Funktionelle Charakterisierung von mesenchymalen stammzell-derivativen lncRNAs
Zusammenfassung der Projektergebnisse
Long noncoding RNAs (lncRNAs) have gene regulatory and genome organizational functions. Our major aim was to study the spatiotemporal dynamics of the lncRNA loci Firre and CISTR-ACT, which are in physical contact with coding genes on non-homologous chromosomes, and both lncRNAs are causally associated with Mendelian disease. Therefore, we combined the CRISPR/dCas9 system with the RNA-aptamer technology of phage coat proteins (MS2, PP7), or Puf1-Pumilio coupling to label individual genomic loci in living cells by CRISPR Live cell imagING (CLING). Using confocal-superresolution microscopy (Airyscan), our studies revealed that FIRRE and CISTR-ACT have frequent inter-chromosomal contacts in > 50 % of the imaged nuclei. 4D time-lapse imaging experiments detected that interchromosomal contacts stayed associated across time and FIRRE and CISTR-ACT moved slower and less than control loci. By using heterozygous SNPs within CRISPR’s PAM motif which is required for specific CRISPR-recognition, we were able to direct the CLING complexes to individual alleles, termed SNP-CLING. With the ability to resolve single alleles in 129S1/CAST living cells of a hybrid cross, we studied spatial allelic properties of Hdac4, Sox9 and Cistr-act, all linked to Mendelian-inherited brachydactyly in heterozygous structural aberrations. We described inter-allelic distances of the labeled alleles to one another and to other loci. Moreover, we labeled the sub-nuclear compartment of the nucleolus in combination with the specific alleles. Every locus harbored a preserved and unique position in the nucleus. Irrespective of the inherited genetic background (129S1 or CAST), the alleles showed no bias in positioning and their distances to either the nucleoli or to the nuclear periphery were stable in a majority of cells. However, the studied loci were closer to the nucleoli when compared to the nuclear periphery distances. Interestingly, we detected that > 60 % of either 129S1 or CAST alleles were closer than 1 µm to the nucleoli, indicating that the functional landmark of the nucleolus could play an important role in allele positioning. We anticipate that CLING and SNP-CLING will be widely used to address a variety of biological questions in the fields of chromatin biology, nuclear architecture, allele-specific disease mechanisms, epigenetic phenomena (i.e. imprinting), and transcriptional control of alleles. Our live cell imaging techniques will yield insights into the genomic, genetic and structural properties of the underpinning nuclear architecture.
Projektbezogene Publikationen (Auswahl)
- Clinical effects of phosphodiesterase 3A mutations in inherited hypertension with brachydactyly, Hypertension. 2015 Oct;66(4):800-8
Toka O, Tank J, Schächterle C, Aydin A, Maass PG, Elitok S, Bartels-Klein E, Hollfinger I, Lindschau C, Mai K, Boschmann M, Rahn G, Movsesian MA, Müller T, Doescher A, Gnoth S, Mühl A, Toka HR, Wefeld-Neuenfeld Y, Utz W, Töpper A, Jordan J, Schulz- Menger J, Klussmann E, Bähring S, Luft FC
(Siehe online unter https://doi.org/10.1161/HYPERTENSIONAHA.115.06000) - PDE3A mutations cause autosomal dominant hypertension with brachydactyly, Nat Genet. 2015 Jun;47(6):647-53
Maass PG, Aydin A, Luft FC, Schächterle C, Weise A, Stricker S, Lindschau C, Vaegler M, Qadri F, Toka HR, Schulz H, Krawitz PM, Parkhomchuk D, Hecht J, Hollfinger I, Wefeld-Neuenfeld Y, Bartels-Klein E, Mühl A, Kann M, Schuster H, Chitayat D, Bialer MG, Wienker TF, Ott J, Rittscher K, Liehr T, Jordan J, Plessis G, Tank J, Mai K, Naraghi R, Hodge R, Hopp M, Hattenbach LO, Busjahn A, Rauch A, Vandeput F, Gong M, Rüschendorf F, Hübner N, Haller H, Mundlos S, Bilginturan N, Movsesian MA, Klussmann E, Toka O, Bähring S
(Siehe online unter https://doi.org/10.1038/ng.3302) - A map of human circular RNAs in clinically-relevant tissues, J Mol Med (Berl). 2017 Nov;95(11):1179-1189
Maass PG, Glažar P, Memczak S, Dittmar G, Hollfinger I, Schreyer L, Sauer AV, Toka O, Aiuti A, Luft FC, Rajewsky N
(Siehe online unter https://doi.org/10.1007/s00109-017-1582-9) - Gene silencing and a novel monoallelic expression pattern in distinct CD177 neutrophil subsets, J Exp Med. 2017, May 30
Eulenberg-Gustavus C, Bähring S, Maass PG, Luft FC, Kettritz R
(Siehe online unter https://doi.org/10.1084/jem.20161093) - Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging: SNP-CLING, in press, Nat. Struct. Mol. Biol. 2017
Maass PG, Barutcu AR, Shechner DM, Weiner CL, Melé M, Rinn JL
(Siehe online unter https://doi.org/10.1038/s41594-017-0015-3)
