Depletion of KNL2 Results in Altered Expression of Genes Involved in Regulation of the Cell Cycle, Transcription, and Development in Arabidopsis.

Anastassia Boudichevskaia,Ales Pecinka,Anne Fiebig,Andreas Houben,Klara Prochazkova,Inna Lermontova

doi:10.3390/ijms20225726

Abstract

Centromeres contain specialized nucleosomes at which histone H3 is partially replaced by the centromeric histone H3 variant cenH3 that is required for the assembly, maintenance, and proper function of kinetochores during mitotic and meiotic divisions. Previously, we identified a KINETOCHORE NULL 2 (KNL2) of Arabidopsis thaliana that is involved in the licensing of centromeres for the cenH3 recruitment. We also demonstrated that a knockout mutant for KNL2 shows mitotic and meiotic defects, slower development, reduced growth rate, and fertility. To analyze an effect of KNL2 mutation on global gene transcription of Arabidopsis, we performed RNA-sequencing experiments using seedling and flower bud tissues of knl2 and wild-type plants. The transcriptome data analysis revealed a high number of differentially expressed genes (DEGs) in knl2 plants. The set was enriched in genes involved in the regulation of the cell cycle, transcription, development, and DNA damage repair. In addition to comprehensive information regarding the effects of KNL2 mutation on the global gene expression, physiological changes in plants are also presented, which provides an integrated understanding of the critical role played by KNL2 in plant growth and development.

Highlights

The centromeres are specialized chromosomal domains that are required for proper separation of chromosomes during mitosis and meiosis
We found that genes representing the Gene Ontology (GO) term “DNA repair” (26 genes) and related categories, such as “double-strand break repair via homologous recombination”, “non-recombinational repair”, and “DNA ligation involved in DNA repair” were overrepresented among knl2 differentially expressed genes (DEGs) (Table 1)
KINETOCHORE NULL 2 (KNL2) (At5g02520) and cenH3 (At1g01370) genes were included for the analysis in both types of tissues

Summary

Introduction

The centromeres are specialized chromosomal domains that are required for proper separation of chromosomes during mitosis and meiosis. The human Mis protein complex localizes to centromeres during late telophase and remains associated with the centromere during early G1 phase when new CENP-A is deposited [2,9,10]. A knockout of mammalian Mis18α resulted in reduced DNA methylation, altered histone modifications, and increased centromeric transcripts in cultured embryos [7]. It was not tested whether a knockout of Mis complex components has an effect on the methylation status of non-centromeric chromatin and on the expression of other repetitive or gene-coding chromosomal regions. The specific pattern of gene expression in response to the inactivation of the KNL2 gene provides a resource for future functional studies to unravel the role of KNL2 in kinetochore assembly and function

Loss of KNL2 Function Leads to Massive Transcriptional Changes

DNA Damage Repair Genes are Downregulated in the knl2 Plants

Real-time Quantitative PCR Confirms RNA-seq Analysis

2.10. Conclusions

RNA Isolation and Illumina Sequencing

RNA-seq Data Processing