mitochondrial fraction treated with proteinase K under various conditions, as indicated, was compared with the localization of marker proteins. The precursor of F-ZFPNARP–meth–NES was found in the mitochondrial fraction but was clearly located outside the mitochondria, because it was accessible to protease digestion. In contrast, the mature form of the chimeric methylase was protected and became accessible to proteolysis only after the mitochondria were lysed with Triton X-100. The following endogenous proteins were used as fractionation markers: (i) GAPDH, previously reported as electrostatically associated with mitochondrial outer membrane (23, 24); and (ii) TFAM, the transcription factor that is localized in the mitochondrial matrix (25). (C) F-ZFPNARP–meth–NES zinc finger methylase colocalizes with mitochondrial nucleoid. The intracellular localization of F-ZFPNARP–meth–NES was analyzed by immunofluorescence in transiently transfected NARP cells. Mitochondria were stained with MitoTracker CMX Red (red), and F-ZFPNARP–meth–NES was detected with antibodies against the HA epitope tag followed by secondary antibodies conjugated to FITC (green). The F-ZFPNARP–meth–NES exhibits a punctate intramitochondrial staining pattern (images 1–3). Moreover, the majority of transiently expressed F-ZFPNARP–meth–NES colocalized with TFAM, a well known protein of the human mitochondrial nucleoid, stained here with polyclonal antibodies and visualized with Texas red (images 4–6). Intramitochondrial foci that were positive for F-ZFPNARP–meth–NES colocalized with mtDNA labeled with BrdU (images 7–9).
Fig. 4.
Fig. 4.
F-ZFPNARP–meth–NES selectively methylates mtDNA in vivo in the vicinity of its predetermined binding site. (A) We determined the methylation status of cytosine residues in the H-strand of mtDNA surrounding the NARP mutation site (positions 8950–9070 of mtDNA as indicated at the top) upon expression of mitochondrial ZFP methylases. For each indicated construct total cellular DNA was subjected to bisulfite conversion (to convert all cytosines to uracils while leaving 5-methylcytosine unchanged). Then the region of interest was amplified by PCR and cloned into Escherichia coli. For each construct a number of clones from two independent experiments (the total number of clones from the two experiments is indicated by N) was randomly chosen, sequenced, and analyzed to identify which cytosine had been methylated. The diagrams represent the mtDNA fragments originated from either the NARP cells or control WT cells, where unmethylated CpN dinucleotides are represented by open squares and the methylated CpN sites (mCpN) are depicted by filled squares and are colored according to the key. The numbers inside the filled squares represent the frequency of mCpN detected for each construct. The ZFPNARP binding site is shaded. (B) For each construct the percentage of clones containing at least one mCpN, mCpG, or methylated non-CpG is presented on the graph. The data presented are combined from two independent experiments that gave very similar results.

Similar articles

See all similar articles

Cited by 40 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback