Single-particle imaging of stress-promoters induction reveals the interplay between MAPK signaling chromatin and transcription factors

摘要

Schematics of the transcriptional response induced by the MAPK Hog1 upon osmotic stress. Under normal growth conditions, the genomic locus is repressed by histones set in place by the Ino80 complex and Asf1/Rtt109. In addition, H3K4 methylated histones mediated by Set1 contribute to the further repression of the locus (upper panel). When Hog1 is active (lower panel), it accumulates in the nucleus with the transcription factors Msn2/4. Hog1 binds to the transcription factors Hot1 and Sko1, allowing the remodeling of the chromatin by Rpd3 and the SAGA complex. The polymerases can be recruited to the locus and the RSC and SWR complexes evict nucleosomes on the ORF. Construction of the transcriptional reporter. The promoter of interest (p ) is cloned in front of 24 stem-loops ( ). This construct is transformed in yeast and integrated in the locus 5′UTR, replacing the endogenous promoter. Upon induction of the promoter of interest, the mRNA stem-loops are transcribed and recognized by the fluorescently-tagged PP7 phage coat proteins. Maximum intensity projections of Z-stacks of microscopy images from the p -PP7sl reporter system in a 0.2 M NaCl osmotic stress time-lapse experiment. The appearance of bright foci (arrow heads) in the nucleus of the cells denotes the active transcription arising from the promoter. Scale bar represents 5 µm. Representative images from at least three biological replicates. Dynamics of the p -PP7sl transcription site intensity (20 brightest pixels in the nucleus minus the median fluorescence of the cell) following hyperosmotic stress. The mean of the population is represented by the solid line. The shaded areas represent the s.e.m. Number of cells for each trace: 0.0 M: 313; 0.1 M: 404; 0.2 M: 229; 0.3 M: 201. Analysis of one representative single-cell trace. The raw trace (gray) is smoothed with a moving average (dark blue) and normalized by subtracting the intensity of the first time point after the stimulus. Multiple quantitative values can be extracted from this trace (see Methods). Source data are provided for .