Probing inducible transcription factor activation at the single molecule level in living cells: Past, present and future challenges
Dr Davide MAZZA
San Raffaele Scientific Institute. Experimental Imaging Center. Milan, Italy
Monday, February 11th 2019 - 11:30 a.m.
- Auditorium, IGBMC
Hosted by Nacho MOLINA
In the last ten years, applications of single-molecule approaches to study the dynamic behavior individual proteins inside living cells have multiplied, especially in the field of transcription and gene expression. Single molecule tracking (SMT) of nuclear factors (NFs), for example, has been used to determine that: (i) Transcription factors bind transiently to their responsive elements in living cells (1, 2); (ii) individual NFs use distinct search mechanisms to scan for their binding sites (3); (iii) longer binding times of transcriptional activators and repressors result in stronger activation/repression of target genes (4, 5).
By using our research on the tumor-suppressor p53 as an example, I will describe the methods we and others used to acquire, analyze and interpret single molecule data of NFs. I will discuss some of the challenges that our field has faced, from the necessity of common grounds for data analysis to the extension of the method to thick 3D samples – e.g. living embryos. More challenges lie ahead, towards a biochemical characterization of the NF/chromatin interactions with single-cell and single-molecule sensitivity.
(1) Mazza D. et al., Nucl. Ac. Res., 2012.
(2) Gebhardt J.C.M. et al., Nat. Meth., 2013.
(3) Izeddin I., et al., eLife, 2014.
(4) Loffreda A.et al, Nat Commun., 2017.
(5) Callegari A. et al., Plos Genetics, in press.