Biology

Dr. Swati Tyagi
Salk Institute, USA

Abstract:

Chromatin organization in the 3D space of a nucleus is maintained through inter-/intra- chromosomal forces, and the external cues from nuclear landmarks like nucleoli, speckles, nuclear envelope (NE) and its components, such as nuclear lamina and nuclear pore complexes (NPCs). While nuclear lamina interacts mainly with heterochromatin, NPCs have been linked to interact with euchromatin regions of chromatin, but much less is known about the identity of these regions and how the spatial and temporal dynamics of these interactions affect the change in the transcriptional state of these gene loci. The conventional antibody-based method like ChIP-seq fails for NPC components due to tight association of NPCs with the nuclear membrane and moreover, the presence of soluble components of NPCs (Nucleoporins) in the nucleoplasm makes it difficult to dissect the interactions that are specifically associated with NPCs. To overcome these problems, we have developed a new method, in vitro DamID, by combing the DamID method with a reconstituted nuclear transport assay in semi-permeabilized cells or purified nuclei. In this technique, we exploit well-established nuclear transport assay to target recombinant expressed and purified Dam fused nuclear transport receptor, Dam-Importin beta (Dam-Ibeta) to the NPCs. Since there is no dependency on the expression of the protein inside the cells, Dam-Ibeta probes the genomic DNA in the vicinity of NPCs through methylation just after the addition of the protein to the cells achieving high temporal resolution. The probed methylated genomic regions are sequenced and imaged through high-resolution microscopy to study the spatial and temporal dynamics of NPC-genome interactions. The assay is performed in non-fixed samples which allows us to not only study the NPC mediated genome organization in cell lines but also in live tissue samples. The results from this method has provided us the new insights of conserved principles of genome organization through NPCs in different cell types. We found that the NPCs associates with super-enhancers genomic regions and we are uncovering exciting mechanisms behind the structural organization of super-enhancers regions at NPCs.

The restricted mobility of Dam-Ibeta at NPCs will increase the effective concentration and thus will likely be able to report on weak interactions that could be missed in in vivo DamID experiments.