Single-molecule super-resolution imaging of the X chromosome using oligopaints FISH technology.

Dr. Asifa Akhtar (Max-Planck-Institute of Immunobiology and Epigenetics Freiburg)

Nuclear organization plays an important role in epigenetic regulation. Recent work in several model organisms has demonstrated that gene loci contact different components of the nuclear membrane depending on their gene activity. For example, association of genes with the nuclear lamina leads to gene repression, while in contrast association with nuclear pore complexes is associated with gene activation. Our lab is working towards gaining a better understanding of how the X chromosome is spatially organized in the nucleus to execute a two-fold upregulation of genes in the milieu of all the other autosomes in flies. Using the HiC chromosome capture technology, performed during the last BIOSS funding period (Ramirez et al. 2016), we now propose a new concept of “conformation based affinity” as the novel mechanism used by the dosage compensation complex to specifically recognize the X chromosome versus autosomes. However, the current model is based on population based genomewide analyses, where chromosomal contact “probabilities” of loci are calculated. For the first time, we are now in a position to study via super resolution microscopy on a single cell level, how the high affinity sites located on the X chromosome cluster in space, compared to other regions on autosomes, thereby contributing to X chromosomal specificity.

Classically, spatial organization of nucleic acids can be visualized by in situ hybridization techniques. Yet, our ability to directly visualize the fine scale structure of the genome in situ, remains constrained by the optical resolution of light microscopy and importantly, our ability to target regions of interest. In future, we are interested to use the latest technologies of visualizing multiple genome locations simultaneously, using barcoded strategies to study how several regions of the X chromosome come in close proximity using super resolution microscopy.

Selected publications

Quinn J.J., Zhang Q.C., Georgiev P., Ilik I.A., Akhtar A. and Chang H.Y. (2016) Rapid evolutionary turnover underlies conserved lncRNA–genome interactions. Genes & Development 30, 191-207.

Ramirez F., Lingg T., Toscano S., Lam K.C., Georgiev P., Chung H.R., Lajoie B., de Wit E., Zhan Y., de Laat W., Dekker J., Manke T. and Akhtar A. (2015). High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila. Molecular Cell 60, 146-162.

Meunier S., Shvedunova M., Van Nhuong N., Avila L., Vernos I. and Akhtar A. (2015). An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis. Nature Communications 6, 7889 doi:10.1038/ncomms8889

Dias J*, Van Nguyen N*, Georgiev P, Gaub A, Brettschneider J,  Cusack S, Kadlec J and Akhtar A (2014). Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex.  Genes & Development 28, 929-942.

Chelmicki T, Dündar F, Turley M, Khanam T, Aktas T, Ramírez F, Gendrel AV, Heard E, Manke T, Akhtar A (2014) MOF complexes use short and long-range interactions to ensure stem cell identity and Xist repression. eLife10.7554, eLife.02024.

Quinn JJ, Ilik AI, Qu K, Georgiev P, Chu C, Akhtar A, Chang HY (2014) Domain ChIRP reveals the modularity of long noncoding RNA architecture, function, and target genes. Nature Biotechnology 32, 933–940

Ilik I, Quinn JJ, Georgiev P, Tavares-Cadete F, Maticzka, Toscano S, Wan Y, Spitale RC, Luscombe NM, Backofen R, Change HY, Akhtar A (2013) Tandem stem loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila. Molecular Cell 51, 156-173.

Hallacli E, Lipp M, Georgiev P, Spielman C, Cusack S, Akhtar A,* Kadlec J*. (2012) MSL1 mediated dimerization of the dosage compensation complex is essential for male X chromosome regulation in Drosophila. Molecular Cell 48, 587-600.* co-corresponding authors.

Conrad T, Cavalli FMG, Vaquerizas JM, Luscombe NM, Akhtar A. (2012)  Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters. Science 337, 742-746. *co-corresponding authors.

Lam KC, Muehlpfordt F, Vaquerizas JM, Raja S, Luscombe NM, Manke T, Akhtar A. (2012) NSL complex regulates housekeeping genes in Drosophila. PLoS Genetics 8, e1002736. Epub 2012 Jun 14.

Conrad T, Cavalli F, Hallacli E, Holz H, Kind J, Iilk I, Vaquerizas JM, Luscombe NM, Akhtar A. (2012) The MOF chromobarrel domain controls genomewide H4K16 acetylation and spreading of the MSL complex. Developmental Cell 22, 610-624.

Kadlec J, Hallacli E, Lipp M, Holz H, Weatherby JS, Cusack S*, Akhtar A*. (2010) The structural basis for the recruitment of MOF and MSL3 into the dosage compensation complex by MSL1. Nature Structural and Molecular Biology 18, 142-149.*co-corresponding authors