Fig. 4.

Chromatin changes induced by starvation stress. (A) Bar graph showing the response to 16 h of starvation stress for either selected chromatin-associated proteins, or negative control proteins (RPS-6, PAR-5, MRPS-5). The y axis indicates each protein. The bar is colored green to indicate whether the p value, derived from a Student's t test across the three biological replicates for each protein, was p < .05. The relative fold change is shown on the x axis using the log₂ ratio L/H (starved/FED), with error bars indicating standard deviation (n = 3). (B) Line graphs showing the mean peptide SILAC ratio versus time after starvation stress, for selected chromatin-associated proteins. Error bars show standard error of the mean for the peptide SILAC ratio. (C) Mass spectra showing precursor intact peptide ions for representative SILAC pairs from peptides detected in the 16 h triplicate starvation experiment as shown in part (A), which are specific for either histone H3.3 (left), or H3.1 (right). (D) An alignment of the highly similar histone-H3-related proteins (HIS-2/71/72/74). The conservation of each sequence motif is indicated by the shade of the blue colored overlay. Red boxes surrounding the peptide sequence indicate the peptides detected in our starvation experiments. (E) Immunoblotting of total C. elegans lysates from nematodes either starved, or fed, for 16 h. The strain of C. elegans used contained a stable transgene with his-71::GFP to express a GFP-tagged histone H3.3 type-1 (HIS-71) protein. Immunoblotting was performed using either an anti-GFP antibody to detect HIS-71-GFP or an antibody specific for alpha-tubulin as a loading control. (F) Plot showing the response to 16 h of starvation stress for either total histone H4 (HIS-1) protein, or the double modified histone H4 peptide containing both acetylated lysine 5 and acetylated lysine 8. The relative fold change is shown on the x axis using the log₂ ratio L/H (starved/fed), with error bars indicating standard deviations (n = 3).