-1- Epigenetic mechanisms of cellular memory Introduction The many mitotic cell divisions following the stage of the fertilized egg to the adult organism require mechanisms ensuring that defined cellular states, determined at specific times during development, are faithfully inherited. Since cellular identities are based on a carefully orchestrated gene expression pattern of developmental regulators, like the HOX genes, maintaining identity is equivalent to maintaining the differential gene expression patterns. This basic cellular function is controlled at the level of chromatin. The proteins of the Polycomb group (PcG) and trithorax group (trxG) generate stable and heritable chromatin structures, maintaining the expression of developmental control genes. Our major research focus in the lab deals with the molecular mechanisms by which the PcG and trxG proteins control chromatin structure and in particular how chromatin-based epigenetic information controlling gene expression is faithfully maintained during DNA replication and cell division. This mechanism of maintaining determined states has been termed “cellular memory”. Epigenetic mechanisms of cellular memory Mechanisms of cellular memory are required to ensure that gene expression patterns, defining particular cell lineages, are faithfully inherited during cell division. The Polycomb (PcG) and the trithorax group (trxG) genes encode proteins that lock a gene expression state at the level of chromatin organization, by generating heritable chromatin structures that are transmitted during DNA replication and mitosis. PcG and TrxG proteins bind to Lab members Renato Paro ZMBH University of Heidelberg Im Neuenheimer Feld 282 69120 Heidelberg Telefone: +49-62 21 4 68 78 Telefax: +49-0 62 21 54 58 91 e.mail: firstname.lastname@example.org Collaborators Postdoctoral fellows: Ph.D. students: Dr. Christian Beisel Nara Lee Dr. Leonie Ringrose Ana-Laura Monqaut Dr. Muhammad Tariq Sabine Schmitt Gero Strübbe Techn. assistants: Ehret, Heidi Hagar, Sylvia Frank, Andrea -2- chromosomal elements termed PREs (PcG Response Elements), and interact with the histones and the basic transcription apparatus to keep their target genes either repressed by the PcG proteins or active by the TrxG proteins. Assessing the heritability of the PRE epigenetic state Changing cellular fates requires changing PRE epigenetic states. What is the process that switches a silenced PRE into a mitotically heritable activated PRE? Our working hypothesis is that PREs are silenced by default. Transcription through PREs remodels chromatin by setting positive epigenetic marks (i.e. acetylated histones) and thus prevents the PcG silencing complexes from binding to particular PREs, thus keeping the associated target gene active (figure 1). To demonstrate a causal relationship of this type of non-coding transcription, we established a set of reporter gene constructs to determine whether transcription through a PRE from a dedicated promoter (Actin 5C flanked by Cre/lox sites for removal by recombination) would inhibit silencing. Indeed, these constructs always gave strong miniwhite reporter expression. Upon removal of the promoter silencing of miniwhite is restored. The attractive hypothesis ensuing from our observation is that the transcriptional memory mechanism would only require the propagation of a positive epigenetic mark (set by the transcription process) through DNA replication and mitosis. This positive mark would prevent reestablishment of PcG-silencing at the particular PRE in the next interphase of the daughter cells and thus ensure an active expression state of the associated target gene. Figure 1: In situ hybridizations to embryos and larval brain and CNS show for the tailless gene that the PRE is expressed in the same tissue as the mRNA. Quantitative studies to determine interactions of epigenetic components Based on the fact that PRE targeting of PcG complexes is dependent on a set of defined DNA binding proteins, we have developed an algorithm capable of predicting PRE sequences in the Drosophila genome. This allowed us to identify over 150 potential candidate genes subjected to PcG/TrxG regulation. The gene categories represented cover early functions involved in segmentation and organogenesis as well as basic cellular regulatory features like cell cycle and cancer control. We are currently developing tools to isolate native PREs in order to identify the bound constituents and assess the tissue specificity of the regulatory complexes. Target gene specificity of PcG-silencing is primarily achieved through the PRE sequence. However, epigenetic marks like histone H3 methylated at K9 (H3K9me) and K27 (H3K27me) contribute to PcG repression, as the Polycomb (PC) protein can interact with these moieties through its chromo domain. Using polytene chromosome immunostainings we show that the two methyl marks colocalize with PC in distinct but overlapping patterns. We find that high levels of methylation and PC binding at the promoter do not prevent strong transcription, suggesting rather that multiple, Figure 2: Schematic model of PC binding to PREs used for the mathematical modeling of peptide competition (top) and simulation of binding shown in the bottom part. -3- regulated interactions between methylated PREs and promoters are required to create a silenced locus. In a cellular assay where we compete PC binding with various modified histone tail peptides, we show that the stability of PC binding is different at different loci and correlates with local differences in histone methylation and transcriptional status. We applied mathematical modeling (figure 2) to assess the kinetic nature of this observed behavior, showing theoretically that weak interactions are sufficient to maintain stable local concentrations of PcG proteins, which are in constant dynamic exchange with their target sites, offering opportunities for locus-specific regulation. Developing a systematic approach to study the epigenetic network on a whole-genome scale We have developed microarray and high-throughput approaches to study PcG/TrxG function at a genome-wide level. We have generated a new gene annotation of the Drosophila genome yielding the number of approximately 17.000 – 17.500 total Drosophila genes. A microarray containing cDNA sequences (PCR fragments of approx. 500 bp in length) has been developed and successfully employed to assess gene expression patterns during various stages of development (for more information see http://hdflyarray.zmbh.uni-heidelberg.de). Material from this study (Oligos, PCR fragments and microarrays) is being provided to the scientific community by the company Eurogentec. In addition, the PCR-set was utilized for the production of dsRNAs applicable in whole genome RNAi screens of Drosophila cells. This collaborative effort with the group of Norbert Perrimon (Harvard Genetics) culminated in the establishment of an RNAi Screening Center (www.flyrnai.org ) funded by NIH and servicing the scientific community utilizing these new tools for high-throughput whole-genome screens. We have used the knowledge gained in this genomics project, to establish the ChIP-on-Chip methodology in order to map protein distributions using microarrays. We have characterized the binding profiles of several members of the PcG/TrxG as well as compared them to the profiles of histones with particular modifications on a tiling array of the BX-C and ANT-C as well as other PRE- containing genes (figure 3). Besides establishing a genomic microarray, we have elaborated methods to produce homogeneous material for ChIP. To this purpose, we have established the BirA ligase technology in Drosophila, allowing particular proteins in defined cells to be marked with a biotin moiety, in order to affinity purify the protein complexes. Figure3: Result of a ChIP-on-Chip experiment comparing the binding profile of the PC protein with acetylated histones in the BX-C. The availability of the microarray and RNAi technology will give us a very powerful tool to systematically assess PcG/TrxG function on a genome wide scale. The questions that can be tackled at this level of complexity are i) Do the candidate genes identified by the PRE algorithm become globally deregulated in PcG or trxG mutants, ii) How is tissue-specific control of these broad classes of different PcG/TrxG target genes achieved, iii) How do chromatin protein distributions in the BX-C/ANT- C relate to other candidate PRE genes, is there a common profile motif. Indeed, it is becoming quite evident that PcG/TrxG regulation is widespread and involved in many diverse processes from plant seed development to mammalian stem cell renewal. Thus, it is important to continue on the large basis of information that has been generated in the model Drosophila to better understand the basic mechanism and, thus, to be able to extrapolate knowledge to more complex related phenomena like tissue remodeling or cancer epigenetics in mammalian organisms.
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