Remodellierung der Chromatinstruktur und Entwicklung therapeutischer Strategien für durch strukturelle Variationen verursachte Veränderung
Allgemeine Genetik und funktionelle Genomforschung
Entwicklungsbiologie
Zusammenfassung der Projektergebnisse
Structural variants that result in ectopic enhancer promoter interactions and enhancer hijacking present a great setting to understand what makes a certain enhancer more likely to activate only some genes rather than all genes it has access to and to elucidate the resultant phenotypes. However, it is not quite feasible to study each one in detail, since many of them involve an enhancer active in complex tissues, such as the limb, and require creation of distinct mouse models. Therefore, we wanted to create an approach where we could study these structural variants in vitro, in a developmental stage in which the enhancer is not active. This approach would enable us to assay the gene expression changes due to ectopic enhancer promoter interactions and understand the possible etiology of the phenotypes without having to create a mouse model first. To this end, we used three mouse embryonic stem cell lines that recapitulate structural variants in the Epha4 locus that result in abnormal limb phenotypes. These include Resp18 inversion, Wnt6 inversion (InvF) and Pax3 deletion (DelB). To probe the ectopic enhancer promoter interactions, we activated the limb enhancer cluster with CRISPRa, using dCas9-VPR. This is a construct with a nuclease deficient Cas9 tethered to 3 activating domains, VP64, p65 and Rta, which makes it an extremely potent activation method and superior to constructs with only one effector. We designed 200 guides that targets the limb enhancer cluster and directed the dCas9-VPR construct, which was already being stably expressed in these cells, to the enhancer domain upon simultaneous lentiviral transfection at the mESC stage. We observed that the ectopic expression profiles in mESCs do not fully recapitulate the E11.5 limb expression ectopic expression profiles. This might be due to the changed chromatin landscape, and the promoters of these genes might have different states. To test this, we can repeat this experiment by differentiation these cells to obtain a different chromatin landscape and observing if the changes correlate with the changed chromatin states. We also might be losing important regulatory information by stage specific transcription factors and cofactors that conferred the enhancers clusters’ affinity for the promoters at E11.5. Moreover, repeating this experiment with constructs like dCas9-P300 might give us better result since endogenous acetyltransferases might recapitulate the enhancer machinery better than VPR. For the second part of the project, we studied polycomb-group proteins which play critical role in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cells (ESCs) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling processive spread of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remains unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes, such as Hmx1, Wnt6 and Hoxa. Genetic deletion of H3K27me3 loop anchors revealed a coupling of Polycomb-associated genome architecture and H3K27me3 deposition evidenced by disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. Further, we find that global alterations in PRC2 occupancy resulting from an EZH2 mutant selectively deficient in RNA binding is accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 acts as a “genomic wormhole”, using RNA binding to enhance long range chromosome folding and H3K27me3 spreading. Further, developmental gene loci have novel roles in Polycomb spreading, emerging as important architectural elements of the epigenome.
Projektbezogene Publikationen (Auswahl)
- Polycomb-mediated Genome Architecture Enables Long-range Spreading of H3K27 methylation
Kraft K, Yost K, Murphy S, Magg A, Long Y, Corces R, Jeffrey M. Granja J, Mundlos S, Cech T, Boettiger A, Chang HY
(Siehe online unter https://doi.org/10.1101/2020.07.27.223438)
