Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation

Abstract

V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR) loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.

Keywords: Igk; Tcrb; enhancer-sharing; gene regulation; localized and long-range contacts; nuclear architecture; super-enhancer; transcription factor binding; transcriptional output.

FIGURE 1. Enhancer hubs and their impact on super-enhancer activity

(A) Top: Scheme showing the location of the Igk AgR locus with its respective enhancers: MiEκ, 3’Eκ and Edκ on murine chromosome 6. Bottom: Outline of the different stages of B cell development. Stages under investigation are highlighted in orange (pre-B and immature B). (B) Left: Distribution of H3K27Ac signal across the peaks identified by MACS in pre-B and immature B cells with super-enhancers containing an exceptionally high amount of H3K27Ac. Right: H3K27Ac signal at the 3’ end of Igk in pre-B and immature B cells with the region defined as the super-enhancer highlighted. ATAC-seq profiles of the region in wild-type pre-B cells. (C) Detailed scheme showing the location of MiEκ and 3’Eκ 4C baits. (D) 4C signal normalized by DESeq2 in 5kb windows sliding by 0.5kb for ~50kb region neighboring the MiEκ and 3’Eκ baits in WT versus enhancer-deficient cells. Filled circles highlight significant differences in 4C-seq counts identified by DESeq2 analysis of the plotted region. Transcriptional output within the region is represented below each plot by RNA-seq profiles. (E) Model showing the organization of the individual enhancer elements within the Igk super-enhancer in wild-type versus MiEκ^−/− and 3’Eκ^−/− pre-B cells. See also Figure S1.

FIGURE 2. The MiEκ regulates T cell development and Tcrb rearrangement

(A) Outline of the different stages of T cell development. Stages under investigation are highlighted in blue (DN2/3 and DP). (B) DNA-FISH combined with immunofluorescence to detect γH2AX-containing repair foci at the Igk and Tcrb loci. Bar graphs represents three independent experiments. Error bars reflect SEM. Examples of confocal sections are shown in the panels below. (C) Germline retention assay, performed on genomic DNA, to assess the proportion of Tcrb loci in germline versus rearranged conformation. Scheme showing the primer design. Plots showing the percentage of retention in wild-type and MiEκ^−/− DN2/3 cells normalized by analysis of RAG1 deficient control cells (a combination of two independent repeats of two wild-type versus two MiEκ^−/− samples). (D) Left: FACS analysis of DN cell development in thymi isolated from wild-type and MiEκ deficient mice. Right: Bar plots show quantification of differences in the absolute numbers of wild-type versus MiEκ^−/− DN cells per thymus from a combination of three independent experiments. Cells were gated on Thy1.2⁺TCRb^loCD4⁻CD8⁻ and the DN population defined by expression of CD44 and CD25 as followed: DN1 CD44⁺CD25⁻, DN2 CD44⁺CD25⁺, DN3 CD44⁻CD25⁺ and DN4 CD44⁻CD25⁻. Error bars represent SEM. See also Figure S2.

FIGURE 3. The presence of Jκ-Cκ transcripts in Igk is linked to a long-range contact with Tcrb

(A) RNA-seq, ATAC-seq and iChIP for H3K27Ac reveal the activity of the 3’ Igk interacting domain. Nc-RNA track displays transcripts identified by transcriptome assembly using cufflinks V2.2.1 (see methods for more details). The bottom track shows the 3’ Igk interacting domain called by 4C-ker from the Eβ viewpoint. (B) Detailed scheme showing the location of the Eβ 4C bait relative to the enhancer. (C) Interaction profile of Eβ for the 6Mb region encompassing Igk in wild-type DN, DP, pre-B and immature B cells using 4C counts in 200kb windows sliding by 20kb. (D) Interaction profile of Eβ with the 3’ Igk interacting domain in WT versus Eβ and MiEκ deficient DN cells using 50kb windows sliding by 5kb. A purple bar delineates a 200kb window, which chromosome-wide DESeq2 analysis identified as having a significantly different 4C signal in Eβ^−/− versus wild-type DN cells. A t-test was performed on 4C counts to assess the same 200kb window for differences between wild-type versus MiEκ^−/− (gold bar). See also Figures S3 and S4.

FIGURE 4. The MiEκ regulates CBFβ binding at Eβ in DN cells

(A) CBFβ ChIP-qPCR for REIR plotted as fold enrichment over negative control. (B) CBFβ ChIP-qPCR for Eβ plotted as fold enrichment over negative control. T-test to determine significance of fold enrichment change at Eβ in Eβ^RUNX (RUNX binding sites at Eβ mutated, p-value = 0.004398) and in MiEκ^−/− (p-value= 0.005621) DN cells. Error bars represent SEM of three experiments. (C) Model implicating the CBFβ-bound REIR and MiEκ in co-operatively regulating transcription factor binding at Eβ. In wild-type T and B cells Eβ and MiEκ respectively regulate rearrangement of Tcrb and Igk. Three enhancers of Igk (MiEκ, 3’Eκ and Edκ) comprise the super-enhancer in developing B cells with individual enhancers interacting and co-operatively regulating transcriptional output. In wild-type DN cells Jκ-Cκ transcripts are expressed at the 3’ end of Igk. The contact between Eβ and the 3’ Igk interacting domain encompassing CBFβ-bound REIR promotes Tcrb recombination in an Eβ and MiEκ dependent manner. An absence of MiEκ leads to a reduction in the contact and transcription at the 3’ end of Igk, which in turn leads to a reduction in the localized concentration of CBFβ and RUNX1 in the proximity of Eβ, thereby impairing its function.