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

Xiao Chen,Weibo Song,Yuanyuan Wang,Yifan Liu,Shan Gao,Yurui Wang

doi:10.1007/s11427-016-5102-x

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.

Highlights

Nucleosome is the basic repeating unit of chromatin in the nucleus of eukaryotic cells (Kornberg, 1974)
Adapting the reported procedure for hydroxyl radical cleavage for mapping MAC nucleosome distribution (Brogaard et al, 2012a, b; Moyle-Heyrman et al, 2013), we showed that DNA from the H4 S47C/H3 C110A strain were extensively fragmented, with a band at ~200 bp emerging from the background smear, while DNA from the H3 C110A strain stayed largely intact (Figure 6D)
Purification of structurally and functionally differentiated MAC and MIC from the ciliated model organism Tetrahymena thermophila provides a unique opportunity for chromatin biology and epigenetic studies

Summary

Introduction

Nucleosome is the basic repeating unit of chromatin in the nucleus of eukaryotic cells (Kornberg, 1974). The nucleosome core particle is comprised of 147 base pairs (bp) of nucleosomal DNA wrapped around a protein complex of two copies of each histone: H2A, H2B, H3 and H4 (Richmond and Davey, 2003; Sahasrabuddhe and Van Holde, 1974). Variable length of linker DNA exists between adjacent core particles (Spadafora et al, 1976). Tetrahymena thermophila is a well-established unicellular eukaryotic model organism (Figure 1A–F) (Chen et al, 2015; Gao et al, 2016; Karrer, 1999). Tetrahymena exhibits nuclear dimorphism, containing one micronucleus (MIC) and one macronucleus (MAC) in the same cell compartment (Figure 1F) (Karrer, 2012). MIC is the germline nucleus that ensures the transmission of genetic information from generation to generation, while MAC is the somatic nucleus that provides “house-keeping” func-

Methods

Results

Conclusion