Journal: Molecular Biology of the Cell

Article Title: H4K20 methylation regulates quiescence and chromatin compaction

doi: 10.1091/mbc.E12-07-0529

Figure Lengend Snippet: Modification status of H4K20 changes as fibroblasts become quiescent. The relative abundances of detectable histone modifications on histones H3 and H4 were determined for P, G 1 -enriched, 14dCI, and 21dCI fibroblasts using (LC-MS/MS). P, G 1 -enriched, and 21dCI histones were compared with 14dCI histones by labeling the N-termini of histones with hydrogen- or deuterium-containing propionic anhydride. The values for all modification states on each peptide were used to determine relative abundance for each individual modification state for both samples analyzed together. (A) The log 2 fold changes of 14dCI vs. proliferating, G 1 -enriched, and 21dCI are shown in heat map format. Data represent averages from three independent experiments. (B) Western blotting shows a similar change in abundance of H4K20me3 in noncycling states and cycling states. A pan-H4 antibody was used as a loading control. (C) Six modifications exhibited statistically significant differences ( t test, p < 0.05) between G 1 -enriched and 14dCI fibroblasts. The data from A are plotted to show the fold change between 14dCI and proliferating, G 1 -enriched, and 21dCI fibroblasts. Error bars indicate SE. (D) Percentage of total histones modified for each of the six significant modifications.

Article Snippet: Other nondividing states, such as differentiation of murine myogenic and neural lineages, also show increased levels of H4K20me3 ( Biron et al. , 2004 ; Tsang et al. , 2010 ), and mass spectrometry analysis of differentiating embryonic stem cells showed a correlation between H4K20 methylation and loss of pluripotency ( Phanstiel et al. , 2008 ).

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy, Labeling, Western Blot, Control

Journal: Molecular Biology of the Cell

Article Title: H4K20 methylation regulates quiescence and chromatin compaction

doi: 10.1091/mbc.E12-07-0529

Figure Lengend Snippet: Suv4-20h1 and Suv4-20h2 knockdown results in decreased compaction. shRNAs were stably integrated and expressed in fibroblasts. Nonspecific sequences were expressed in the control cells, and sequences targeting Suv4-20h1 and Suv4-20h2 were used to reduce H4K20me2 and H4K20me3. (A) Histones were analyzed by mass spectrometry, and the fold change was plotted for shSuv4-20h1/h2 cells vs. shControl. (B) Dual-colored FISH was used to measure the distance between both arms of chromosome 16 in shControl and shSuv4-20h1/h2 cells ( p = 1.2 × 10 −6 ; ANOVA). (C) Representative images from FISH experiments depicting both copies of chromosome 16 with each arm identified with a different color. Right, shSuv4-20h1/h2, an example of more extreme decompaction. (D) siRNA sequences were used to reduce the expression of Suv4-20h1 or Suv4-20h2 individually. FISH was used to measure chromosome 16 compaction in siControl, siSuv4-20h1 ( p = 0.055; paired t test), and siSuv4-20h2 cells ( p = 0.01; paired t test).

Article Snippet: Other nondividing states, such as differentiation of murine myogenic and neural lineages, also show increased levels of H4K20me3 ( Biron et al. , 2004 ; Tsang et al. , 2010 ), and mass spectrometry analysis of differentiating embryonic stem cells showed a correlation between H4K20 methylation and loss of pluripotency ( Phanstiel et al. , 2008 ).

Techniques: Knockdown, Stable Transfection, Control, Mass Spectrometry, Expressing