Differentiation Dependent Dynamics of Histone Modifications during Myelopoiesis.

Covalent modifications on histones are epigenetic changes that play critical roles in control of gene expression. Most studies correlating specific histone modifications with transcriptional activity have been performed in yeast, and little is known about their dynamics during hematopoietic differentiation. We examined the dynamics of histone modifications and chromatin accessibility during all-trans retinoic acid (ATRA) induced differentiation of promyelocytes down the neutrophil lineage. As a model system, we used the human promyelocytic NB4 cell line, which undergoes neutrophil differentiation in response to ATRA. Using chromatin immunoprecipitation (ChIP) and quantitative PCR, we measured changes in dimethyl K4 (2MeH3K4), trimethyl K4 (3MeH3K4) and acetyl lysine 9 (Ac9H3K9) of histone H3 in the promoters of 3 genes that undergo transcriptional activation (Defensin-a, C/EBP-b and RAR-b), 1 gene that undergoes transcriptional downregulation (Myeloperoxidase), one gene that is constitutively active (GAPDH) and 1 gene that is silent (Albumin) during ATRA-induced differentiation. We correlated the changes in histone modifications with the gene expression pattern of these genes. AcH3K9 levels correlated with active gene transcription. At time 0, levels of AcH3K9 were enriched 50-fold and 100-fold over input in the MPO and GAPDH promoters, respectively, but only 5-fold on the C/EBP-b, Def-a and RAR-b promoters. Consistent with this finding, levels of AcH3K9 increased to 40-fold over input within 24h of differentiation in the Def-a and C/EBP-b promoters. On the silent albumin promoter, AcH3K9 levels never increased over input after ATRA. For the methylation patterns on H3K4, however, the findings were quite revealing. As expected, on the active MPO promoter, 2MeH3K4 was enriched 60-fold. However, 2MeH3K4was also present at high levels (15-30-fold) on the promoters of unexpressed Def-a, C/EBP-b and RAR-b suggesting that silent genes that are “primed” for activation are enriched for 2MeH3K4, consistent with previous data in yeast. After differentiation with ATRA, 2MeH3K4 went up only 3-fold for Def-a and C/EBP-b. The most surprising changes were found in 3MeH3K4 levels and in chromatin modification at the RAR-b promoter. Consistent with previous data showing that 3MeH3K4 is associated with gene activation, time 0 levels of 3MeH3K4 were enriched 80-fold and 150-fold over input in the MPO and GAPDH promoters, respectively, and were at background levels in the C/EBP-b, Def-a, and RAR-b promoters. After differentiation, however, although there was a significant increase in 3MeH3K4 levels within 12 hours in the Def-a promoter, 3MeH3K4 never was present on the C/EBP-b promoter, despite a huge increase in gene transcription, as well as significant and rapid increases in AcH3K9 and 2MeH3K4. Perhaps the most significant findings, however, were at the promoter of the RAR-b gene. RAR-b is unique amongst the genes studies in that it is directly bound by the PML-RAR-a fusion protein. This promoter was not detectable by PCR in ChIP assays after ATRA addition. To test the hypothesis that histones were lost on the RAR-b promoter during ATRA-induced differentiation, we used MNase digestion of chromatin. Nucleosome loss was confirmed by a decrease in precipitated RAR-b promoter DNA within 24 hours of ATRA addition. In contrast, exon1 of RAR-b was modified in a manner similar to Def-a and C/EBP-b. These findings indicate that despite the seemingly coordinately regulated increase in transcription of multiple genes upon myeloid differentiation, the chromatin modifications on the promoters of these genes are regulated quite differently.