Published online 22 May 2019 Nucleic Acids Research, 2019, Vol. 47, No. 12 6519–6537 doi: 10.1093/nar/gkz406 DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region Anna Cuppari1,†, Pablo Fernandez-Mill´ an´ 1,†, Federica Battistini2,†, Aleix Tarres-Sol´ e´ 1, Sebastien´ Lyonnais1, Guillermo Iruela3, Elena Ruiz-Lopez´ 1, Yuliana Enciso1, Anna Rubio-Cosials1, Rafel Prohens4, Miquel Pons3, Carlos Alfonso5, Katalin Toth´ 6, 5 2,7 1,* German´ Rivas , Modesto Orozco and Maria Sola` Downloaded from https://academic.oup.com/nar/article/47/12/6519/5494731 by guest on 28 September 2021 1Structural MitoLab, Structural Biology Department, Maria de Maeztu Unit of Excellence, Molecular Biology Institute Barcelona (IBMB-CSIC), 08028 Barcelona, Spain, 2Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain, 3BioNMR Laboratory, Inorganic and Organic Chemistry Department, Universitat de Barcelona, 08028 Barcelona, Spain, 4Unitat de Polimorfisme i Calorimetria, Centres Cient´ıfics i Tecnologics,` University of Barcelona, 08028 Barcelona, Spain, 5Centro de Investigaciones Biologicas,´ Consejo Superior de Investigaciones Cient´ıficas (CSIC), 28040 Madrid, Spain, 6Deutsches Krebsforschungszentrum, Division Biophysics of Macromolecules, Heidelberg, Germany and 7Department of Biochemistry and Biomedicine, University of Barcelona, Barcelona 08028, Spain Received August 14, 2018; Revised April 30, 2019; Editorial Decision May 01, 2019; Accepted May 15, 2019 ABSTRACT merization, which at a very high concentration trig- gers disruption of preformed complexes. Therefore, Human mitochondrial DNA (h-mtDNA) codes for 13 our results suggest that mtDNA sequences induce subunits of the oxidative phosphorylation pathway, non-uniform TFAM binding and, consequently, direct the essential route that produces ATP. H-mtDNA tran- an uneven distribution of TFAM aggregation sites scription and replication depends on the transcrip- during the essential process of mtDNA compaction. tion factor TFAM, which also maintains and com- pacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at INTRODUCTION the mtDNA control region where DNA regulatory el- Human mitochondria contain hundreds of copies of a 16.5 ements cluster. Previous studies identified still un- kb circular genome (h-mtDNA) that encodes 13 out of characterized, additional binding sites at the control the ∼80 subunits of the oxidative phosphorylation pathway region downstream from and slightly similar to LSP, (OXPHOS), the main source of ATP. Mutations, deletions, namely sequences X and Y (Site-X and Site-Y) (Fisher or misregulation of mtDNA impair the OXHPOS, which leads to considerable alterations of cell functions that are et al., Cell 50, pp 247–258, 1987). Here, we explore at the basis of rare diseases and syndromes (1), have been TFAM binding at these two sites and compare them associated with aging (2) and can aggravate major diseases to LSP by multiple experimental and in silico meth- such as diabetes (2), cancer (2), obesity (3) and neurologi- ods. Our results show that TFAM binding is strongly cal disorders such as Alzheimer’s and Parkinson’s diseases modulated by the sequence-dependent properties of (4). Therefore, mtDNA integrity must be well-preserved for Site-X, Site-Y and LSP. The high binding versatility correct cell function. Apart from OXPHOS proteins, tR- of Site-Y or the considerable stiffness of Site-X tune NAs and rRNAs are encoded by both h-mtDNA strands, TFAM interactions. In addition, we show that increase termed the heavy (HS) and light (LS) strands. Addition- in TFAM/DNA complex concentration induces multi- ally, h-mtDNA contains a non-coding control region (CR) of 1.1 kb that harbors mtDNA control elements, including *To whom correspondence should be addressed. Tel: +34 934034950; Email: [email protected] †These authors contributed equally to the paper as first authors. Present addresses: Anna Cuppari, ALBA Synchrotron Light Source, 08290 Cerdanyola del Valles,` Spain. Pablo Fernandez-Mill´ an,´ Department of Biochemistry and Molecular Biology, Autonomous University of Barcelona (UAB), 08193 Cerdanyola del Valles,` Spain. Sebastien´ Lyonnais, UMS 3725 CNRS – Universite´ de Montpellier, Montpellier Cedex 05, France. C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 6520 Nucleic Acids Research, 2019, Vol. 47, No. 12 the heavy and light strand promoters (HSP and LSP, re- chondrial TFAM levels, i.e. lower TFAM concentrations spectively) and the heavy strand origin of replication (OH), stimulate transcription, whereas high amounts lead to com- which is downstream from LSP (5,6). In addition, down- paction (25–28). stream from LSP there are three conserved sequence blocks TFAM binds to sequences that, albeit similar to LSP, named CSB- I (mtDNA coordinates nt 213–235), II (nt 299– are not involved in transcription activation but subjected 315) and III (closest to LSP, nt 346–363) that are preserved to TFAM phased binding. This poses the question of in mammals (7)(Figure1A). In addition to transcription, whether the distortions induced by TFAM to LSP would LSP together with CSB-II is further involved in mtDNA also occur in Site-X and Site-Y during compaction at replication. Former studies showed that RNA fragments the mtDNA control region. To this end and gain insight originated at LSP occasionally terminate downstream of into the DNA properties underlying dsDNA recognition CSB-II, creating a ∼120 nt RNA (R-loop) whose free 3 by TFAM, we characterized Site-X and Site-Y free or in ends prime the DNA polymerase so that RNA to DNA complex with the protein, by X-ray crystallography, molec- transitions are generated (6,8,9). This premature RNA ter- ular dynamics (MD) simulations, FRET and other bio- Downloaded from https://academic.oup.com/nar/article/47/12/6519/5494731 by guest on 28 September 2021 mination depends on a poly-guanine sequence at CSB-II physical methods. We compared these sites with binding that forms a hybrid G-quadruplex DNA structure between to LSP, which is the best characterized binding site as the displaced HS and the transcribed RNA, which presum- shown by several X-ray structures, FRET, SAXS and muta- ably destabilizes the RNA polymerase (mtRNAP) (10–13). tional analyses (17,18,29). Our results show that sequence- Directly downstream of CSB-II is a six-adenine tract (A- dependent DNA conformation modulates TFAM binding, tract, Figure 1A) whose mutations decrease the termina- which does not always occur as predicted by sequence align- tion effect at CSB-II (11,13). This A-tract is at the centre ment. In addition, we show that a simple concentration ef- of Site-X, a motif that overlaps 13 nucleotides from CSB- fect of TFAM/DNA complexes induces their multimeriza- II and 15 nucleotides downstream (Figure 1A). Site-X to- tion. Mutations at TFAM interfaces identified in the crystal gether with Site-Y, which is right upstream of CSB-I and structures disrupt this effect. These results suggest a mech- partially overlaps it (thus, both Site-X and Site-Y sequences anism of mtDNA compaction based on DNA-sequence de- lay between CSB-II and CSB-I Figure 1A), were both iden- pendent binding modes, and that multimerization of the tified as sequences similar to the sites that are bound bythe complexes is mediated by protein-protein interactions in- mitochondrial transcription factor A (TFAM or mtTFA) volving distant surfaces. within promoters LSP and HSP1. From these latter, the protein recruits the transcription machinery (14), but both MATERIALS AND METHODS Site-X and Site-Y are not close to any transcription initi- TFAM–DNA complex preparation ation site. TFAM is also involved in mtDNA compaction, and studies by in organello mtDNA methylation protection Mature full-length TFAM (residues 43–246; and hypersensitivity showed an organized phased binding UniProtQ00059) in complex with double-stranded of TFAM along the control region that was related to the DNA (dsDNA) fragments was prepared as reported compaction mechanism at this area (15). In particular, these previously (18). Based on the alignment of the crystal- studies specifically showed binding of TFAM to 28 bp pre- lized DNA sequence from the TFAM/LSP complex, cisely at Site-X (nt 276–303) and Site-Y (nt 233–260). In oligonucleotides harboring 22 bp from both Site-X addition, recent ChIP-seq analysis in HeLa cells showed (5-TAACAAAAAATTTCCACCAAAC-3;5-TTTGG that the Site-Y sequence is a TFAM enriched site, whereas TGGAAATTTTTTGTTAG-3, with overhanging ends) Site-X sequence is partially enriched (16). Interestingly, the and SITE-Y (5-TAACAATTGAATGTCTGCACAG-3, slight similarity of Site-X and Site-Y to LSP (14,15)(Figure and the complementary sequence that generated blunt ends) 1A and B) suggests a similar binding mechanism. were purchased (Biomers) and annealed. TFAM:DNA The crystal structure of TFAM in complex with LSP complexes were assembled by mixing the protein and DNA shows that binding at this promoter occurs at a 22 bp se- in the gel filtration buffer (50 mM HEPES pH 7.5, 750 quence upstream of the transcription initiation site (14,17– mM NaCl, 5 mM DTT) and the mixture was dialyzed 19)(Figure1A). The protein contains two HMG-box do- stepwise at 4◦C in buffer A (50 mM HEPES pH7.5, 500 mains (HMG-box1 and HMG-box2, roughly 60 aa each) mM NaCl, 5 mM DTT), buffer B (50 mM HEPES pH 7.5, separated by a linker (30 aa) and followed by a flexible C- 250 mM NaCl, 5 mM DTT) and buffer C (50 mM HEPES terminal tail.