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SCIENCE CHINA Life Sciences, Volume 59, Issue 9: 909-919(2016) https://doi.org/10.1007/s11427-016-5102-x

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

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  • ReceivedMay 21, 2016
  • AcceptedJun 12, 2016
  • PublishedAug 23, 2016

Abstract

Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.


Funded by

Natural Science Foundation of China(31522051,31470064)

funding awarded to Weibo Song(15-12-1-1-jch)

and the Qingdao National Laboratory for Marine Science and Technology

China. Yifan Liu was supported by National Sanitation Foundation(MCB 1411565)

National Institute of Health(R01 GM087343)

and the Department of Pathology at the University of Michigan.


Acknowledgment

Acknowledgements We are grateful to Dr. Sean D. Taverna, Department of Biochemistry and Molecular Genetics, University of Virginia Health System for sharing a protocol for MIC purification of Tetrahymena. Many thanks are due to Mr. Mingjian Liu and Ms. Yalan Sheng, Institute of Evolution & Marine Biodiversity, OUC, for generously providing photos of Tetrahymena thermophila. This work was supported by the Natural Science Foundation of China (31522051, 31470064), the funding awarded to Weibo Song (15-12-1-1-jch), and the Qingdao National Laboratory for Marine Science and Technology, China. Yifan Liu was supported by National Sanitation Foundation (MCB 1411565), National Institute of Health (R01 GM087343), and the Department of Pathology at the University of Michigan.


Open access

This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


Interest statement

Compliance and ethics The author(s) declare that they have no conflict of interest.


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  • Figure 1

    Purification of structurally and functionally differentiated MAC and MIC from Tetrahymena thermophila. A–F, Morphology of Tetrahymena thermophila. A–C, Phase-contrast imaging of live cells, arrow in (B) indicates the membranelles. D, Silverline system. E, Infraciliature on ventral side, arrow marks the oral apparatus. F, 4′,6-diamidino-2-phenylindole (DAPI) staining of MAC (Ma) and MIC (arrows). Scale bar=20 μm. G, Images of pellet 1 (600 g) and pellet 9 (5,000 g), after methylene blue staining. Pellet 1 shows many MACs as well as a few MICs; pellet 9 shows many MICs but no MAC. H, Number of MAC and MIC in each pellet, based on triplicate experiments.

  • Figure 2

    A workflow for enzymatic and chemical mapping of nucleosome distribution in Tetrahymena. These include MAC/MIC purification, MNase digestion or hydroxyl radical cleavage of chromatin, and mono-nucleosome sized DNA purification (by sucrose gradient ultracentrifugation or agarose gel purification).

  • Figure 3

    Different factors affecting MNase digestion of MAC. A, Hierarchical organization of chromatin. Mono-nucleosomes and oligo-nucleosomes can be generated by limited MNase digestion. Mono-nucleosomes may contain only DNA within a nucleosome core particle (147 bp), while di-nucleosomes, generated by insufficient MNase digestion (indicated by the hollow pattern), contain DNA within two nucleosome core particle as well as the linker DNA between them (~350 bp). B, Digestion pattern with 0.63% 1-octanol and 20, 50, 100 Kunitz units mL-1 MNase. C, Digestion pattern with 0.32% 1-octanol and 50, 100, 200 Kunitz units mL-1 MNase. D, Effect of nonidet P-40 and β-mercaptoethanol (+, -), and T150 buffer with Triton X-100 and protease inhibitor (+,-), on chromatin fragmentation and nucleosome recovery. E, RNase A treatment (+, -).

  • Figure 4

    MIC MNase digestion and mono-nucleosomal DNA sequencing (MNase-Seq). A, MIC MNase digestion pattern with 0.32% 1-octanol and 10, 20, 50 Kunitz units mL-1 MNase. B, Phasogram of mono-nucleosomal DNA in MIC and MAC. The MIC sample was digested in 50 Kunitz units mL-1 MNase, while the MAC sample was digested in 100 Kunitz units mL-1 MNase. C, Fragment size distribution of MIC mono-nucleosomal DNA produced by MNase-Seq with 50 Kunitz mL-1 MNase.

  • Figure 5

    Mono-nucleosome purification by ultracentrifugation in a sucrose gradient. A, A260 values of each fraction after sucrose gradient ultracentrifugation (from top to bottom) identified those containing nucleosomes. The MAC sample was digested with 50 Kunitz units mL-1 MNase. B, Coomassie brilliant blue staining confirmed the presence of only core histones in mono-nucleosome fractions. C, 2% of each fraction was analyzed by agarose gel electrophoresis. Input was labeled as “In”. D, Fractions containing mono-nucleosome sized DNA after MNase digestion under different concentrations were collected and sequenced. Input was labeled as “In”. E, Fragment size distribution of MAC mono-nucleosomal DNA produced by MNase-Seq with different MNase digestion concentration. C20: 20 Kunitz units mL-1 (CU428), peaking at 168 bp; C50: 50 Kunitz units mL-1 (CU428), peaking at 165 bp; C100: 100 Kunitz units mL-1 (CU428), peaking at 159 bp. F, Mapping results of mono-nucleosomal DNA produced by MNase-Seq with different MNase digestion concentration shown in Gbrowse.

  • Figure 6

    Hydroxyl radical cleavage of Tetrahymena chromatin. A, Comparison between enzymatic and chemical mapping of nucleosome distribution. Left: MNase digests linker DNA between nucleosomes, and leaves mono-nucleosomes with ~150 bp DNA; right: chelated copper I (red asterisk) catalyzes local production of hydroxyl radicals, which cleave nucleosomal DNA precisely at sites adjacent to the dyads, and generate nucleosome repeat length DNA, at ~200 bp. B, gDNA of MAC and MIC purified with 0.63% 1-octanol and 20 mmol L-1 EDTA (+, -). Red arrows indicated intact gDNA. C, gDNA of MAC and MIC purified with 0.32% 1-octanol and 20 mmol L-1 EDTA (+, -). D, Hydroxyl radical cleavage of MAC chromatin from H3 C110A (left, negative control) and H4 S47C/H3 C110A cells (right). Note the limited fragmentation in H3 C110A sample, and the extensive fragmentation as well as the ~200 bp band in H4 S47C/H3 C110A sample. Red arrows indicated intact gDNA.

  • Table 1   Different factors that may affect the MNase digestion pattern in MAC.

    Factors

    Options

    Description

    Centrifugal force

    1,500 g

    sufficient to collect most MACs according to Figure 1H

    MNase concentration

    50/100/200 Kunitz units mL-1

    heavy digestion promoting the yield of mono-nucleosomes

    1-octanol

    0.32%/0.63%

    no significant influence

    Nonidet P-40 and β-mercaptoethanol

    +/-

    no significant influence

    T150 buffer with Triton X-100 and protease inhibitor

    +/-

    will promoting yield if exists

    RNase A treatment

    +/-

    no significant influence

  • Table 2   Sequencing information of MAC/MIC mono-nucleosomal DNA samples after MNase digestion.

    Sample

    MNase digestion (Kunitz units mL-1)

    Strain

    Purification

    Input

    Mapped reads

    Unique mapped reads

    non-MDS unique mapped reads

    Percentage of non-MDS reads

    MIC

    50

    CU428

    Gel purification

    131,754,966

    102,186,811

    99,659,285

    29,611,205

    29.71%

    MAC1

    20

    CU428

    Sucrose gradient

    56,868,076

    54,422,059

    52,264,892

    382,320

    0.67%

    MAC2

    50

    CU428

    Sucrose gradient

    52,076,454

    49,336,271

    47,118,038

    308,826

    0.59%

    MAC3

    100

    CU428

    Sucrose gradient

    55,796,824

    52,607,335

    50,248,546

    347,728

    0.62%

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