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Phase separation drives the self-assembly of mitochondrial nucleoids for transcriptional modulation

An Author Correction to this article was published on 01 December 2021

This article has been updated

Abstract

Mitochondria, the only semiautonomous organelles in mammalian cells, possess a circular, double-stranded genome termed mitochondrial DNA (mtDNA). While nuclear genomic DNA compaction, chromatin compartmentalization and transcription are known to be regulated by phase separation, how the mitochondrial nucleoid, a highly compacted spherical suborganelle, is assembled and functions is unknown. Here we assembled mitochondrial nucleoids in vitro and show that mitochondrial transcription factor A (TFAM) undergoes phase separation with mtDNA to drive nucleoid self-assembly. Moreover, nucleoid droplet formation promotes recruitment of the transcription machinery via a special, co-phase separation that concentrates transcription initiation, elongation and termination factors, and retains substrates to facilitate mtDNA transcription. We propose a model of mitochondrial nucleoid self-assembly driven by phase separation, and a pattern of co-phase separation involved in mitochondrial transcriptional regulation, which orchestrates the roles of TFAM in both mitochondrial nucleoid organization and transcription.

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Fig. 1: Phase separation of TFAM and DNA drives nucleoid formation.
Fig. 2: Structural features of TFAM in determining phase separation.
Fig. 3: Liquid droplet formation of TFAM-mtDNA recruits the transcription initiation complex of mitochondria.
Fig. 4: Co-phase separation of the elongation complex and termination factor with mitochondrial nucleoids in transcription.

Data availability

The data for this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We are grateful to all members of the Liu laboratory for useful discussions. We also thank the entire staff of the Public Instrument Center at Guangzhou Institutes of Biomedicine and Health, CAS. This study was funded by the National Key Research and Development Program of China (2018YFA0107100), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030505), the National Key Research and Development Program of China (2017YFA0106300, 2017YFA0102900, 2017YFC1001602, 2019YFA09004500, 2016YFA0100300), the National Natural Science Foundation projects of China (32025010, 31801168, 31900614, 31970709, 81901275, 32070729, 32100619, 32170747), the Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-SMC001), and International Cooperation Program, CAS (154144KYSB20200006), Guangdong Province Science and Technology Program (2020B1212060052, 2018A030313825, 2018GZR110103002, 2020A1515011200, 2020A1515010919, 2020A1515011410, 2021A1515012513), Guangzhou Science and Technology Program (201807010067, 202002030277, 202102021250, 202102020827, 202102080066), Open Research Program of Key Laboratory of Regenerative Biology, CAS (KLRB201907, KLRB202014) and CAS Youth Innovation Promotion Association (to Y.W.).

Author information

Authors and Affiliations

Authors

Contributions

X.L. designed and supervised the project. Q.L. and Y.Z. designed and carried out most of the experiments, and performed the data analysis. X.L., Q.L. and Y.Z. wrote the manuscript. H.W. carried out most of the protein purification and participated in experiments. M.H. carried out TEM imaging. S.D., W.L. and J.Q. participated in cell culture. S.Y., J. Wen and H.M. participated in the Hessian-SIM imaging work. Y.C. provided the Xenopus tropicalis. Y.W., G.X., L.Y., L.W., P.L., H.Z, W.-Y.C., J. Wang, J.G. and J.L. participated in data analysis and manuscript revision.

Corresponding author

Correspondence to Xingguo Liu.

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The authors declare no competing interests.

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Peer review information Nature Structural and Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phase separation of TFAM in vitro.

a, SDS-PAGE analysis of purified human recombinant TFAM. b, Representative images of TFAM (20 µM) forming liquid droplets. Scale bar, 20 µm. c,d, Comparison of droplet occupied area (c) and droplet number (d) illustrated in (Fig. 1b). The value represents the area of droplet occupied in a defined space (total droplets from 6 frames of images were counted in each group in one experiment, and similar results from 3 independent experiments were obtained). The orange squares indicate strong droplet formation; blue squares indicate weak droplet formation or no droplets. e, Droplet formation of TFAM (20 µM) in the presence of indicated concentrations of 1,6-Hexanediol. Scale bar, 20 µm. f, SDS-PAGE analysis of the removal 6×His-tag from recombinant TFAM protein. g, The droplet formation of TFAM with 6×His-tag or not. 20 µM protein and 10 nM 820 bp D-loop DNA were used. Scale bar, 10 µm.

Source data

Extended Data Fig. 2 Phase separation of TFAM and DNA in vitro.

a, The electrophoresis of ND1 DNA in the presence of TFAM at the indicated concentrations. b,c, Occupied area (b), diameter and droplet number (c) of TFAM-DNA droplets illustrated in (Fig. 1c) (Data are present with mean value of total droplets from 6 frames of images in 1 experiment were counted in each group. Similar results from 3 independent experiments were obtained). d, The concentration dependence of phase separation of TFAM protein and ND1 DNA. Scale bar, 20 μm. The images of TFAM conjugated with Alex Fluor 488 are shown. e, The analysis of droplet size in (d). The area of the orange circles indicates the occupied area of droplets, while grey round showed no droplets. f, A small region of the TFAM-DNA droplet was bleached and time-lapse images were recorded as shown on the top panel. The average fluorescence recovery traces of the region are displayed at the bottom panel (mean ± s.e.m., n = 23 regions from 3 independent experiments were counted). Scale bar, 1 µm.

Source data

Extended Data Fig. 3 Phase separation of TFAM-mtDNA in vitro and in vivo.

a, TEM image of extracted circular mtDNA. Scale Bar, 200 nm. b,c, The occupied area (b) and diameter (c) of TFAM-mtDNA droplets in the conditions illustrated in (Fig. 1h). Data are presented as mean ± s.d., n = 3 independent experiments. d, Representative images of mitochondrial nucleoids in vitro and in cell. Droplet formation of self-assembled nucleoid with TFAM (10 µM) and mtDNA in vitro. MEF cells expressing TFAM-mApple were labeled with mtDNA and mitochondria by PicoGreen (Pico) and MitoTracker Deep red (Mito), respectively. Scale bar, 2 µm. e, Quantification of the diameter of mitochondrial nucleoids in (d). The experiments of phase separation in vitro were performed with 10 mM HEPES, pH 7.6, 100 mM NaCl. Data are presented as mean ± s.e.m., 500 nucleoids in cell and 260 droplets in vitro from 3 independent experiments were counted. Two tailed unpaired Student’s t-test was used in (b) (comparing to no DNA group). The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Extended Data Fig. 4 Identify the domain of TFAM responsible for phase separation.

a,b, Comparison of relative droplet number (a) and occupied area (b) of TFAM-DNA droplets illustrated in (Fig. 2c). c,d, Comparison of droplet number (c) and occupied area (d) of TFAM-DNA droplets illustrated in (Fig. 2d). e, The percentage of nucleoid-like TFAM mutants were counted in (Fig. 2f). f, Representative images of mitochondrial nucleoid in MEF cells labeled with TFAM or TFAM truncation mutants fused with mCherry. The TFAM mutants overexpressed alongside with shTFAM targeting UTR region of endogenous TFAM. mtDNA was labeled with PicoGreen and mitochondria were labeled with MitoTracker Deep Red. Scale bar, 2 µm. g, The percentage of nucleoid-like TFAM mutants were counted in (f). Data in this figure are present with mean ± s.d., and n = 3 independent experiments. two-tailed unpaired Student’s t-test was used. The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Extended Data Fig. 5 TFB2M or POLRMT alone could not undergo phase separation.

a, SDS-PAGE analysis of purified human recombinant POLRMT, TFB2M and TFAM. b, Representative images of POLRMT (2.4 µM) conjugated with Alexa Fluor 647 with or without ND1 DNA (20 nM) under 100 mM NaCl. Scale bar, 10 µm. c, Representative images of TFB2M (10 µM) conjugated with Alexa Fluor 568 with or without ND1 DNA (20 nM) under 100 mM NaCl. Scale bar, 10 µm. d, The incorporation of UTP into the TFAM/DNA droplets. 10 µM UTP-Atto 488 was mixed with 20 µM TFAM and 20 nM D-loop DNA. Scale bar, 5 µm. e, The co-phase separation of UTP into the transcription initiation complex. 10 µM UTP-Atto 488 was mixed with 20 µM TFAM and 20 nM D-loop DNA. Scale bar, 5 µm. f, The phase separation of TFAM truncation mutants, 43-152, 43-222 and 122-246, after adding all the components of transcription initiation complex, including D-loop DNA labeled with YOYO-1, TFB2M and POLRMT conjugated with Alexa Fluor 647. The TFAM (43-246) is set as control. Scale bar, 5 µm. g, The percentage of layered structure of POLRMT of all droplets was counted in left images (f). Data are present with mean ± s.d., and n = 3 independent experiments. Two-tailed unpaired Student’s t-test was used. The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Extended Data Fig. 6 Mitochondrial transcription is affected by TFAM expression level.

a, The in vitro transcription of D-loop DNA under the droplet formation condition. The transcription level detected by QPCR under different concentration of TFAM. The bottom panel showed the droplets formation in vitro transcription. Scale bar, 1 µm. b, The transcription level of mitochondria after TFAM knockdown with shRNA (Data are present with mean ± s.d., n = 3 independent experiments). The shA, shB and shC are 3 shRNA targeting TFAM. c, TFAM protein level after TFAM knockdown. d, The mtDNA level after TFAM knockdown by shRNA in Hela cells. Data are present with mean ± s.d., n = 3 technical repeats. e,f, The transcription level (e) and mtDNA copy (f) of mitochondrial after TFAM knockdown by siRNA. g, The distribution of POLRMT after TFAM knockdown by siRNA. POLRMT was labeled by fusion with DsRed, and mtDNA was labeled by SYBR GOLD, while mitochondria were labelled by MitoTracker Deep Red. Scale bar, 10 µm. Data are present with mean ± s.d., and n = 3 independent experiments. Two-tailed unpaired Student’s t-test was used. The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Extended Data Fig. 7 The multi-phase separation of nucleoids is remodeled by TFB2M in promoter melting.

a, The multi-phase separation of transcription initiation complex after promoter melting. The paired two strand 52 bp LSP promoter DNA (LSP-WT) or a melted LSP DNA (LSP-melt) containing a 7 bp unpaired sequence was used in droplet formation with transcription initiation complex. All the sample contains 200 nM DNA, 20 µM TFAM and 100 nM POLRMT. Mock, without TFB2M and ATP; + TFB2M, add TFB2M; + TFB2M + ATP, add TFB2M and 200 µM ATP in phase separation. Scale bar, 5 μm. b, The percentage of layered structure of POLRMT of all droplets was counted in left images (a). c, The multi-phase separation of transcription initiation complex after promoter melting. The paired two strand 50 bp HSP promoter DNA (HSP-WT) or a 50 bp melted HSP DNA (HSP-melt) containing a 7 bp unpaired sequence was used in transcription initiation complex droplet formation. All the sample contains DNA, TFAM and POLRMT. Mock, without TFB2M and ATP; + TFB2M, add TFB2M; + TFB2M + ATP, add TFB2M and 200 µM ATP in phase separation. Scale bar, 5 μm d, The percentage of layered structure of POLRMT of all droplets was counted in left images (c). Data in (b), (d) are present with mean ± s.d. e,f, The mitochondrial transcription level after the different dose overexpression of TFB1M (e) or TFB2M (f). Data are present with mean ± s.d., and n = 3 independent experiments. Two-tailed unpaired Student’s t-test was used. The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Extended Data Fig. 8 TEFM and MTERF1 alone cannot undergo phase separation.

a, SDS-PAGE analysis of purified human recombinant TEFM. b, Representative images of TEFM conjugated with Alexa Fluor 568 with or without ND1 DNA under 100 mM NaCl. Scale bar, 5 µm. c, SDS-PAGE analysis of purified human recombinant MTERF1. d, Representative images of MTERF1 conjugated with Alexa Fluor 568 with or without 820 bp Leu DNA containing MTERF1 binding site (tRNA-Leu) under 100 mM NaCl. Scale bar, 10 µm. e,f, The percentage of layered POLRMT after adding TEFM (e) illustrated in (Fig. 4b) or MTERF1 (f) illustrated in (Fig. 4d). g, The super-resolution images of TEFM-DsRed in MEF cells by Hessian-SIM. The mtDNA is labeled with PicoGreen. TFAM is tracked by fusion with EGFP. Scale bar, 0.5 µm. Data are present with mean ± s.d., and n = 3 independent experiments. More than 400 droplets were counted in each group in each experiment. Two-tailed unpaired Student’s t-test was used. The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Extended Data Fig. 9 Mitochondrial transcription after the overexpression of transcription factors or the disturbance of phase separation.

a, The RNA transcripts in mitochondria was labeled with SYTO RNASelect after the overexpression of TEFM-DsRed or MTERF1-DsRed. Mito, MitoTracker Deep Red. Scale bar, 10 μm. b, The transcription level of mitochondria after the overexpression of TEFM (up panel) or MTERF1 (bottom panel) in Hela cells. Transcription level was detected with QPCR 48 hours after the transfection of 0-0.5 ng plasmids. Transfection with pCAG-FH plasmid was set as control. c,d, The transcription level of mitochondria (c) and TFAM (d) after the overexpression of TFAM truncates in Hela cell meanwhile endogenous TFAM knockdown with siRNA (siTFAM-2). Transcription level was detected with QPCR 48 hours after transfection. The statistical analysis was done by comparation to wild type Hela cells. e, The morphology of TFAM-GFP before or after 10% 1,6-hex treatment for 10 min. Scale bar, 10 µm. f, The transcription level of mitochondria after 1,6-hex treatment. Data are presented as mean ± s.d., n = 3 independent experiments. Two-tailed unpaired Student’s t-test was used. The p values were noted on the panel. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Source data

Supplementary information

Supplementary Information

Supplementary Discussion and Tables 1–6.

Reporting Summary

Supplementary Video 1

Fusion of two TFAM-DNA droplets.

Supplementary Video 2

The time-lapse images of TFAM-GFP in MEF cells after photobleaching. The TFAM-GFP labeled nucleoid is marked with a yellow circle.

Supplementary Video 3

Mitochondrial nucleoid fusion and fission in vivo.

Supplementary Video 4

The dynamics of POLRMT in MEF cells under super-resolution imaging with Hessian-SIM.

Supplementary Video 5

The dynamics of MTERF1 and mitochondria under super-resolution imaging with Hessian-SIM. MTERF1 is fused with DsRed and mitochondria is labeled with MitoTracker Green.

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Long, Q., Zhou, Y., Wu, H. et al. Phase separation drives the self-assembly of mitochondrial nucleoids for transcriptional modulation. Nat Struct Mol Biol 28, 900–908 (2021). https://doi.org/10.1038/s41594-021-00671-w

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