Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome.

Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (approximately 25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9/14 of histone H3 were both highly localized to the 5' regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes.

Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-aza-2'-deoxycytidine.

Epigenetic modifications of cytosine residues in DNA and the amino termini of histone proteins have emerged as key mechanisms in chromatin remodeling, impacting both the transcriptional regulation and the establishment of chromosomal domains. Using the chromatin immunoprecipitation (ChIP) assay, we demonstrate that aberrantly silenced genes in cancer cells exhibit a heterochromatic structure that is characterized by histone H3 lysine 9 (H3-K9) hypermethylation and histone H3 lysine 4 (H3-K4) hypomethylation. This aberrant heterochromatin is incompatible with transcriptional initiation but does not inhibit elongation by RNA polymerase II. H3-K9 methylation may, therefore, play a role in the silencing of tumor-suppressor genes in cancer. Treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), previously known for its ability to inhibit cytosine methylation, induced a rapid and substantial remodeling of the heterochromatic domains of the p14ARF/p16INK4a locus in T24 bladder cancer cells, reducing levels of dimethylated H3-K9 and increasing levels of dimethylated H3-K4 at this locus. In addition, 5-Aza-CdR increased acetylation and H3-K4 methylation at the unmethylated p14 promoter, suggesting it can induce chromatin remodeling independently of its effects on cytosine methylation.