Claes M. Gustafsson

Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA (mtDNA) is of fundamental biological interest and may also lead to therapeutic interventions for human diseases associated with mitochondrial dysfunction. Here we report that mitochondrial transcription factors B1 (TFB1M) and B2 (TFB2M) are necessary for basal transcription of mammalian mitochondrial DNA (mtDNA). Human TFB1M and TFB2M are expressed ubiquitously and can each support promoter-specific mtDNA transcription in a pure recombinant in vitro system containing mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A. Both TFB1M and TFB2M interact directly with POLRMT, but TFB2M is at least one order of magnitude more active in promoting transcription than TFB1M. Both factors are highly homologous to bacterial rRNA dimethyltransferases, which suggests that an RNA-modifying enzyme has been recruited during evolution to function as a mitochondrial transcription factor. The presence of two proteins that interact with mammalian POLRMT may allow flexible regulation of mtDNA gene expression in response to the complex physiological demands of mammalian metabolism.

MTERF3 Is a Negative Regulator of Mammalian mtDNA Transcription

Regulation of mammalian mtDNA gene expression is critical for altering oxidative phosphorylation capacity in response to physiological demands and disease processes. The basal machinery for initiation of mtDNA transcription has been molecularly defined, but the mechanisms regulating its activity are poorly understood. In this study, we show that MTERF3 is a negative regulator of mtDNA transcription initiation. The MTERF3 gene is essential because homozygous knockout mouse embryos die in midgestation. Tissue-specific inactivation of MTERF3 in the heart causes aberrant mtDNA transcription and severe respiratory chain deficiency. MTERF3 binds the mtDNA promoter region and depletion of MTERF3 increases transcription initiation on both mtDNA strands. This increased transcription initiation leads to decreased expression of critical promoter-distal tRNA genes, which is possibly explained by transcriptional collision on the circular mtDNA molecule. To our knowledge, MTERF3 is the first example of a mitochondrial protein that acts as a specific repressor of mammalian mtDNA transcription initiation in vivo.

TRAP230/ARC240 and TRAP240/ARC250 Mediator subunits are functionally conserved through evolution

In Saccharomyces cerevisiae Mediator, a subgroup of proteins (Srb8, Srb9, Srb10, and Srb11) form a module, which is involved in negative regulation of transcription. Homologues of Srb10 and Srb11 are found in some mammalian Mediator preparations, whereas no clear homologues have been reported for Srb8 and Srb9. Here, we identify a TRAP240/ARC250 homologue in Schizosaccharomyces pombe and demonstrate that this protein, spTrap240, is stably associated with a larger form of Mediator, which also contains conserved homologues of Srb8, Srb10, and Srb11. We find that spTrap240 and Sch. pombe Srb8 (spSrb8) regulate the same distinct subset of genes and have indistinguishable phenotypic characteristics. Importantly, Mediator containing the spSrb8/spTrap240/spSrb10/spSrb11 subunits is isolated only in free form, devoid of RNA polymerase II. In contrast, Mediator lacking this module associates with the polymerase. Our findings provide experimental evidence for recent suggestions that TRAP230/ARC240 and TRAP240/ARC250 may indeed be the Srb8 and Srb9 homologues of mammalian Mediator. Apparently Srb8/TRAP230/ARC240, Srb9/TRAP240/ARC250, Srb10, and Srb11 constitute a conserved Mediator submodule, which is involved in negative regulation of transcription in all eukaryotes.

Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro

The mitochondrial transcription machinery synthesizes the RNA primers required for initiation of leading-strand DNA synthesis in mammalian mitochondria. RNA primers are also required for initiation of lagging-strand DNA synthesis, but the responsible enzyme has so far remained elusive. Here, we present a series of observations that suggests that mitochondrial RNA polymerase (POLRMT) can act as lagging-strand primase in mammalian cells. POLRMT is highly processive on double-stranded DNA, but synthesizes RNA primers with a length of 25 to 75 nt on a single-stranded template. The short RNA primers synthesized by POLRMT are used by the mitochondrial DNA polymerase γ to initiate DNA synthesis in vitro. Addition of mitochondrial single-stranded DNA binding protein (mtSSB) reduces overall levels of primer synthesis, but stimulates primer-dependent DNA synthesis. Furthermore, when combined, POLRMT, DNA polymerase γ, the DNA helicase TWINKLE, and mtSSB are capable of simultaneous leading- and lagging-strand DNA synthesis in vitro. Based on our observations, we suggest that POLRMT is the lagging-strand primase in mammalian mitochondria.

A family of putative transcription termination factors shared amongst metazoans and plants

The human mitochondrial transcription termination factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.

A genome-wide role for CHD remodelling factors and Nap1 in nucleosome disassembly

Chromatin remodelling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here, we show that Schizosaccharomyces pombe CHD remodellers, the Hrp1 and Hrp3 paralogs physically interact with the histone chaperone Nap1. Genome‐wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements revealed that the CHD factors and Nap1 colocalized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions, where they have redundant roles to stimulate transcription. Previously, DNA replication‐dependent and ‐independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation.

Mediator – a universal complex in transcriptional regulation

The Mediator complex is essential for basal and regulated expression of nearly all RNA polymerase II‐dependent genes in the Saccharomyces cerevisiae genome. Mediator acts as a bridge, conveying regulatory information from enhancers and other control elements to the promoter. It is now clear that Mediator‐like complexes also exist in higher eukaryotic cells and that they have an important role in metazoan transcriptional regulation. However, the exact mechanism of Mediator‐dependent transcriptional regulation remains unclear. We review here some recent advances in our understanding of Mediator structure and function. We also discuss a model to account for the functional and evolutionary relationship between yeast and metazoan Mediators. As an appendix to this review, we have created a database, MEDB, in which we have compiled information about all the S. cerevisiae Mediator subunits and their homologues in other eukaryotic cells (http://bio.lundberg.gu.se/medb/).

Mediator influences Schizosaccharomyces pombe RNA polymerase II-dependent transcription in vitro.

The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.

Purification and Characterization of RNA Polymerase II Holoenzyme from Schizosaccharomyces pombe

We have purified the RNA polymerase II holoenzyme from Schizosaccharomyces pombe to near homogeneity. The Mediator complex is considerably smaller than its counterpart inSaccharomyces cerevisiae, containing only nine polypeptides larger than 19 kDa. Five of these Mediator subunits have been identified as the S. pombe homologs to Rgr1, Srb4, Med7, and Nut2 found in S. cerevisiae and the gene product of a previously uncharacterized open reading frame, PMC2, with no clear homologies to any described protein. The presence of Mediator in a S. pombe RNA polymerase II holoenzyme stimulated phosphorylation of the C-terminal domain by TFIIH purified fromS. pombe. This stimulation was species-specific, becauseS. pombe Mediator could not stimulate TFIIH purified fromS. cerevisiae. We suggest that the overall structure and mechanism of the Mediator is evolutionary conserved. The subunit composition, however, has evolved to respond properly to physiological signals.

Analysis of Schizosaccharomyces pombe Mediator reveals a set of essential subunits conserved between yeast and metazoan cells

With the identification of eight new polypeptides, we here complete the subunit characterization of the Schizosaccharomyces pombe RNA polymerase II holoenzyme. The complex contains homologs to all 10 essential gene products present in the Saccharomyces cerevisiae Mediator, but lacks clear homologs to any of the 10 S. cerevisiae components encoded by nonessential genes. S. pombe Mediator instead contains three unique components (Pmc2, -3, and -6), which lack homologs in other cell types. Presently, pmc2+ and pmc3+ have been shown to be nonessential genes. The data suggest that S. pombe and S. cerevisiae share an essential protein module, which associates with nonessential speciesspecific subunits. In support of this view, sequence analysis of the conserved yeast Mediator components Med4 and Med8 reveals sequence homology to the metazoan Mediator components Trap36 and Arc32. Therefore, 8 of 10 essential genes conserved between S. pombe and S. cerevisiae also have a metazoan homolog, indicating that an evolutionary conserved Mediator core is present in all eukaryotic cells. Our data suggest a closer functional relationship between yeast and metazoan Mediator than previously anticipated.