Histone Modifications Surrounding DMRs

Because histone modifications provide information about the chromatin accessibility and the gene expression status, the question arises, if there is also a link to active DNA demethylation. By means of reporter constructs, it was demonstrated that histone acetylation and transcription are necessary for active demethylation (D'Alessio et al., 2007; Detich et al., 2003). Promoter regions of active genes are characterized by the acetylation of various histone H3 and H4 residues as well as by histone 3 lysine 4 (H3K4) methylation (Barski et al., 2007; Bernstein et al., 2005; Kim et al., 2005; Pokholok et al., 2005; Schubeler et al., 2004). Here, it is important that methylation of H3K4 occurs in three different forms: mono-, di- and trimethylation (H3K4me1, H3K4me2 or H3K4me3) which are interdependent.

Heintzman et al. as well as Barski et al. identified different chromatin signatures of promoter and enhancer regions, providing new insights into correlations between chromatin modifications and transcriptional regulation (Barski et al., 2007; Heintzman et al., 2007).

Despite of several similarities in their histone modification profiles, Heintzman et al.

distinguished enhancers from promoters by an enrichment of H3K4me1 but missing H3K4me3. Promoter areas, however, were characterized by strong H3K4 trimethylation but marked depletion of H3K4me1. In order to determine the timing of ongoing events and the relevance of activating histone marks during DNA demethylation in dendritic cells, chromatin immunoprecipitations were performed at different differentiation time points. Corresponding to the bisulphite data, seven demethylated sites covering upstream, downstream and promoter regions were selected for real time PCR analysis.

Results Every analysed activating histone modification was detected quite early within gene promoter regions, although acetylation of histone H3 and H3K4me3 seemed to be slightly delayed (Figure 5-21). Trimethylation of histone 3 lysine 4, a modification usually connected to actively transcribed genes, successively increased with culture time corresponding to the CCL13 mRNA data shown in Figure 5-9A.

Figure 5-21 Correlation of histone modifications with CpG demethylation at promoter regions

Activating histone marks were analysed at differentially methylated promoter regions during dendritic cell differentiation using ChIP. Grey lines represent the background control IgG. DNA enrichment of the indicated time points is normalised to 5% input DNA and shown relative to monocyte (0 h) enrichment. Data represent mean values ±SD of at least three independent ChIP experiments.

ChIP primers for the transcriptionally active genes CLEC10A and P2RY6 (see Figure 5-20) cover a region less than 1000 bp downstream their TSSs. Within this area high levels of H3K4me3 were detected whereas H3K4me1 was completely absent (Figure 5-22).

H3K4me2 was detected at the P2RY6 locus but not at CLEC10A. This is in line with recently published genome-wide, high resolution data describing a significant dip in H3K4me3 signals between -200 to +50 bp but strong signal peaks at +50, +210 and +360 bp of active genes (Barski et al., 2007). In that study, two major peaks for each modification were detected: -900 and +1000 for H3K4me1, -500 and +700 for H3K4me2 and -300 as well as +100 for H3K4me3.

Signals of mono- and dimethylation downstream the TSS of highly active genes, that were analysed in the context of the present thesis, decreased, probably due to the high levels of trimethylation (Figure 5-22). The H3K4me2 signal at the CLEC10A locus seems to flare up only at the beginning, suggesting that the mono- and dimethylated states were not captured due to the rapid and complete methylation of lysine 4.

Figure 5-22 Correlation of histone modifications with CpG demethylation at intragenic regions

Activating histone marks were analysed at DMRs downstream TSSs during dendritic cell differentiation using ChIP. Grey lines represent the background control IgG. DNA enrichment of the indicated time points is normalised to 5 % input DNA and shown relative to monocyte (0 h) enrichment. Data represent mean values ±SD of at least three independent ChIP experiments.

As expected, H3K4 trimethylation was only measured at loci near the transcription start sites (TSS) whereas this mark was undetectable in upstream regions (Figure 5-23). The pattern of histone modifications at the intergenic sites reflected some characteristics of enhancers as described by Heintzman et al.. Certainly, to confirm this hypothesis, further experiments like transfection assays have to be done.

Dimethylated H3K4 (H3K4me2) was the most abundant mark compared to the other methylation states because it was detected quite early and at every demethylated site irrespective of the investigated genomic region. Acetylated histone 3 and histone 4 (AcH3 and AcH4) were also found at every demethylated region with AcH3 showing weaker signals except for the downstream regions.

Results

Figure 5-23 Correlation of histone modifications with CpG demethylation at intergenic regions

Activating histone marks were analysed at DMRs upstream or upstream/downstream (regarding to the C9ORF78-Usp20 locus; see also Figure 5-17) from TSSs during dendritic cell differentiation using ChIP. Grey lines represent the background control IgG. Samples were normalised to the 5 % input DNA and evaluated relative to monocytes. Data represent means ±SD obtained from at least three independent donors.

Time course experiments analysing DNA methylation patterns and histone modifications demonstrated that CpG demethylation succeeded or occurred simultaneously with alterations in the histone code. Downstream of the P2RY6 transcription start site, DNA demethylation started after approximately 42 hours in culture. Histone marks were already detected after 6 hours when the locus was still completely methylated. Likewise, histone modifications at the DNASE1L3 promoter or at the intragenic CLEC10A region appeared before first DNA demethylation events were detected. At other loci like the CCL13 promoter or the intragenic C9ORF78 region, it was difficult to determine, whether the appearance of histone marks coincided with DNA demethylation or preceded it, on the basis of the present data.