Abstract
Transcription factors (TFs) are core players in the control of gene expression, evolutionarily selected to recognise a subset of specific DNA sequences and nucleate the recruitment of the transcriptional machinery. How TFs assemble and move in the nucleus to locate and bind their DNA targets and cause a transcriptional response, remains mostly unclear. NF-Y is a highly conserved, heterotrimeric TF with important roles in both housekeeping and lineage-specific gene expression, functioning as a promoter organiser. Despite a large number of biochemical, structural and genomic studies of NF-Y, there is a lack of experiments in single living cells; therefore, basic assumptions of NF-Y biology remain unproven in vivo. Here we employ a series of dynamic fluorescence microscopy methods (FLIM-FRET, NB, RICS and FRAP) to study NF-Y dynamics and complex formation in live cells. Specifically, we provide quantitative measurement of NF-Y subunit association and diffusion kinetics in the nucleus that collectively suggest NF-Y to move and bind chromatin as a trimeric complex in vivo.
Highlights
Orchestration of gene expression underlies the differentiation of cells and development of organisms, and the corruption of transcriptional regulation is a central feature of diseases such as c ancer[1]
Within the transfected population of cells, where the transfected NF-Y subunits are present in excess to their endogenous counterparts, we selected cells exhibiting medium to low eGFP and mCherry fluorescence (Fig. S1), as this condition most accurately reflects NF-Y biology and is a requirement of Fluorescence Fluctuation Spectroscopy (FFS) based methods of analysis (e.g. Number and Brightness (NB) and Raster Image Correlation Spectroscopy (RICS))[9,26]
Our results suggest that NF-Y moves and acts as a trimeric complex in the nucleus
Summary
Orchestration of gene expression underlies the differentiation of cells and development of organisms, and the corruption of transcriptional regulation is a central feature of diseases such as c ancer[1]. NF-Y displays early binding to promoters and enhancers during mouse preimplantation development (2–8 cell stage), where it contributes to zygotic genome activation[21] These data suggest a second role of NF-Y in collaborating with cell-type specific pioneer TFs to establish and reinforce an open chromatin state at cis regulatory sequences of genes important for cell identity. Sequence-specific DNA binding is provided by the YA subunit, which trimerises with YB/YC and directs high affinity binding to the CCAAT-box in the DNA minor groove, resulting in an 80° DNA b end[22,23] Despite this abundance of structural, biochemical and genomic data, to our knowledge there are only two published studies of fluorescently tagged NF-Y subunits in mammalian cells: (1) a microscopy study on the intrinsic nuclear localization of YA and YB, as well as the YB-mediated nuclear import of YC24 and (2) a FRET study suggesting the association of YB-CFP to YC-YFP25. Our FLIM-FRET and NB results confirmed the presence of the NF-Y trimer in live cells, while FRAP, RICS and biochemical experiments suggested the trimeric form to be the active, DNA bound species
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
