Accumulating evidence demonstrates that epigenetic changes, including DNA methylation play a central role in differentiation, providing cellular memory and stabilizing lineage choice in hematopoiesis1–3. DNA methylation is an important epigenetic mechanism involved in transcriptional regulation, heterochromatin formation and the normal development of many organisms.
In this study we investigated the DNA methylome and transcriptome of human cells in four separate differentiation stages in granulopoiesis, ranging from the multipotent Common Myeloid progenitor (CMP) to terminally differentiated bone marrow neutrophils (PMN). To this end we employed HumanMethylation 450 BeadChip (450K array) from Illumina with extensive genomic coverage and mRNA expression arrays (Illumina).
Differential methylation between two cell stages was defined as an average difference in β value of at least 0.17 (p ≤ 0.05). We detected 12132 DMSs during granulopoiesis. Of these the majority showed decreased methylation during granulopoeisis (10771 CpGs) and a smaller set gained methylation (1658 CpGs). Strikingly, increases in methylation predominantly occur between CMP and GMP, the two least mature cell types. Some CpGs also show increased methylation in the GMP-PMC transition, while very few CpG sites increase at the final stage of differentiation from PMC to PMN. Although reduction of methylation occurs at all stages of granulopoiesis, the greatest change is between GMP and PMC. It is striking that the DNA methylation patterns preferentially change at points of lineage restriction, and that the greatest change occurs upon loss of oligopotency between GMP and PMC.
DMSs within CGIs were greatly underrepresented (p<0.001 with chi-square test), while DMSs were overrepresented in shelves (p<0.001) and open sea (p<0.001). Thus, methylation appears to be more dynamic outside of CGIs during granulocytic development. For all regions the variation within enhancers was greater than outside of enhancers indicating greater methylation changes in enhancers compared to non-enhancers. In addition, CpGs in enhancer regions are significantly enriched in the list of DMSs (p<0.001, chi-square test) further supporting the observation that enhancer regions display dynamic DNA methylation changes during granulopoiesis.
There was a significant overlap between genes showing decreased methylation and genes with increased expression as well as for the reverse comparison between genes with increased methylation and decreased expression. Thus, support a general anticorrelation between DNA methylation and gene expression. Azurophilic granule proteins showed increased expression peaking in PMC and a rapid decrease toward PMN. CpG methylation levels for those genes decreased concomitantly with the peak in expression.
We report cell population specific changes of DNA methylation levels. The main reduction of CpG methylation coincides with the loss of oligopotency at the transition from GMP-PMC. This suggests a role of DNA methylation in regulating cell plasticity and lineage choice.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.