Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites

Abstract

DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and erythroblasts (ERYs). Methyl binding domain protein 2 (MBD) enrichment of DNA followed by massively parallel sequencing (MBD-seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSCs, with a 40% reduction in CMPs, and a 67% reduction in ERYs. Only 3% of peaks arise during differentiation, demonstrating a genomewide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20%-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSCs and CMPs. Elevated genome-wide DNA methylation in HSCs and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSCs. Using de novo motif discovery, we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPA and ELF1, are overrepresented in methylated regions. Our genomewide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis.

Hematopoietic stem cells: concepts, definitions, and the new reality

“…These analyses have uncovered numerous types of chromatin regulators, including members of many transcription factor complexes and their downstream targets as well as regulators of the metabolome, protein synthesis, and mediators of signaling responses to external cues. [47][48][49][50][51][52] Indeed, the multiplicity of entities that appear to have nonredundant roles has been disconcerting in suggesting the lack of a singular pathway or state that regulates the maintenance of LTRC potential, or that distinguishes these cells from derivatives that appear irreversibly destined to differentiate in available assay systems. Rather, current findings suggest the involvement of highly complex and possibly alternative molecular networks controlling LTRC self-renewal that may require systems approaches to elucidate.…”

The role of chromatin modifiers in normal and malignant hematopoiesis

“…50 The MBD domain MBD2 was recently used to affinity-purify methylated DNA fragments from mouse HSCs, CMPs, and erythroblasts to determine changes in 5mC during myeloid differentiation. Using this method, a high level of 5mC was detected in mouse HSCs, with a dramatic loss of global methylation occurring during myeloid differentiation, 51 similar BLOOD, 18 APRIL 2013 x VOLUME 121, NUMBER 16 …”

Signatures of DNA target selectivity by ETS transcription factors

“…Detailed information on the localization, sequence characteristics of DNA targets, and associated binding partners is now available for ETS transcription factors in a range of cell types and developmental contexts. 27,[50][51][52][53][54][55] Although various levels of redundancy and specificity are observed that correlate with the ontology of the genes involved, one recurring feature is the close correspondence between in vivo and in vitro DNA sequence preferences. Moreover, in the case of PU.1 and Ets-1, the information contents (a direct informatic measure of target specificity) of the in vivo sequences preferences shown by both proteins are over 15% higher (> 3 bits over a 10-bp sequence space) than their in vitro counterparts.…”

“…Genomic surveys have found that hematopoietic ETS transcription factors are over-represented in hypermethylated regions. 53 While several close class I ETS paralogs (such as Ets-1 and GABPa) have been reported to be inhibited by CpG methylation at their cognate sites, whether inhibition is a universal property of ETS proteins remains unknown. We have directly studied the binding properties of PU.1 and Ets-1 to hemi-methylated and fully methylated cognate DNA harboring a site-specific CpG dinucleotide (5 0 -CGGAA-3 0 ) that frequently occurs in cognate ETS binding sites.…”

“…The established molecular pathophysiology associated with some of these mechanisms are listed. The literature on ETS proteins is vast and this summary is only intended to be illustrative; readers interested in specific aspects or paralogs mentioned in this figure are referred to reviews and studies such as the following: 26,49,51,53,66,[87][88][89][90][91][92][93] Note that auto-inhibition (indicated by the cartoon helix) is not a universal feature of ETS proteins; several paralogs, such as PU.1, are not auto-inhibited. Abbreviations: AML, acute myeloid leukemia; Ca, cancer; cHD, classical Hodgkin's disease; MM, multiple myeloma.…”