SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

Abstract

The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh—Shh-brain-enhancer-6 (SBE6)—that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.

PARP mediated chromatin unfolding is coupled to long-range enhancer activation

“…We have shown previously that SBE6 is involved in the proper induction of Shh expression during NPC differentiation, and that there is a prominent gain of H3K4me1 and H3K27ac at this regulatory element during differentiation to Sox1+ve NPCs (Benabdallah et al, 2016). FISH showed that the increased Shh-SBE6 inter-probe distances are abolished in NPCs derived from SBE6 -/-ESCs ( Figure 2A).…”

“…This suggests that a large-scale chromatin unfolding occurs upon Shh neural induction and that this is limited to the region 300kb 5' of the Shh TSS. It is noteworthy that, within this 300kb region, the most prominent peaks of H3K3me1 and H3K27ac gained during this NPC differentiation programme occur at SBE6, and that SBE6 is required for full induction of Shh during this differentiation programme (Benabdallah et al, 2016). The increased separation between Shh and SBE6/SBE4 upon Shh induction does not seem compatible with the formation of a chromatin loop between Shh and these two neural enhancers.…”

“…To analyse the spatial relationship of Shh with its known brain enhancers in the nucleus we differentiated 46c ESCs (Ying et al, 2003) into NPCs. Efficient differentiation was monitored through Sox1-GFP fluorescence (Benabdallah et al, 2016).…”

“…We performed 3D FISH on 46c ESCs that do not express Shh, and on NPCs obtained after seven days of differentiation, when Shh is expressed (Benabdallah et al, 2016) and we imaged the slides by 3D-Structured Illumination Microscopy (3D-SIM). Surprisingly, upon Shh activation there was a significant increase in inter-probe distances between Shh and both the SBE6 and SBE4, located 100 and 350 kb 5' of Shh respectively ( Figures 1B and 1C).…”

“…Shh expression is important for several aspects of brain development. Shh-Brain-Enhancers-5 (SBE5), SBE2/3, SBE4 and SBE6 have been demonstrated to drive expression in the midbrain and anterior domains of the developing brain and are located 780, 450, 350 and 100 kb upstream of the Shh TSS, respectively (Jeong, 2006;Benabdallah et al, 2016;Yao et al, 2016). As distal enhancers, the SBE elements might be expected to loop into spatial proximity of Shh during its activation in neural progenitors cells (NPCs).…”