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<h3><font style="color:#F8B603;">Genome and epigenome maintenance</font></h3>


The goal of Pecinka lab is to expand knowledge on the molecular basis of mechanisms shaping plant genomes.
<h3><font style="color:#F8B603;">Plant Genome Biology</font></h3>


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.
Genetic information of a living organism is constantly exposed to internal and external DNA damaging factors, such as reactive oxygen species, solar UV radiation or cleavage by endogenous transposases. Robust protection of genetic information may be particularly important for sedentary organisms with late separation of germline cells, such as plants. Therefore, controlling genome integrity by the repair of damaged DNA and epigenetic silencing of transposable elements are the key molecular functions needed for a long term plant survival.  
The goal of our laboratory is to understand the molecular mechanisms of plant genome protection and repair under ambient and stress conditions.


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.
<h3><font style="color:#F8B603;">Current Projects</font></h3>
 
====DNA damage repair mechanisms in plants – the key to genome integrity====
 
 
'''Mutagenic effects of solar UV-B radiation.''' Plants require sunlight for photosynthesis. However, UV-B (280-312 nm) may cause damage of membranes, proteins and nucleic acids. In DNA, UV-B introduces non-native bonds between two pyrimidine sites, which can result in mutations if not repaired. Although the key repair components are known, it remains unknown how many UV-B mutations are induced in meristems and transmitted to next generations by solar doses of UV-B radiation. We address this question in collaboration with the sun simulator facility at the Helmholtz Zentrum in Munich and Schneeberger lab at MPIPZ. This will elucidate what is the number and spectrum of mutations caused by UV-B radiation under 'natural' conditions and how does this contribute to plant genome evolution.
 
 
[[Image:HU-175px.jpg | left]] '''Arabidopsis natural variation in response to DNA replication stress.''' DNA replication is regulated in response to developmental and environmental effects and is essential for cell division, expansion and differentiation. We analyzed survival of ''A. thaliana'' accessions in response to DNA replication stress induced by hydroxyurea (HU), which revealed both sensitive and resistant accessions. A combination of genome-wide association studies and quantitative trait locus mapping suggested several candidate genomic regions responsible for a large part of this variation. Currently, we perform fine mapping and validation of the candidate loci.


<h3><font style="color:#F8B603;">Current Projects</font></h3>


====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.
[[Image:atmatr_oww6.jpg | right]] '''Cytidine analog zebularine-induces DNA damage in plants.'''  
Cytidine analogs, zebularine or 5-aza-cytidine, are used in epigenetic research as suppressors of gene silencing. However, their molecular effects are only incompletely understood. We found that zebularine treatment triggers DNA damage response that is signaled additively by ATR and ATM kinases and is repaired preferentially by synthesis-dependent strand annealing-type of homologous recombination. Our current efforts are focused on understanding the mechanism of zebularine DNA damage induction and repair in plants.
 
 
 


[[Image:UV-B-800px2.jpg | center | Figure 1]]
====Epigenetic control of transposon silencing under ambient and stress conditions and role of transposons in genome evolution====


====Natural variation in response to DNA replication stress====
Transposons occur in all known eukaryotic genomes. Although considered as excessive genomic fraction, there is growing evidence on their importance for specific biological processes. However, spurious transposon activity may have deleterious effects on genome stability. Therefore, transposons are suppressed by transcriptional gene silencing (TGS), an epigenetic control mechanism that creates and maintains constitutively repressive chromatin state.


[[Image:HU-300px2.jpg | right | Figure 2]] DNA replication is needed for somatic cell division and expansion, two processes required for proliferation, increasing metabolic activity of a cell in particular during stress or development of special structures (e.g. trichomes). The frequency of DNA replication can be regulated in a spatial and environment dependent manner. Recent study revealed natural variation of ''A. thaliana'' in the degree of DNA endoreplication, e.i. replication not followed by division, under standard growth conditions (Sterken et al., 2011). This prompted us to analyze whether ''A. thaliana'' accessions show different performance under DNA replication stress. To this end, we treated seedlings of ca. 350 accessions by mock and hydroxyurea (HU), which blocks production of deoxyribonucleotides (dNPTs) required for new DNA synthesis, and scored relative survival of stressed plants. In general, ''A. thaliana'' accessions were resistant to HU with exception of several lines. Association mapping by GWAs revealed several close to be significant peaks. This could be potentially caused by a low frequency of the sensitive allele(s). Therefore, GWAs results are currently complemented by a classical quantitative trait locus (QTL) mapping.




====Analysis of epigenetic control of repetitive DNA and its role in genome maintenance====
[[Image:synopsis-EMBO_R.gif | right]] '''Apical meristems are hotspots of epigenetic silencing.''' Recently, we have discovered a novel RNA directed DNA Methylation (RdDM) function that reinforces silencing of repetitive elements during early vegetative growth. In apical meristems, ''de novo'' DNA methylation activity of RdDM compensates for zebularine-reduced silencing and thus prevents repetitive element transcription. Loss of this activity allows inheritance of activated repeats in somatic tissues and partially also into the progeny. Increased transcription of many chromatin-related genes in apical meristems suggests that they function as vegetative epigenetic checkpoints. To find out more read our recent paper [http://embor.embopress.org/content/15/4/446 Full Text]


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''.


[[Image:TSI-200px.jpg | right | Figure 3]] We have identified ''A. thaliana'' accession that shows activation of TRANSCRIPTIONALLY SILENT INFORMATION (TSI; Steimer et al., 2001) – an ''ATHILA'' related retrotransposon (Figure 3). Mutations in Arabidopsis genes ''DECREASED DNA METHYLATION 1'' (''DDM1'') and ''METHYLTRANSFERASE 1'' (''MET1'') leading to transcriptional activation of ''TSI'' are severely affected in their life-span and fitness (Kakutani et al., 1996; Mathieu et al., 2007). We are therefore interested to identify causal polymorphisms and to test their influence on the survival. Genetic mapping revealed one dominant ''TSI'' activating QTL and a recessive 'additive effect' QTL. Further mapping is in progress.


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.
'''''ONSEN'' – transposable element exploring host necessity for defense.''' Successful life cycle of a retrotransposon includes its amplification by the copy-paste mechanism. However, this is strongly opposed by epigenetic silencing. Recent studies have identified a heat-responsive ''COPIA''-type retroelement named ''ONSEN'' that can transpose in the Pol IV defective background. This activation is enabled by the presence of several heat-responsive elements in its long terminal repeats. We use ''Arabidopsis lyrata'' and other related species in order to understand evolution of ''ONSEN'' heat-responsiveness in ''Brassicaceae''.
''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.
[[Image:Ballant-800px2.jpg | center | Figure 4]]
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.




<h3><font style="color:#F8B603;">Bibliography</font></h3>


#Atwell, s., Huang, Y.S., Vilhjalmsson, B.J., Willems, G. et al. Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465:627-631 (2010).
[[Image:retro_175px.jpg | right]] '''Transposons contribute to the emergence of new genes.''' Animal data suggest that ''LINE'' retrotransposons play an important role in gene duplication. Occasionally, retrotransposon reverse transcriptase processes a gene-derived mRNA and integrates it into the genome as an intron and promoter-less copy. Although such retrogenes have been found in plants, their biology and evolution are poorly understood. We identified 251 retrogenes in ''A. thaliana'', corresponding to 1% of protein-coding genes. They are frequently transcribed in tissues other than their parental genes and many reach their transcription maximum in pollen, the tissue analogous to animal spermatocytes, where up-regulation of retrogenes has been found previously. However, the underlying mechanisms and function are currently unknown. See original publication [http://www.plantcell.org/content/26/8/3299.full?sid=e0a1463c-99cd-4be8-8f70-0effce112566 Full text]
#Berr, A., Pecinka, A., Meister, A., Kreth, G. et al. Chromosome arrangement and nuclear architecture but not centromeric sequences are conserved between ''Arabidopsis thaliana'' and ''Arabidopsis lyrata''. Plant Journal 48:771-783 (2006).
#Devos, K.M., Brown, J.K.M., Bennetzen, J.L.. Genome Size Reduction through Illegitimate Recombination Counteracts Genome Expansion in Arabidopsis. Genome Research 12:1075–1079 (2002).
#He, F., Zhang, X., Hu, J.Y., Turck, F. et al. (2012): Widespread interspecific divergence in cis-regulation of transposable elements in the Arabidopsis genus. Molecular Biology and Evolution 29:1081-1091 (2012).
#Hollister, J.D., Smith, L.M., Guoc, Y.-L., Ott, F. et al. Transposable elements and small RNAs contribute to gene expression divergence between ''Arabidopsis thaliana'' and ''Arabidopsis lyrata''. PNAS 108:2322-2327 (2011).
#Hu T.T., Pattyn, P., Bakker, E.G., Cao, J. et al. The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nature Genetics 43:476–481 (2011).
#Kakutani, T., Jedelloh, J.A., Flower, S.K., Munakata, K. and Richards, E.J. Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proceedings of the National Academy of Sciences USA 93:12406-12411 (1996).
#Mandakova, T., Joly, S., Krzywinski, M., Mummenhoff, K. and Lysak, M.A. Fast Diploidization in Close Mesopolyploid Relatives of Arabidopsis. Plant Cell 22:2277-2290 (2010).
#Mathieu, O., Reinders, J., Čaikovski, M., Smathajitt, C. and Paszkowski, J. Transgenerational Stability of the Arabidopsis Epigenome Is Coordinated by CG Methylation. Cell 130:851-862 (2007).
#Rizzini L, Favory JJ, Cloix C, Faggionato D et al. Perception of UV-B by the Arabidopsis UVR8 protein. Science 332:103-106 (2011).
#Soppe W.J.J, Jacobsen S.E., Alonso-Blanco, C., Jackson, J.P. et al. The Late Flowering Phenotype of fwa Mutants Is Caused by Gain-of-Function Epigenetic Alleles of a Homeodomain Gene. Molecular Cell 6:791–802 (2000).
#Steimer, A., Amedeo, P., Afsar, K., Fransz, P., Mittelsten Scheid, O. and Paszkowski, J. Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis. Plant Cell 12:1165–1178 (2000).
#Sterken, R., Kiekens, R., Boruc, J., Zhang, F. et al. Combined linkage and association mapping reveals CYCD5;1 as a quantitative trait gene for endoreduplication in Arabidopsis. PNAS doi: 10.1073/pnas.1120811109 (2012).
#The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796-815(2000).
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Latest revision as of 01:31, 24 March 2015


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Plant Genome Biology

Genetic information of a living organism is constantly exposed to internal and external DNA damaging factors, such as reactive oxygen species, solar UV radiation or cleavage by endogenous transposases. Robust protection of genetic information may be particularly important for sedentary organisms with late separation of germline cells, such as plants. Therefore, controlling genome integrity by the repair of damaged DNA and epigenetic silencing of transposable elements are the key molecular functions needed for a long term plant survival. The goal of our laboratory is to understand the molecular mechanisms of plant genome protection and repair under ambient and stress conditions.


Current Projects

DNA damage repair mechanisms in plants – the key to genome integrity

Mutagenic effects of solar UV-B radiation. Plants require sunlight for photosynthesis. However, UV-B (280-312 nm) may cause damage of membranes, proteins and nucleic acids. In DNA, UV-B introduces non-native bonds between two pyrimidine sites, which can result in mutations if not repaired. Although the key repair components are known, it remains unknown how many UV-B mutations are induced in meristems and transmitted to next generations by solar doses of UV-B radiation. We address this question in collaboration with the sun simulator facility at the Helmholtz Zentrum in Munich and Schneeberger lab at MPIPZ. This will elucidate what is the number and spectrum of mutations caused by UV-B radiation under 'natural' conditions and how does this contribute to plant genome evolution.


Arabidopsis natural variation in response to DNA replication stress. DNA replication is regulated in response to developmental and environmental effects and is essential for cell division, expansion and differentiation. We analyzed survival of A. thaliana accessions in response to DNA replication stress induced by hydroxyurea (HU), which revealed both sensitive and resistant accessions. A combination of genome-wide association studies and quantitative trait locus mapping suggested several candidate genomic regions responsible for a large part of this variation. Currently, we perform fine mapping and validation of the candidate loci.



Cytidine analog zebularine-induces DNA damage in plants.

Cytidine analogs, zebularine or 5-aza-cytidine, are used in epigenetic research as suppressors of gene silencing. However, their molecular effects are only incompletely understood. We found that zebularine treatment triggers DNA damage response that is signaled additively by ATR and ATM kinases and is repaired preferentially by synthesis-dependent strand annealing-type of homologous recombination. Our current efforts are focused on understanding the mechanism of zebularine DNA damage induction and repair in plants.



Epigenetic control of transposon silencing under ambient and stress conditions and role of transposons in genome evolution

Transposons occur in all known eukaryotic genomes. Although considered as excessive genomic fraction, there is growing evidence on their importance for specific biological processes. However, spurious transposon activity may have deleterious effects on genome stability. Therefore, transposons are suppressed by transcriptional gene silencing (TGS), an epigenetic control mechanism that creates and maintains constitutively repressive chromatin state.


Apical meristems are hotspots of epigenetic silencing. Recently, we have discovered a novel RNA directed DNA Methylation (RdDM) function that reinforces silencing of repetitive elements during early vegetative growth. In apical meristems, de novo DNA methylation activity of RdDM compensates for zebularine-reduced silencing and thus prevents repetitive element transcription. Loss of this activity allows inheritance of activated repeats in somatic tissues and partially also into the progeny. Increased transcription of many chromatin-related genes in apical meristems suggests that they function as vegetative epigenetic checkpoints. To find out more read our recent paper Full Text


ONSEN – transposable element exploring host necessity for defense. Successful life cycle of a retrotransposon includes its amplification by the copy-paste mechanism. However, this is strongly opposed by epigenetic silencing. Recent studies have identified a heat-responsive COPIA-type retroelement named ONSEN that can transpose in the Pol IV defective background. This activation is enabled by the presence of several heat-responsive elements in its long terminal repeats. We use Arabidopsis lyrata and other related species in order to understand evolution of ONSEN heat-responsiveness in Brassicaceae.


Transposons contribute to the emergence of new genes. Animal data suggest that LINE retrotransposons play an important role in gene duplication. Occasionally, retrotransposon reverse transcriptase processes a gene-derived mRNA and integrates it into the genome as an intron and promoter-less copy. Although such retrogenes have been found in plants, their biology and evolution are poorly understood. We identified 251 retrogenes in A. thaliana, corresponding to 1% of protein-coding genes. They are frequently transcribed in tissues other than their parental genes and many reach their transcription maximum in pollen, the tissue analogous to animal spermatocytes, where up-regulation of retrogenes has been found previously. However, the underlying mechanisms and function are currently unknown. See original publication Full text
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