Systematic analysis of DNA methylation of unrearranged and partially rearranged IgH alleles in primary cells revealed several interesting features. First, the lack of correlation between histone modifications and DNA methylation status indicates that these marks are independently regulated. This was most clearly evident in the JH
region that is marked with the highest levels of histone acetylation and H3K4me3 in the unrearranged state, yet remains hypermethylated. Furthermore, germline DFL16.1 and the DSP gene segments were comparably methylated despite being differentially marked at the level of histone modifications. These observations appear to be at odds with genomic studies that show a correlation between histone acetylation and DNA demethylation, and H3K9 methylation and DNA methylation 
. We suggest that the results from genome-wide studies may be skewed towards promoter-based CGIs. Instead, the sites we analyzed were largely non-CGIs and included enhancers (such as Eμ), promoters (such as DQ52), cryptic promoters (such as DSP and DFL16.1, see below), and regions that cannot be categorized as any of these.
An important corollary of this observation is that pre-rearrangement DNA methylation status correlates poorly with recombination potential. Recent studies identified the JH
region as a RAG1/2-rich recombination center 
. Our observation suggests that DNA demethylation is not required to generate the recombination center. Additionally, DFL16.1 and DQ52 gene segments that rearrange most frequently 
have very different levels of DNA methylation prior to rearrangement; conversely, DSP gene segments that rearrange relatively less frequently have comparable levels of DNA methylation as DFL16.1. Taken together, our working model is that DNA methylation status does not guide the first step of IgH gene assembly.
We note, however, that we cannot unequivocally rule out the possibility that selective demethylation of specific DH and JH segments occur in a subset of cells just prior to recombination. For example, while the JH1 region that we analyzed was largely methylated in pro-B cells prior to rearrangement, we observed that a minority of alleles were demethylated in both RAG-sufficient and RAG-deficient pro-B cells. These demethylated JH1 alleles may represent a subset of cells in which DH recombination will occur preferentially to the JH1 gene segment. It will be interesting to determine whether other JH gene segments are similarly singled out for demethylation in subsets of pro-B cells.
Second, sites of maximal tissue-specific CpG demethylation in the germline IgH locus corresponded to the two strongest DNase 1 hypersensitive sites in the 70-kb DH
–Cμ domain, DQ52 and Eμ. Because these regions share transcriptional regulatory properties, it is likely that their demethylation is linked to transcription or transcription-associated chromatin changes. Additionally, we noted highly selective CpG demethylation of some sequences and partial demethylation of others 5′ of DFL16.1 where three additional DNase 1 HSs have been recently identified. Though these observations link DNase 1 hypersensitivity to DNA methylation, this doesn't seem to be always the case. For example, JH
region is known to be highly sensitive to DNase 1 digestion 
(without being associated with “classical” DNase 1 hypersensitive sites), yet it is not demethylated. A possible model for this pattern of methylation emerges from the recent demonstration that Eμ is in spatial proximity to the cluster of DHSs 5′ of DFL16.1 
. Since Eμ itself has a demethylating potential (Eμ flanking regions are methylated on Eμ-deficient alleles), perhaps Eμ looping to 5′ DFL16.1 sites “delivers” demethylating activities to this region. The partial demethylation observed at 5′ DFL16.1 sites may reflect that such loops are present in only a fraction of pro-B cells at any given time. Consistent with the idea that Eμ induces demethylation in a subset of cells, the form of partial demethylation seen at DFL16.1 (−6.5) involves extensive demethylation of some alleles and essentially complete methylation of others (). Eμ has also been proposed to loop to DHS5-7 of the 3′ regulatory region at the 3′ of the IgH locus. Interestingly, earlier studies by Giambra et al. 
showed that DHS5-7 was partially demethylated in a pro-B cell line in a manner similar to the methylation state 5′ of DFL16.1. This pattern may be the consequence of Eμ looping to DHS5-7 in a subset of pro-B cells. Overall, we propose that the pattern of DNA demethylation in the germline IgH locus is determined by spatial proximity to Eμ.
Third, we found that DJH junctions were extensively demethylated. Though we cannot discount the possibility that germline DH and JH gene segments were demethylated just before recombination, we favor the hypothesis that demethylation of DJH junction occurred after recombination. The reasons for this are two-fold: first, DJH junctions remain methylated in Eμ-deficient pro-B cells and in CD4+CD8+ thymocytes. Our interpretation is that these DJH alleles are caught in an intermediate stage where recombination has occurred but demethylation has not. Second, we think it is unlikely that demethylation occurred prior to recombination followed by re-methylation of DJH junctions based on the state of DQ52/JH1 junctions. In both Eμ-deficient pro-B cells and DP thymocytes the methylation status of DQ52/JH1 junctions was such that the DQ52 portion was demethylated and the JH1 portion was methylated. If demethylation preceded rearrangement, generation of each hybrid junction would require re-methylation of a subset of closely associated CpGs.
Though our data clearly demonstrate that DNA demethylation of associated gene segments is not essential for DH
recombination, we note that the two instances of methylated DJH
junctions identified in this study involve circumstances where the frequency of DH
recombination is lower than in wild-type pro-B cells. DH
recombination has been estimated to be 5- to 10-fold lower in Eμ-deleted pro-B cells 
and only about 30%–50% of IgH alleles in DP thymocytes 
junctions. We cannot rule out, therefore, that gene segment demethylation may increase the efficiency of DH
recombination in wild-type pro-B cells. Because Eμ deletion also leads to loss of other accessibility-associated epigenetic marks in the unrearranged locus, it is difficult to deconvolute the contribution of each mark to recombination efficiency.
How might DNA demethylation be targeted to DJH
junctions? We have previously shown that DH
recombination activates cryptic bi-directional promoters associated with most DH
gene segments 
. Transcriptional activity and associated RNA polymerase II recruitment is restricted to the DJH
junctions, and drops off before the unrearranged DH
segments located 5′ of the junction. These changes require Eμ and the simplest interpretation is that recombination places cryptic DH
promoters under the influence of Eμ. Our working model is that DH
-promoter/Eμ interaction brings Eμ-associated demethylating activity to DJH
junctions, thereby resulting in Eμ-directed demethylation. Because Eμ's influence does not extend to the next upstream DH
gene segment, DJH
demethylation is highly localized. In this model the substantial demethylation of DJH
junctions compared to partial demethylation of looping sites may be due to stronger interaction of Eμ with DJH
promoters compared to Eμ interaction with the 3′ RR or 5′ of DFL16.1 sequences.
demethylation serve a function? In this regard it is interesting to note that VH
recombination is significantly reduced in both instances where DJH
junctions remain methylated (Eμ-deleted alleles and in DP thymocytes). Though this is consistent with the view that DJH
demethylation facilitates VH
recombination, we think that the regulation of VH
recombination is more complex. The highly localized Eμ-dependent DJH
demethylation that we describe in this report adds to the emerging evidence that DJH
junctions are distinguished from un-rearranged DH
gene segments by several forms of epigenetic changes 
. These include activation-associated histone modifications, such as H3 acetylation and H3K4me3 and increased sensitivity to DNase I. Like DJH
demethylation, these alterations are also restricted to DJH
junctions and are Eμ dependent. Taken together, our working model is that all these DJH
-restricted epigenetic changes work in concert to promote the timing and precision of VH