Genome and epigenome evolution
The goal of Pecinka lab is to expand knowledge on the molecular basis of mechanisms shaping plant genomes.
Genetic information of an organism is written in its DNA. Although DNA is amplified in a conservative manner, its occasional mutations can occur due to replication-associated errors or in connection with DNA damage and repair. DNA mutations are important for introducing novelty into the genome and thus fueling its evolution but can have also negative effects. Therefore, appearance of pre-mutation structures and DNA lesions is counteracted by genome maintenance mechanisms such as proofreading activity of DNA polymerases or DNA damage repair. Defective or incomplete function of these mechanisms may result in rapid accumulation of mutations, chromosomal breakage or rearrangements that will be phenotypically manifested as reduced fitness, fertility and development of diseases.
Other prominent force leading to potentially massive DNA sequence and genome size changes is transposition activity of repetitive elements. These elements amplify predominantly by copy-paste or cut-paste mechanisms and one family can occur in large numbers of thousands of copies in a genome. Plants prevent invasion of transposable elements by their inactivation using specific chromatin marks such as e.g. DNA methylation and histone H3 lysine 9 di-methylation, resulting in 'transcriptional gene silencing' (TGS). Since these marks represent 'heritable information not stored in DNA sequence', they are considered to be epigenetic. Lack of TGS leads to genome deterioration within several generations and the plants are generally not viable anymore.
For our research we use mainly the model species Arabidopsis thaliana, however, several other Brassicaceae such as A. lyrata, Ballantinia antipoda, Stenopetalum nutans and Arabis alpina are also used. The question of the smallest plant genomes is addressed using carnivorous plant Genlisea pygmaea. To answer our questions we apply mixture of genetic, molecular, biochemical, cytogenetic and bioinformatic methods.
The effects UV-B light on genome evolution and analysis of natural variation in response to UV-B stress
Plants require light for growth and photosynthesis. UV-B ligth (280-312 nm) is a natural component of sunlight that is important for photomorphogenesis and accumulation of protective polyphenolic compounds. High doses of UV-B or exposure of non-acclimated plants cause damage of various cellular components including DNA. UV-B modifies DNA molecule by introducing non-native bonds between two pyrimidine sites (C and T) the so called 'pyrimidine dimers'. These can result in a transition of Cs into Ts during DNA replication. In a collaborative project between our lab, the sun simulator facility at the Helmholtz Institute in Munich and the Schneeberger lab at the MPIPZ we ask for genome-wide mutagenic effects of different natural UV-B light doses on A. thaliana. This will elucidate what is the number and spectrum of mutations caused by UV-B light under 'natural' conditions and whether UV-B mutagenesis could have made a significant contribution to the plant genome evolution.
Great progress has recently been achieved in understanding the UV-B 'specific' signalling pathway, including identification of the UV-B photoreceptor UVR8 (Rizzini et al., 2011). Contrary to this, deciphering of the UV-B 'stress and damage' signaling pathway is much less advanced. To identify potential novel genes involved in UV-B signaling in natural Arabidopsis populations we have set up a screen in which 345 A. thaliana accessions were phenotyped for several growth related traits under mock and UV-B stress conditions (Figure 1A-B). This revealed great differences in response of some lines (Figure 1B) and the data were used to identify underlying genetic components using Genome-Wide Association (GWA) studies. GWA revealed a major association peak on chromosome 1 (Figure 1C) in a position which does not contain genes known to be involved in UV-B signaling. Analysis and validation of the candidates is in progress.
Natural variation in response to DNA replication stress
Analysis of epigenetic control of repetitive DNA and its role in genome maintenance
A large part of eukaryotic genome consists of repetitive DNA. Repeats have important functions in e.g. constituting centromeres, telomeres and rDNAs and developmental regulation of gene expression (Soppe et al., 2000). In addition, they contribute to genome instability and chromosomal rearrangements due to transposition activity and as frequent targets of homologous recombination machinery (Devos et al., 2002). To preserve genome integrity, repeat activity is suppressed by transcriptional gene silencing (TGS), an epigenetic control mechanism that brings DNA into a less accessible "heterochromatic" state via small interfering RNA directed deposition of silencing chromatin marks, e.g. DNA methylation and histone H3 lysine 9 di-methylation. Our aim is to explore variability in the control of TGS in the natural variants of A. thaliana, its close relatives and other Brassicaceae.
The knowledge on TGS in plants is biased towards A. thaliana model system. However, A. thaliana has greatly reduced genome with only about 13% of repetitive DNA (The Arabidopsis Genome Initiative, 2000) and therefore is rather an exception in the plant kingdom. We are interested in understanding TGS and epigenetic genome regulation also in other Brassicaceae with larger genomes and higher content of repetitive DNA. Current publications suggested that A. lyrata, a close relative of A. thaliana contains more transposable elements and differs in the TGS (Hu et al., 2011; Hollister et al., 2011; He et al., 2012). Applicability of many A. thaliana-based tools makes A. lyrata an ideal model for comparative epigenome studies. We focus on analysis of its TGS components as well as genome-wide distribution of specific chromatin marks.
A. thaliana and its close relatives have largely uniform pattern of genome-wide distribution of repetive DNA, with majority of repeats being clustered in pericentromeric regions and generally repeat depleted chromosome arms (Berr et al., 2006; Hu et al., 2011). Recent study revealed an unusual distribution of heterochromatin in Ballantinia antipoda (2n = 12), an Australian Brassicaceae species closely related to A. thaliana (Mandakova et al., 2010). In addition to the typical pericentromeric heterochromatin present in A. thaliana (Figure 4A, black arrows), B. antipoda contains large heterochromatic segments (HSs) covering 30-100% of six chromosome arm (Figure 4A, white arrows). Another striking phenotype of the HSs is an absence of DNA methylation albeit there are normal levels of DNA methylation signal in pericentromeric regions (Figure 4B). Hybridization of B. antipoda chromosomes with a fluorescently labeled A. thaliana-derived probes suggested that HSs are not consisting of telomeric or rDNA sequences and do contain interspersed single copy sequences (Mandakova et al., 2010). Thus the nature of B. antipoda's HSs segments is challenging the A. thaliana-based models of heterochromatin establishment.
Currently, the principal sequence of HSs in B. antipoda is isolated in collaboration with the Lysak lab (Masaryk University, Brno, Czech Republic) and will be characterized with respect to DNA methylation (using single base-pair resolution methods), histone modifications and transcriptional activation pattern during different developmental stages and stress situations.