The Dynamics of Chromatin Modification during RA Induced Promyelocyte Differentiation.

Epigenetic histone modifications have recently been shown to be important in gene regulation and to be correlated with gene expression profiles. In particular, particular modifications at Lysine 4 and Lysine 9 of Histone H3 may be highly indicative of both active and silenced chromatin. Promoters of highly active genes are generally tri-methylated at Lysine 4 and acetylated at Lysine 9 whereas silenced chromatin is unmethylated or mono methylated at Lysine 4 and di or tri-methylated at Lysine 9. NB4 is a human acute promyelocytic leukemia (t15:17) cell line that can be differentiated down the neutrophil lineage by induction with supraphysiological levels of retinoic acid (RA). We have examined post-translational modifications at Lysine 4 and Lysine 9 of Histone H3 at specific promoters using chromatin immunoprecipitation (ChIP) during differentiation of NB4 cells. We have performed quantitative (q) RT-PCR for the Myeloperoxidase (MPO) and Defensin-alpha (Def-a) genes after 0,12, 24, 48 and 72 hours of RA induction of NB4 cells. Concurrently we performed ChIP followed by qPCR for Di- and Tri-methylated Lysine 4 of Histone H3 and acetyl- and di-methylated Lysine 9 of Histone H3 at the promoters of these genes. MPO mRNA is expressed at time 0, and decreases progressively starting at 24 hours such that by 48 hours mRNA levels are negligible. This correlates nicely with a sharp decrease in tri-methyl Lysine 4 and acetyl Lysine 9 levels by 12 hours, with a smaller decrease again at 24 hours on the MPO promoter. By 72 hours a concurrent increase in dimethyl Lysine 9 levels is seen, indicating chromatin silencing. Conversely, def-a is initially not expressed in NB4 cells. After 24 hours of induction with RA, an increase in mRNA levels is seen. This correlates well with a sharp increase of acetyl Lysine 9 in the promoter and a concurrent decrease in dimethyl lysine 9 levels. In conclusion we have shown that during RA induced differentiation of promyelocytic leukemia cells, chromatin modifications slightly precede changes in mRNA expression levels and recapitulate other studies linking Lysine 4 methylation with active transcription and Lysine 9 methylation with gene silencing.