Nuclear and chromatin mechanics in cell fate specification

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

We have made significant progress in all 3 research areas of interest this year, especially as it pertains to setting up the tools and pipelines with which to interrogate key open questions of interest to the lab and fields of nuclear and chromatin mechanics and cell fate specification. Specifically we further optimized LADome proteomics approaches in a human cell line and designed strategies to transition the system to hiPSC system. We further engineered constructs allowing us to manipulate spatiotemporal composition of LADs and CRISPred them into hiPSC system. We have generated two large proteomics datasets for this project analysis of which will generate key targets and provide a molecular handle on mechanistic role of LAD composition and architecture during cell fate transitions. In a collaborative effort with NCIFrederick MHL unit we performed an imaging screen on a panel of colorectal cancer TMA samples accessing nuclear shape, chromatin state, mechanontrunsduction, and stemness at single cell-level resolution and redesigned pipelines for all image analyses studies which are currently ongoing. This year we finished all analyses and set up a collaborative CosMx spatial transcirptiomics analysis of full CRC tumor biopsies in collaboration with the NCI genomics laboratory. In addition we set up new in vitro cellular systems (2-and 3-dimensaional colon/colorectal cancer cultures with modulated wnt signaling in collaboration with Jason Spence at U Michigan) and tools (antisense oligonucleotide- and si-RNA-mediated silencing, CRISPRlenti approaches to module APC etc) with which to validate our imaging screen. Furthermore, we have initiated mouse work aimed at deciphering the role of APC to genome rewiring and nuclear/chromatin mechanics in vivo using tamoxifen inducible APC knockout in LGR5-positive intestinal stem cells. In the next year with aim to cross this mouse with a LAD/transcription reporter mouse (MTA is ongoing).