Histone modifications are implicated in regulating chromatin condensation but it 
is unclear how they differ between constitutive heterochromatin and unexpressed 
euchromatin. Chromatin immunoprecipitation (ChIP) assays were done on various 
human cell populations using antibodies specific for acetylated or methylated 
forms of histone H3 or H4. Analysis of the immunoprecipitates was by 
quantitative real-time PCR or semi-quantitative PCR (SQ-PCR). Of eight tested 
antibodies, the one for histone H4 acetylated at lysine 4, 8, 12, or 16 was best 
for distinguishing constitutive heterochromatin from unexpressed euchromatin, 
but differences in the extent of immunoprecipitation of these two types of 
chromatin were only modest, although highly reproducible. With this antibody, 
there was an average of 2.5-fold less immunoprecipitation of three constitutive 
heterochromatin regions than of four unexpressed euchromatic gene regions and 
about 15-fold less immunoprecipitation of these heterochromatin standards than 
of two constitutively expressed gene standards (P <0.001). We also analyzed 
histone acetylation and methylation by immunocytochemistry with antibodies to H4 
acetylated at lysine 8, H3 trimethylated at lysine 9, and H3 methylated at 
lysine 4. In addition, immunocytochemical analysis was done with an antibody to 
heterochromatin protein 1alpha (HP1alpha), whose preferential binding to 
heterochromatin has been linked to trimethylation of H3 at lysine 9. Our 
combined ChIP and immunocytochemical results suggest that factors other than 
hypoacetylation of the N-terminal tails of H4 and hypermethylation of H3 at 
lysine 9 can play an important role in determining whether a chromatin sequence 
in mammalian cells is constitutively heterochromatic.