Chromatin insulator and the promoter targeting sequence modulate the timing of long-range enhancer-promoter interactions in the Drosophila embryo

Abstract

The homeotic genes are essential to the patterning of the anterior-posterior axis along the developing Drosophila embryo. The expression timing and levels of these genes are crucial for the correct specification of segmental identity. The Abdominal-B (Abd-B) gene is first detected in the most posterior abdominal segments at high levels and gradually appears in progressively anterior abdominal segments in lower amounts. Regulatory mutations affecting this expression pattern produce homeotic transformations in the abdomen. The promoter targeting sequences (PTS) from Abd-B locus overcome the enhancer blocking effect of insulators and facilitate long-range enhancer-promoter interactions in transgenic flies (1, 2). In this study, we found that transgene activation by the IAB5 enhancer can be delayed by inserting a 9.5 kb 3' Abd-B regulatory region containing the Frontabdominal-8 (Fab-8) insulator and the PTS element. We found that the delay is caused by the PTS and an insulator, and it is not specific to the enhancer or the promoter tested. Based on these findings, we hypothesize that the delay of remote enhancers is responsible for the Abd-B expression pattern, which is at least in part due to the regulatory activities of the PTS elements and chromatin boundaries.

Figure 1. Expression pattern of IAB5, IAB7 and IAB8 enhancers when they activate transgenic promoters

A. Activation of lacZ by the 1.0 kb IAB5 in a broad stripe encompasses PS10, 12 and 14 when it is inserted at the 5’ of Tp promoter (1). Blastoderm embryos are shown. B. A 1.1 kb IAB7 enhancer activates the lacZ promoter in two stripes (PS12, 14) when inserted at the BamHI site of C4PLZ vector. C. The 1.6 kb IAB8 activates lacZ in a single band in the most posterior region of the embryo. D. LacZ activation by endogenous iab-7 domain enhancers in stage 5 P-element enhancer trap line fs05369. This P-element is inserted 4 kb away from the IAB7 enhancer.

Figure 2. Abd-B expression during embryogenesis

A–C. IAB5, 7 and 8 enhancer activities in stage 10. These embryos carry the same transgenes as in Fig 1AA–1C. DA–F. Abd-B expression in stage 5, 10 and 11. It first appeared in PS13 and occupies PS13 and 14 by stage 9. In stage 10 Abd-B becomes detectable in PS10–12 with sequential delay from PS12 to PS10. D. Stage 5 embryos, showing the initial expression of Abd-B in PS13. E. In stage 9, Abd-B expression is seen in PS13 and PS14. F. Abd-B is expressed in PS10–14 in stage 10. A comparison of Abd-B expression in these stages with lacZ activation by IAB enhancers indicates delayed of Abd-B activation in PS10–12.

Figure 3. The tmr region from Abd-B delays the IAB5 enhancer activity

eve-GFP expression in stage 5, 6 and 8 embryos. The transgene contains a 9.5 kb tmr sequence from the 3’ of Abd-B, which includes a proximal IAB8 enhancer, the Fab-8 insulator, the PTS and the distal IAB7 enhancer. IAB5 is approximately 14kb away from eve GFP, where as IAB8 and IAB7 are 5 and 11 kb away, respectively. A. Stage 5 embryos showing IAB8 expression (posterior band) and trace amount of IAB5 activity (arrow). B. A gastrulating embryo showing elevated IAB5 activity (arrow), which is still weaker than that of IAB8 (posterior solid band). C. Stage 8 embryos showing stronger IAB5 activity. Compared to that of IAB8 (PS13), IAB5 activity is at least as strong, if not stronger (see PS10).

Figure 4. Promoter targeting of IAB8 by the PTS delays IAB8 lacZ interaction

All embryos are RNA in situ hybridization showing transcription activation of the lacZ transgene. A–D. lacZ activation by PTS-targeted, lacZ 3’ end located IAB8 and lacZ 5’ located NEE. IAB8 activity starts weak but grows stronger in during stage 5, while NEE activity remain constant. E–H. lacZ activation by a 1.6 kb 3’ located IAB8. No obvious difference in IAB8 activity could be seen. A and E. early stage 5 (2hr 30 min). B and F. at mid stage 5 (2hr 45min). C and G. Late stage 5 (3hr), when embryonic cells are elongating. D and H. later stage 5 (3hr 15 min) when embryos are about to gastrulate.

Fig. 5. Promoter targeting delays the heterologous NEE enhancer

Embryos from three time points, early stage 5, 2hr 30 min (A, D, G), mid stage 5, 2hr 45min (B, E, H), and late stage 5, 3hr 15 min (C, F, I) were photographed. A–C. Embryos carrying control transgene HZλN showing similar staining pattern in all three time points. D–F. When the spacer was replaced by suHw insulator (HSN) the distal NEE enhancer is selectively blocked. G–I. When the 625 bp PTS was also added, NEE overcomes the enhancer blocking activity of suHw and becomes targeted to the lacZ promoter. However, compared to A–C, NEE is barely detectable in early stage 5 (G), visible but weak in mid to late stage 4 (H), and become very strong at late stage 5 (I), suggesting a delay of NEE-lacZ interaction by about 30 minutes.

Fig. 6. Insulator is essential for enhancer delay by PTS

Similar to Fig. 3, embryos from three time points were photographed for each of the three constructs. For A, D and G early stage 5 embryos were shown; For B, E and H mid to later stage 5 were shown; For C, F and I, late stage 5 and early stage 6 (gastrulation) were shown. All embryos shown were hybridized using the anti-sense probe to the w gene. A–C. NEE activity comes on in early stage 5 and remains robust through late stage 5. At the end of stage 5, it is slightly reduced. D–F. When PTS alone is inserted between the 3’ of lacZ and NEE, no promoter targeting is observed, and NEE activates w similar to control transgenes in A–C. G–I. Two copies of 1.6 kb tmr sequence containing Fab-8 and PTS (1) were inserted to flank the lacZ gene. Due to promoter targeting by PTS, NEE is able to activate w. However, the onset of w activation by NEE is delayed. NEE-w interaction could not be detected in early stage 5. In mid stage 5, it becomes detectable. Peak interaction is reached in gastrulating embryos.

Figure 7. Enhancer delay model for the Abd-B locus

We propose that the boundary elements such as Fab-7 and Fab-8 act as timing devices to slow down the activation of Abd-B by downstream enhancers. A. The 3’ regulatory region of Abd-B contains four regulatory domains, iab-5 iab-6, iab-7 and iab-8. Enhancers from the first three domains must overcome the enhancer blocking activity of insulators, possibly with help from the PTS elements. Since each of the boundaries may delay bypassing enhancers, more downstream enhancers will be delayed further because they have to overcome more insulators. B. The differential delay of enhancers could generate a gradient with step-wise increment of Abd-B from PS10 to PS13. For example, a delayed IAB7 would result in less accumulation of Abd-B transcript in PS12 when compared to that in PS13 where IAB8 activates Abd-B without delay. C. Enhancer delay model could explain the gain of function phenotypes boundary elements deletions (Fab mutations). As a prediction of the enhancer delay model, the deletion of Fab-7 removes a delay step, as a result both IAB6 and IAB7 are equally timed, thus a similar levels of Abd-B in PS11 and PS12. In fact, Fab-7 mutations result in transformation of PS11 into a copy of PS12 (roughly A6 to A7), and similar level and timing of Abd-B expression both segments (48).