Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

Elisa Pesenti,Hiroshi Masumoto,William C Earnshaw,Oscar Molina,Joan Aurich-Costa,Vladimir Larionov,Mikhail Liskovykh,Natalay Kouprina

doi:10.1021/acssynbio.8b00018

Abstract

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

Highlights

C entromeres define the site of the assembly of the kinetochore, a multiprotein complex that directs chromosome segregation during cell division.[1]
As the basis for the kinetochore domain, we designed a 343 bp alphoid 21-I dimer from high-order repeats (HORs) of chromosome 21 in which sequences corresponding to the CENP-B box in one monomer were replaced by a 42 bp tetracycline operator sequence, the binding site for E. coli tetracycline repressor (Figure 1)
An artificial alphoid 21-I (α21-I) dimer and a synthetic α21-II-lacOgal4 12-mer were extended by rolling circle amplification (RCA) using phage φ29 DNA polymerase followed by yeast Transformation-associated recombination (TAR) cloning (Figure 3).[24,25]

Summary

Introduction

C entromeres define the site of the assembly of the kinetochore, a multiprotein complex that directs chromosome segregation during cell division.[1]. Research Article with precisely defined DNA sequence variation and the possibility of manipulating alphoid DNA arrays.[24] This approach involves rolling circle amplification (RCA) of alphoid DNA oligomers as small as a dimer followed by assembly of the amplified fragments by transformation-associated recombination (TAR) in yeast.[25,26] Using the RCA-TAR method, synthetic alphoid DNA arrays up to 140 kb have been generated and used for de novo HAC formation.[24] The method permitted generation of a synthetic HAC with a conditional centromere, the alphoidtetO-HAC,[27] which has been instrumental in resolving a role for chromatin structure in kinetochore function.[23] The alphoidtetO-HAC is based on a dimeric alphoid DNA array that contains alternating monomers with either CENP-B boxes or tetracycline operator (tetO) sequences. Results from different groups showed that disruption of pericentromeric heterochromatin is associated with chromosome mis-segregation and tumorigenesis.[35−37]

Methods

Results

Conclusion