Global histone profiling by LC-FTMS after inhibition and knockdown of deacetylases in human cells.
Global histone modifications and their putative relevance to short and long term cellular programming have drawn substantial interest in the study of chromatin. Here we describe the use of reverse-phase liquid chromatography coupled to Linear Ion Trap-Fourier Transform Mass Spectrometry (RPLC-LTQ-FTMS) to quickly profile post-translationally modified isoforms and variants for core histone proteins from as few as 5x10(4) cells at isotopic resolution. Such LC-MS profiling greatly facilitated the detection of histones from HeLa S3 or 293T cells experiencing shRNA- or siRNA-knockdown of histone deacetylase (HDAC) 1, 2, 3 or 1 and 2 together. In no case was significant global histone hyperacetylation relative to control cells observed, suggesting possible compensation of deacetylation activity by partially redundant enzymes in the 18-member HDAC family. This contrasts sharply with yeast where genetic deletion of HDAC rpd3 causes massive hyperacetylation. Treatment of cells with TSA and class I selective HDAC inhibitors had similar ability to induce global histone hyperactylation, though to different extents in HeLa S3 vs. 293T cells.
Li
Mingxi
M
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Jiang
Lihua
L
Kelleher
Neil L
NL
D000107:Acetylation / Q000187:drug effects; D002460:Cell Line; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D015870:Gene Expression / Q000187:drug effects; D055785:Gene Knockdown Techniques; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D020033:Protein Isoforms / Q000737:chemistry
D056572:Histone Deacetylase Inhibitors; D006657:Histones; D020033:Protein Isoforms; D006655:Histone Deacetylases