H3K4 tri-methylation provides an epigenetic signature of active enhancers

Abstract

Combinations of post-translational histone modifications shape the chromatin landscape during cell development in eukaryotes. However, little is known about the modifications exactly delineating functionally engaged regulatory elements. For example, although histone H3 lysine 4 mono-methylation (H3K4me1) indicates the presence of transcriptional gene enhancers, it does not provide clearcut information about their actual position and stage-specific activity. Histone marks were, therefore, studied here at genomic loci differentially expressed in early stages of T-lymphocyte development. The concomitant presence of the three H3K4 methylation states (H3K4me1/2/3) was found to clearly reflect the activity of bona fide T-cell gene enhancers. Globally, gain or loss of H3K4me2/3 at distal genomic regions correlated with, respectively, the induction or the repression of associated genes during T-cell development. In the Tcrb gene enhancer, the H3K4me3-to-H3K4me1 ratio decreases with the enhancer's strength. Lastly, enhancer association of RNA-polymerase II (Pol II) correlated with the presence of H3K4me3 and Pol II accumulation resulted in local increase of H3K4me3. Our results suggest the existence of functional links between Pol II occupancy, H3K4me3 enrichment and enhancer activity.

Figure 1

Comparative epigenetic profiles in ΔRag and ΔRagCD3 thymocytes. (A) Epigenetic profiles at the Cd3 loci. Each track gives the results of one ChIP-Seq experiment on the histone modification specified in the ΔRag or ΔRagCD3 cell type. The genomic location and transcriptional orientation of the corresponding genes is indicated at the bottom of the panel. The positions of enhancer regions are indicated by a black rectangle and the associated epigenetic profiles are highlighted by a dotted rectangle. (B) Genome-wide distribution and stage specificities of enriched regions (peaks) for each epigenetic mark. Bar plots give the relative percentages of promoter-proximal and promoter-distal peaks found either in ΔRag or ΔRagCD3 thymocytes (stage specific; Sp.) or in both cell populations (common peaks; Com.). (C) Epigenetic profiles at three additional lymphoid-specific loci for which enhancer elements are known, including Ikzf1 (top-left panel), Dntt (top-right panel) and Gata3 (bottom panel).

Figure 2

Epigenetic profiles of bona fide distal regulatory elements known to be activated via pre-TCR signalling. (A–C) Epigenetic profiles at the Cd8 (A) and Cd4 (B) loci and around the Tcra-associated enhancer Eα (C). In the later case, only the 3′ end of the Tcra locus is shown. The names of the associated enhancers are indicated. Legends are as in Figure 1. (D) Relative gain of H3K4me1 and H3K4me3 at indicated enhancer regions. The average enrichment was quantified by qPCR for each H3K4 methylation mark in ΔRag and ΔRagCD3 thymocytes (Supplementary Figure S3) and the differences between the two cell populations were plotted as a ratio of the signal obtained in ΔRag thymocytes. The relative changes are indicated at the top of each bar.

Figure 3

Loss of H3K4me3 in the Eβ¹⁶⁹ knock-in mouse model. (A) Schematic diagram of the wild-type Tcrb gene enhancer (Eβ) and associated TF binding sites. The Eβ¹⁶⁹ region is indicated at the bottom. (B) ES cell line and thymocytes from ΔRag and ΔRag mice crossed onto the Eβ^169/169 (ΔRag;Eβ¹⁶⁹) genetic background (Bonnet et al, 2009) were analysed in replicate by ChIP and qPCR in order to determine the level of H3K4me1 and H3K4me3 enrichment occurring within the Eβ sequences. Values were normalized by the enrichment level recorded in the Actb control promoter and expressed as percentages of the total input. The promoter of the Cd3e gene is shown as a positive control. No significant values were obtained with isotypic IgG controls. (C) ChIP samples were hybridized to a custom 15k array covering the Tcrb locus. ChIP-on-chip signals were normalized by the enrichment levels obtained in the Actb promoter. The Eβ-surrounding region is shown. To avoid confusion and prevent any bias due to differences between the wild-type and mutated Eβ sequences, the signals originating from the probes overlapping the core Eβ region (grey rectangles) were removed.

Figure 4

Epigenetic changes in distal regulatory regions are correlated with the patterns of transcriptional regulation. (A) Pie diagram indicating the rates of overlap between the intergenic H3K4me1 peaks observed in both ΔRag and ΔRagCD3 thymocytes and the H3K4me3 peaks found either in both thymocyte populations (stables) or in a single population (dynamics). (B) Percentage of dynamic H3K4me1 domains associated with either repressed (red) or induced (blue) genes, depending on whether they lost or gained H3K4me3 peaks upon pre-TCR signalling. (C, D) Examples of dynamic H3K4me1 domains (highlighted regions) associated with either induced (C) or repressed (D) genes. The legend for epigenetic profiles is as in Figure 1.

Figure 5

Pol II occupancy in distal regulatory regions is correlated with changes in H3K4me3 levels. (A) Pol II profiles directly assessed from ChIP-Seq data using ΔRag and ΔRagCD3 thymocytes (top panel), and relative changes in H3K4me3 and H3K36me3 levels in the Cd8 locus following KM05283 treatment of P5424 cells (middle and bottom panels). H3K4me3 ChIPs from KM05283- and mock-treated P5424 cells were hybridized to the same 15k custom array and the experiment is shown as either bar plots (middle-bottom panel) or a colour coding bar (middle-upper panel; green and red indicates gain and loss of H3K4me3, respectively). (B) Results of RT–qPCR analysis showing the rates of intergenic transcription occurring at the Cd8 locus in ΔRag and ΔRagCD3 thymocytes. Samples treated or not with reverse transcriptase (RT+ and RT−, respectively) were analysed by qPCR to study the intergenic regions (IGR1-3) indicated in (A). Relative transcript levels are expressed as percentages of Hprt expression. (C) Average Pol II association profiles in H3K4me1 domains overlapping Pol II peaks and that lost or acquired H3K4me3 in ΔRagCD3 thymocytes. The distance (in kb) from the middle of the H3K4me3 peak is indicated. (D) Heat maps of Pol II ChIP-Seq signal from ΔRagCD3 thymocytes on the intergenic H3K4me1 domains that overlap (+) or not (−) the H3K4me3 peaks. Data were ordered according to Pol II levels and centred on the middle of the H3K4me1 peak.