Table 1 Techniques used to investigate nuclear transcription factors in mitochondria

Subcellular fractionation

Centrifugation separates the mitochondrial, nuclear and cytoplasmic fractions of a cell sample. Immunoblotting for known mitochondrial, nuclear and cytoplasmic proteins assesses the efficiency of the fractionation process. Further immunoblotting establishes presence of factor of interest in mitochondrial fraction.

CREB [38], ER [4, 18, 90]

Immunoelectron microscopy

Labeling of intact cell preparations with specific antibodies conjugated to a heavy metal, such as gold. Transmission electron microscopy is used to visually analyze co-localization of the gold with the distinctly identifiable mitochondria.

CREB [38]

Mitochondrial import analysis

Observing the uptake of an in vitro translated radiolabeled protein of interest into isolated intact mitochondria, in the absence of a nucleus or any external stimulation.

CREB [39], p43 [15]

Co-immunoprecipitation

Inference of mitochondrial localization for a protein by characterization of a physical interaction with a known mitochondrial protein.

p53 [56]

Experimental question: Is the putative mitochondrial role independent from the factor's nuclear role?

Method

Summary

References

In organello systems

Isolation of intact mitochondria and observation of their response to stimulation of the factor of interest (for example, by addition of a hormone ligand).

p43 [30]

Mitochondrion-specific overexpression

Overexpression of a mitochondrion-specific isoform of a transcription factor or fusion of a transcription factor with a constitutive mitochondrial localization signal. Altered transcription from the mitochondrial genome in the absence of altered nuclear target expression suggests direct regulation of mitochondrial gene expression by that factor.

CREB [40], p43 [91]

Experimental question: Does the factor bind to the mitochondrial DNA and/or regulate mitochondrial gene expression?

Method

Summary

References

Chromatin immunoprecipitation (ChIP)

Assay of specific protein-DNA interactions by the crosslinking of proteins to DNA followed by antibody-based enrichment of a protein of interest. The DNA bound to that factor can then be assayed site-specifically by quantitative PCR, or on a genome-wide scale by microarray analysis or high-throughput sequencing. Used to detect direct binding of a factor to mtDNA.

CREB [40], p53 [54]

DNA footprinting

Assay of protein-DNA interactions nonspecifically by crosslinking protein to DNA followed by DNase digestion or dimethylsulfate treatment. Protein-bound DNA sequences are protected from digestion or methylation. Regions of mtDNA are then assayed for a change in protection pattern following stimulation of a specific protein or pathway.

p43 [30, 32], CREB [40]

Electro-mobility shift assay (EMSA)

Detects the capacity of a pool of proteins (for example, a mitochondrial extract) to bind to a short sequence of synthesized DNA, causing it to run slower (and thus 'shift' higher) than non-complexed DNA on a non-denaturing polyacrylamide gel. The addition of antibodies against the protein of interest gives specificity. Antibodies can be applied before mixing extract with DNA, resulting in loss of 'shift', or afterwards resulting in increased 'shift'.

p43 [15], CREB [38], ERβ [67]

Transcriptional reporter assays

Use of nuclear transcription constructs containing a putative regulatory sequence derived from mtDNA, upstream of a nuclear promoter and a reporter gene such as luciferase. Changed expression of the construct following stimulation of the transcription factor of interest shows that these mitochondrial sequences can act as regulatory elements when placed in a nuclear context.

p43 [15], p53 [56]