Processes we study
Chloroplast chromatin
In many plant cells the majority of cellular DNA is present in chloroplasts, which originate from endosymbiotic cyanobacteria. The chloroplast genome encodes several photosynthetic proteins which are responsible for the majority of global energy capture. Despite the importance of chloroplasts and their genomes, almost nothing is known about structure and function of the chloroplast equivalent of chromatin. The packaging of the chloroplast genome does not resemble anything we know and uses a unique protein apparatus. Therefore, by deciphering the structure and function of chloroplast chromatin we hope to discover conceptually novel genetic regulatory processes.
RNA mediated transcriptional silencing
We are interested in determining the molecular mechanisms of RNA mediated transcriptional silencing, which in plants is known as RNA-directed DNA methylation. In this conserved process transposons and repetitive DNA elements give rise to ncRNAs, which mediate the establishment of repressive chromatin modifications. These chromatin modifications inactivate transposable elements, control gene expression and affect other aspects of genome activity.
RNA mediated transcriptional silencing involves two categories of ncRNAs. One is small interfering RNA (siRNA), which then recognizes complementary loci and mediates chromatin modifications. Second is long non-coding RNA (lncRNA), which is necessary for siRNAs to recognize their targets in the genome. siRNA and lncRNA work together to recruit several proteins, which establish DNA methylation, repressive posttranslational histone modifications, position nucleosomes and control chromosome organization.
Questions we ask
Chloroplast chromatin
1. How is chloroplast DNA organized and packaged?
2. What is the role of nucleoid-associated proteins in chloroplast DNA packaging?
3. How is chloroplast DNA packaging connected to gene expression?
RNA mediated transcriptional silencing
1. How is lncRNA production regulated and how does it contribute to the regulation of gene expression?
2. What are the molecular mechanisms used by lncRNA to recruit chromatin modifying enzymes?
3. How is lncRNA controlling structural aspects of chromatin including nucleosome positioning and chromosome organization?
Our model organisms
Our favorite model organism is a flowering plant Arabidopsis thaliana, which provides a unique advantage for studying the conserved mechanisms of RNA-mediated transcriptional silencing. In plants siRNA and lncRNA involved in this process are produced by two specialized RNA polymerases. Pol IV produces siRNA precursors and Pol V produces lncRNA needed for siRNA to recognize their targets. The involvement of the specialized RNA polymerases allows to easily distinguish and manipulate ncRNA production by just mutating RNA polymerase subunits.
For studies of chloroplasts we take advantage of genetic tools available in Arabidopsis. However, certain questions are easier to answer using other plant species, therefore we also work with maize, cucumber and tomato.
Methods we use
We are using a combination of genetic, biochemical and molecular biology approaches. We are obtaining and studying mutants, we analyze RNA production and structure, we assay interactions between RNA, DNA and proteins, we analyze chromatin modifications and nucleosome positioning. Much of our work is performed on the genome-wide scale using high throughput sequencing technologies. Therefore, bioinformatic analysis is one of the primary tools in the lab.
