In the present study, we compared the methylation profiles of early progenitor cells starting from a point prior to lineage commitment (predominantly MPPs and CLPs) through pre-B-I, pre-B-II and immature B-cells, and have comprehensively cataloged methylation changes associated with pre-B-cell development. The dense probe placement enabled us to examine methylation changes within and outside CGIs and in various locations within individual genes to gain insights on loco-regional epigenetic control of human early B-cell development. Our analysis created a reference set for those studies investigating malignancies originating from B-cell progenitors which are the most common cancer in children. In combination with genome-wide RNA expression profiling, we have identified a distinct development-dependent demethylation signature which has gene expression regulatory properties. Altered DNA methylation occurred predominantly within gene bodies outside of CGIs, but less frequently at annotated gene promoters or with CGIs.
The canonical view on epigenetic gene regulation is that DNA methylation in CGIs in gene promoters has a principle role for repressing gene expression, and the loss of methylation in this region is associated with gene activation (7
). Recently, several groups have provided evidence linking gene body methylation and transcription, but outstanding questions remain (24
). Our data suggest a complex mechanism of epigenetic gene regulation during pre-B-cell development via DNA methylation. We observed that DNA methylation changes more frequently occur at gene body and remote upstream regions than promoter regions during early B-cell development although those at promoters still possess the most potent effect on gene expression. It is notable that at S1, before complete lineage commitment, the promoters of the majority of the genes are hypomethylated (β
< 0.3), especially those having CGIs in their promoters (Supplementary Figure S2A
). Our data indicate that, during progression through pre-B-cell stages, these promoter regions maintained their hypomethylation status whereas alterations in methylation occurred specifically to the gene body regions of many genes. Many of the DMRs in the body regions may correspond to cryptic TSS sites or enhancer regions, which need to be suppressed or activated for efficient gene transcription (25
). We also observed demethylation of remote upstream regions in some up-regulated genes such as NH1R3
. In fact, there is evidence that enhancer methylation in some genes is functionally critical for gene regulation (26
). This may be understood in line with a previous model that primary TFs prime their target gene promoters at earlier stage in association with cell fate decisions and the gene expression is ‘triggered’ with a time lag after accumulation of secondary reinforcing events (5
). On the contrary, some genes had their promoter methylated at baseline and were activated almost simultaneously with promoter demethylation, suggesting an instant triggering role of these genes. We provided one example of this in the pertinent role of demethylation at low-density CpG (non-Island) sites in the promoter regions having the strongest functional impact in triggering expression changes (A) in the establishment of pre-B-cell lineage. We also found that these non-CGI promoters display a histone modification profile that is more similar to CGI promoters than to the average non-CGI promoters.
Strikingly, only demethylation processes were noted from stages S2 to S4, although many genes were down-regulated during this period. Furthermore, there were only seven genes that were altered in expression and in methylation from stages S3 to S4 (Supplementary Table S3
); these genes included a negative regulator of BCR signaling, BTLA (which was increased in expression in S4) and the adenosine deaminase ADARB1, a gene distinct from the hypermutation and switch recombinase gene AICDA or activation-induced cytidine deaminase (Supplementary Table S3C
). DNA methyltransferases including DNMT1, DNMT3A and DNMT3B were down-regulated as the stages progressed, supporting the diminished role of methylation in later stages. Therefore, it may be postulated that mechanisms other than methylation have the primary role in down-regulating genes in later stages, which may include changes in TF activity from protein modification, alterations in TF interactions and combinatory co-binding properties, changes in histone modification, effects of cytokines and miRNAs, and altered RNA decay speeds from RNA interference (28–32
We noted that the de novo
and demethylation processes occurred in regions enriched for specific TF binding sites, and these were identified in silico
data using motif finding algorithms (Supplementary Table S1
) as well as in in vivo
data using ChIP-Seq data from the ENCODE project (). From S1 to S2, EBF binding motifs were most significantly demethylated corresponding to its central role in B-cell development (3
). This was followed by Ets, RUNX1, TCF3 and ELF5 motifs (in silico
) and E2F family members and PAX5 (in vivo
). The Ets family TFs consists of over 30 members sharing common DNA binding (ETS) domain and have significant but heterogeneous roles on hematopoiesis (33
). Accordingly, de novo-
methylated regions were also enriched for Ets family TFs. SPI1 (PU.1), an Ets family TF involved in the myeloid versus B-lymphoid lineage decision, were also enriched both in hyper- and hypomethylated regions in vivo
suggesting its complex roles (3
). The TCF3 (E2A) is putatively known to activate the B-cell lineage-specific gene program synergistically with EBF1 and its motifs were significantly enriched here () (3
). Gene families associated with hypermethylation include targets of the myeloid TFs CEBPB and P300 (), a marker of lineage commitment. The SPIB motif was also enriched in hypermethylated regions; the molecule is specific to plasmacytoid dendritic cells and HSCs (35
) suggesting that its repression is associated with binding site hypermethylation.
Using expression analysis, we found that many pathway-related genes were up-regulated from S1 to S2 (Supplementary Table S2
). As expected, antigen presenting, BCR and B-cell developmental pathways were most significantly enriched. The PI3K pathway was suggested to be crucial for BCR/pre-BCR signaling (36
), and protein ubiquitination was recently highlighted in NFkB signaling (37
). The HGF β
-chain was found to form a pre-pro-B-cell growth-stimulating factor with interleukin-7 (38
), but may need further exploration. The role of NFAT family, HGF, IL-3, protein kinase A and glucocorticoid and estrogen receptor pathways were somewhat redundant although some were previously implicated in B-cell development (39–44
). From S3 to S4, a number of genes related to cell division and mismatch repair were down-regulated with BRCA1-related pathway being most significant. The ATM, CHK, PLK and cyclin pathways are also related to cell cycle control and DNA repair. This may be due to the reduced need for cell division but may also be related to preparations for ‘hypermutation’ in mature and activated B-cells. Interestingly, a study using Brca−/−
cell line found increased somatic hypermutation in the immunoglobulin genes (45
Recently a global DNA demethylation signature was discovered in mouse erythropoiesis (46
). This demethylation is associated with DNA replication and occurs at all DNA elements. This process is in contrast with the demethylation signature that Calvanese et al.
) observe in non-erythropoietic cells, which are specific and targeted demethylation events with functional relevance, e.g. F. In contrast to erythrocytes, a functional and specific DNA methylation pattern is critical for differentiated cell function in B-cells which remain nucleated and many of which are long-lived.
In summary, we have investigated DNA methylation changes in early human B-cell development in association with expression changes. DNA methylation changes were associated with profound effects on gene expression during early lineage commitment, especially DNA methylation changes in regions other than promoters. The changes were non-randomly located in terms of CGIs, alternative TSSs and TF binding sites. The impact of DNA methylation on gene regulation was reduced in later stages of B-cell development, suggesting that mechanisms other than DNA methylation may have a principal role after lineage commitment.