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Stalled Hox promoters as chromosomal boundaries - PubMed

  • ️Thu Jan 01 2009

Stalled Hox promoters as chromosomal boundaries

Vivek S Chopra et al. Genes Dev. 2009.

Abstract

Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Here, we present evidence that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. We propose that stalled promoters help organize gene complexes within chromosomal loop domains.

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Figures

Figure 1.
Figure 1.

Paused/stalled Hox gene promoters display insulator activity. The Hox gene promoters were tested for their ability to block enhancer promoter interactions in transgenic assays. The Hox/lacZ fusions were cloned between IAB5 enhancer and white reporter gene. The IAB5-lacZ interaction was seen for all of the transgenic lines for Abd-B/lacZ (A), Ubx/lacZ (C), abd-A/lacZ (E), Antp/lacZ (G), lab/lacZ (I), and Scr/lacZ (K) as the classical PS10-14 staining was observed during lacZ probe in situ. The IAB5–white interaction was blocked in all stalled promoter-lacZ fusions like Abd-B/lacZ (B), Ubx/lacZ (D), Antp/lacZ (H), and lab/lacZ (J) lines as no white in situ signal was observed in the IAB5 pattern. The nonstalled promoter-lacZ fusions like abd-A/lacZ (F) and Scr/lacZ (L), however, did show the IAB5 pattern when stained for white probe, as they did not show any insulator activity. The weak staining in the head regions is due to the P-transformation vector used in these experiments (Small et al. 1992). The embryos are aligned anterior to the left and posterior to the right.

Figure 2.
Figure 2.

Abd-B and Ubx promoter insulator activity does not involve tethering. The Abd-B and Ubx stalled promoters were tested for their ability to display promoter competition in a transgenic assay in which the IAB5 enhancer was cloned in between the white reporter and prom/lacZ fusion. As a control, the white/lacZ fusion (A) and white (B) were driven equally well by IAB5 enhancer in a white/lacZ (A,B) transgene. The IAB5-lacZ interaction (C,E) as well as the IAB5–white (D,F) interaction were facilitated in Abd-B/lacZ (C,D) and Ubx/lacZ (E,F) lines, respectively.

Figure 3.
Figure 3.

Negative elongation factors are required for Hox gene promoter insulator activity. The stalled promoter insulator lines were tested in elongation factor mutant backgrounds. (A–H) The Abd-B/lacZ line was tested in Nelf-E/+ (A,B), Nelf-A/+ (C,D), Spt4/+ (E,F), and Spt5/+ (G,H) backgrounds, and there was loss of insulator activity of the Abd-B promoter in these mutant backgrounds as seen by the appearance of the IAB5 pattern in situ signal when white probe was used (B,D,F,H). (I–P) Similar disruption of the insulator activity was observed for the Ubx/lacZ transgene in different mutant backgrounds of Nelf-E/+ (I,J), Nelf-A/+ (K,L), Spt4/+ (M,N), and Spt5/+ (O,P). (J,L,N,P) The white probe displayed the IAB5 pattern staining in these elongation mutant backgrounds.

Figure 4.
Figure 4.

Negative elongation factors are required for enhancer-blocking activity of putative insulators like Fab7 and Fab8. Enhancer-blocking lines containing Fab7 (A–J) and Fab8 (K–T) insulators, cloned between divergently placed IAB5/lacZ and 2XPE/white reporters, were tested in elongation factor mutant backgrounds. The Fab7 (A,B) and Fab8 (K,L) line shows lacZ staining in IAB5 pattern (A,K) and white in twist (2XPE) pattern (arrow) (B,L), respectively. In Nelf-E/+ (C,M), Nelf-A/+ (E,O), Spt4/+ (G,Q), and Spt5/+ (I,S) backgrounds the lacZ in situ now displays both the IAB5 as well as the 2XPE patterns for both Fab7 and Fab8 lines, respectively. A similar dual IAB5 and 2XPE pattern was observed for the white in situ in Nelf-E/+ (D,N), Nelf-A/+ (F,P), Spt4/+ (H,R), and Spt5/+ (J,T) backgrounds for Fab7 and Fab8 lines, respectively. The embryos are aligned anterior to the left, posterior to the right, dorsal up, and ventral down.

Figure 5.
Figure 5.

Stalled Hox promoters may help promote higher-order chromatin organization within the Hox loci. Our results suggest that the stalled promoters contain intrinsic insulator activity that requires NELF (N) and DSIF (D) proteins, and this may help define higher-order loops within gene complexes such as the Hox complex. The stalled Pol II (P) along with the NELF and DSIF complex may interact with putative insulator (In) sequences, as seen for the Abd-B promoter (stalled) and the Fab7 (insulator). Our experiments also suggest that that putative insulator sequences also require NELF and DSIF proteins, and this could be due to sharing of these proteins via the formation of higher-order loops. Such loop domains may help in proper regulation of genes and prevent any aberrant activation from neighboring enhancers (En) (shown by dashed arrow), thus favoring proper gene regulations at the higher-order level.

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