Periodic Report Summary 1 - CHROMORPHO (Chromatin Dynamics of Stem Cells and Lineage Commitment in Plant Development) In recent years chromatin has emerged as an extremely dynamic platform for establishing and maintaining gene expression programs. Chemical alterations of DNA and histone proteins, nucleosome positioning and histone variant usage collectively allow the formation of complex combinatorial codes of chromatin modifications that determine local DNA accessibility to transcription and transcript processing machineries. As in animals, plant stem cell identity and lineage commitment are controlled by unique transcription factor networks and conserved chromatin factors including nucleosome remodelers, histone variants (such as H2A,Z) and histone modifiers (for example the polycomb silencing complex, PcG). My project involved the establishment of a high-throughput pipeline for analyzing chromatin dynamics in differentiating Arabidopsis roots. I have successfully integrated fluorescence-assisted nuclear sorting of individual cell types from the Arabidopsis root with high-throughput ChIP-Seq analysis. Currently bioinformatics and genetics/biochemistry are being undertaken in the lab to complete the work for publication.Objectives for this period:-Create the remaining constructs and transgenic lines that will be used as marker lines in fluorescence-assisted nuclei sorting. -Continue to test and optimize the pipeline for high throughput genome-wide ChIP-Seq analysis of specific cell types from the developing Arabidopsis root.-Initial results will be used to identify interesting loci for further analysis using chromatin analysis as well as genetics and biochemistry.-Study the putative role of H2A.Z histone variants in root stem cell maintenance.Established significant results within this period:-Completion of all transgenic lines that are needed for continuation of the project (see figure).-By using a combinatorial marker approach I managed to isolate single cell-types with a resolution so far not seen in the plant field.-Successful ChIP-Seqs have been performed on several transcription factors studied by the host lab as well as chromatin marks (total H3, H3K4me1 and H3K27me3). A new group member currently performs chiP-Seq analysis: Dr. Luca Santuari (a well trained bio-informatician). So far we have seen interesting dynamics in H3K27me3 levels in stem cells compared to differentiated cell types. We have isolated some putative novel stem cell factors based on these data and are currently studying these in more detail (see figure).- Contrary to animal systems I could -using a genetic approach- not detect a pivotal role for H2A.Z in root stem cell maintenance or differentiation. I’m continuing the analysis in differentiated cell typesDeviation:-By using cell-type specific and inducible knock out of the polycomb system (PcG) I have managed to show that root stem cell maintenance depends on polycomb-mediated mechanisms. -By analyzing regions of interest from the initial ChIP-Seq data we found a highly conserved region in the promoter of PLT2 (a master regulator of root development studied in the host lab). This region shows characteristics of enhancer functionality (i.e. H3K4me1) and possesses autonomous enhancer activity when used in heterologous promoter systems. Controlling plant architecture remains a major target of breeding strategies. Since all post-embryonic organs formed during a plants life cycle originate from selected groups of root and shoot stem cells, knowledge on stem cell functioning and maintenance is of utmost importance. This project provided a very useful new tool that already is generating a wealth of data on root stem cell maintenance and differentiation. Knowledge gained can subsequently be translated to the shoot system and eventually will provide breeders with new insights in how plant architecture is established and can be controlled to optimize and secure future crop yields.