"Multiple Promoter Targeting Sequences exist in Abdominal-B to regulate long-range gene activation".

Qi Chen, Lan Lin, Sheryl Smith, Qing Lin and Jumin Zhou ^@

Gene Expression and Regulation Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA

^@ Corresponding author. Fax: +1 215 898 0663.  E-mail: zhouj@wistar.upenn.edu

In complex genomes, insulators set up chromatin domain boundaries and protect promoters from inappropriate activation by enhancers from neighboring genes. The Drosophila Abdominal-B locus uses insulator elements to organize its large regulatory region into several body segment-specific chromatin domains. This organization leads to a problem in enhancer–promoter communication, that is, how do distal enhancers activate the Abd-B promoter when there are several insulators in between? This issue is partially resolved by the Promoter Targeting Sequence, which can overcome the enhancer blocking effect of an insulator. In this study, we describe a new Promoter Targeting Sequence, PTS-6, from the Abd-B 3' regulatory region. PTS-6, comprised of approximately 200 bp, was found to bypass both homologous Abdominal-B insulators, such as Fab-7 and Fab-8, and a heterologous insulator, suHw. Most importantly, it also overcomes a combination of two insulators such as Fab-7/Fab-8. Thus, PTS-6 could, in principle, target remote enhancers that are separated from the Abd-B promoter by multiple insulators. In addition, PTS-6 selectively targets the distal enhancer to only one transgenic promoter, and it strongly facilitates Abd-B enhancers. These results suggest that promoter targeting is necessary for long-range enhancer–promoter communication in Abd-B, and PTS elements could be a common occurrence in large, complex genetic loci.

Abbreviations: PTS, Promoter Targeting Sequence; Abd-B, abdominal-B; Fab-7, Frontabdominal-7; iab, infraabdominal

In higher eukaryotes, developmentally regulated genes often contain a large number of transcriptional enhancers that are located many kilobases away from the promoter. The Drosophila Bithorax gene complex (BX-C), which controls the body plan along the anterior–posterior axis (in the posterior of the embryo), is comprised of more than 300 kb of DNA. Yet, it encodes only three homeotic genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) (Lewis, 1978, Martin et al., 1995, McGinnis and Krumlauf, 1992 and Morata et al., 1986). Each of these genes contains a large complex regulatory region that is organized into body Parasegment (PS)-specific domains. Abd-B contains five such domains, infraabdominal-5 (iab-5), iab-6, iab-7, iab-8, and iab-9, which function in PS10, 11, 12, 13, and 14, or roughly the 5th through the 9th abdominal segments (Boulet et al., 1991, Celniker et al., 1989, Duncan, 1987, Karch et al., 1985, Morata et al., 1986 and Sanchez-Herrero et al., 1985). Each of these iab domains is believed to contain enough cis-regulatory information to control Abd-B in a specific abdominal segment. Several studies have led to the identification of three early embryonic enhancers, IAB5, IAB7, and IAB8, from these regions (Barges et al., 2000, Busturia and Bienz, 1993 and Zhou et al., 1999). The Abd-B most 3' IAB5 enhancer is positioned at equal distances, more than 50 kb away, from both abd-A and Abd-B promoters, yet, it normally activates the latter (Martin et al., 1995). Thus, a central question regarding complex genetic loci, such as the BX-C, is how an enhancer element consistently finds the right promoter.

Specialized DNA elements, other than enhancers and promoters, have been found in the BX-C to either regulate the activity of tissue-specific enhancers or to modulate long-range enhancer–promoter communications. For example, Polycomb and/or Trithorax Response Elements (PRE/TREs) (Barges et al., 2000, Busturia and Bienz, 1993, Busturia et al., 1997, Chan et al., 1994, Hagstrom et al., 1997, Muller et al., 1999 and Zhou et al., 1999) have been found to recruit protein complexes containing products from the Polycomb and Trithorax group genes to repress or activate the chromatin and, therefore, maintain the activities of early embryonic enhancers. As a result, these enhancers are either “on” or “off” in specific cells during late embryogenesis and throughout adulthood (Paro et al., 1998, Pirrotta, 1998 and Pirrotta et al., 2003).

Chromatin boundary elements, such as Miscadastral Pigmentation (MCP) (Karch et al., 1994), Frontabdominal-7 (Fab-7) (Hagstrom et al., 1996, Karch et al., 1994, Mihaly et al., 1997 and Zhou et al., 1996), or Fab-8 (Barges et al., 2000 and Zhou et al., 1999), are also found in the Abd-B locus (see Fig. 1). A boundary element, also known as an insulator, usually has two activities; first, it provides barrier function to prevent the spreading of silencing activities such as the formation of heterochromatin. This activity is responsible for protecting transgenes from position effect variegation (PEV) due to their insertion near heterochromatin. Insulators also exhibit enhancer blocking activity by preventing transcription activation when inserted between an enhancer and a promoter. Insulators have been discovered in species from yeast to humans; notable examples include the human Igf2/H19 imprinting control region (Hark et al., 2000 and Kanduri et al., 2000), the chicken b-globin HS4 element (Bell et al., 1999 and Chung et al., 1993), the Drosophila suppressor of hairy wing (suHw) from the gypsy insulator (Dorsett, 1993 and Geyer and Corces, 1992), and the scs/scs' elements from the Drosophilahsp70 locus (Gaszner et al., 1999, Kellum and Schedl, 1992, Udvardy et al., 1985 and Zhao et al., 1995).

Fig. 1. A summary of cis-interactions in the Abd-B locus.

Fig. 1. A summary of cis-interactions in the Abd-B locus.

(A) The Drosophila Abd-B locus consists of four 3' abdominal parasegment (PS)-specific regulatory domains, termed infraabdominal-5 (iab-5), iab-6, iab-7 and iab-8, which regulate Abd-B function corresponding to ps10, ps11, ps12, and ps13, or roughly abdominal segment A5, A6, A7, and A8, respectively. These domains are separated by boundary elements such as Frontabdominal-7 (Fab-7) and Fab-8. In transgenic flies, the Fab-7 and Fab-8 elements function similarly to insulator elements and block enhancer–promoter interactions. However, in the endogenous locus, they do not interfere with enhancers such as IAB5 and IAB7, possibly due to the presence of the PTS elements. The PTS possesses an anti-insulator activity and may target these enhancers to the Abd-B promoter. In doing so, it converts the Fab elements into local chromatin boundary elements.

(B) Diagram showing mutant allele Fab-7^R73 removing approximately 800 bp DNA from the PTS region and a 3.7 kb DNA from the Fab-7 region. This mutant exhibits loss of Abd-B function in the A5, A6, and A7 segments when hemizygous for this mutation (Zhou and Levine, 1999).

(C) A female sterile P-element insertion fs(05369) into the PTS region. This mutation exhibits mild Abd-B loss of function in the 7th abdominal segment (Zhou and Levine, 1999).

It has been proposed that the Fab-7 and Fab-8 elements function as chromatin domain boundaries in the Abd-B locus to restrict chromatin regulatory events, such as the function of PREs/TREs so that each iab domain is functionally “isolated” from its neighbors (Mihaly et al., 1998 and Vazquez et al., 1993). However, both of the Fab elements also block enhancer–promoter interactions when tested in transgenic flies (Barges et al., 2000, Hagstrom et al., 1996, Zhou et al., 1996 and Zhou et al., 1999). This activity creates a problem for enhancers located within iab-5, iab-6, and iab-7 elements because these enhancers must communicate with the Abd-B promoter at the appropriate time and in the appropriate segments during development. The iab elements must overcome the enhancer blocking activity of the Fab-7, Fab-8, and other potential insulators in order to activate Abd-B. Thus, an additional mechanism(s) is necessary to mediate long-range enhancer–promoter interactions over intervening insulator elements in Abd-B.

The recently identified Promoter Targeting Sequence (PTS) may provide insight into such a mechanism (Zhou and Levine, 1999). The PTS has an anti-insulator activity: it allows an enhancer to activate its promoter despite an intervening insulator. The PTS also facilitates long-distance enhancer–promoter interactions and selectively activates a single promoter when two are included in the same transgene (Lin et al., 2003 and Lin et al., 2004). These studies support the model that the PTS may mediate long-distance gene activation in Abd-B by overcoming intervening insulators such as Fab-7 and Fab-8, facilitating more 3' enhancers, and specifically activating the Abd-B promoter. Consequently, the PTS converts the Fab elements into local chromatin boundary elements to restrict the active or repressed chromatin within each regulatory domain (Fig. 1).

Mutations in the PTS region lead to loss of function of Abd-B, supporting the role of PTS in facilitating enhancer–promoter interactions (Zhou and Levine, 1999). A P-element insertion mutation in the PTS region leads to a moderate loss of Abd-B function in the 7th abdominal segment (Fig. 1). However, when a mutation of PTS is combined with a 3.7 kb deletion in the Fab-7 boundary region (Fig. 1), a much stronger loss of function is observed: abdominal segments from the 5th through the 7th are transformed into copies of the 4th, suggesting that PTS may be functionally redundant with additional PTS elements removed from the Fab-7 region (Zhou and Levine, 1999). To test this possibility, we analyzed the DNA near the Fab-7 boundary for DNA sequences with promoter targeting function. In this paper, we report the identification of a new PTS element, PTS-6, located just next to the Fab-7 insulator. This element permits an enhancer to selectively activate a transgenic promoter, bypassing the intervening Fab-7 and other insulators. In addition, it can overcome a combination of two insulators such as Fab-7 plus Fab-8. We found that both PTS elements could overcome multiple insulators and function from a number of positions relative to the enhancer and the insulator. These results strongly support the promoter targeting model of long-distance transcription activation in Abd-B and further suggest that multiple PTS elements may work synergistically to regulate enhancer–promoter interactions in Abd-B.

Methods:

Plasmid constructions

To generate the P-transgenes shown in Fig. 2, we inserted either 1.6 kb of l DNA (HZlN), a 0.8 kb Fab-7 insulator (HZFN) (Hagstrom et al., 1996, Mihaly et al., 1997 and Zhou et al., 1996), or a 3.7 kb Fab-7 region (Karch et al., 1994) (W170) into the BglII site downstream of the Transposase (Tp)-lacZ gene of the HZGN vector (Lin et al., 2003). For the P-transgenes in Fig. 4, different BamHI–BglII truncated fragments (position shown in Fig. 4) from the 3.7 kb Fab-7 boundary (for W263, the DNA fragment was flanked by FRT sites to form a BglII insert) were inserted into the BglII site downstream of the Transponsase (Tp)-lacZ gene and SuHw insulator of #125 construct, respectively. A 1.6 kb PstI IAB-8 enhancer was inserted into the PstI site of C4PLZ vector to generate the W76 construct. Thereafter, a 0.7 kb BamHI–BglII Fab-8 and a 0.8 kb BamHI–BglII Fab-7, either with or without the 200 bp BamHI–BglI PTS-6, were sequentially inserted into the BamHI site of a modified pBluescript that contains an additional NotI site converted from the KpnI site. A NotI fragment including Fab-8 plus Fab-7, respectively, with or without PTS-6, was inserted into the NotI site between Tp-lacZ gene and IAB-8 enhancer of W76 vector to generate W267 and W270. A 1.6 kb BamHI ME fragment (including Fab-8 and PTS-7) was inserted into the BglII site of #125 vector in different orientations to generate W114 and W115.

Fig. 2. An anti-insulator activity exists within the 3.7 kb Fab-7 region.

Fig. 2. An anti-insulator activity exists within the 3.7 kb Fab-7 region.

Transgenic embryos carrying different transgenes were hybridized with Digoxigenin-labeled antisense RNA to either the white or the lacZ gene and were stained with alkaline phosphatase conjugated anti-Digoxigenin antibody (Roche) followed by reactions in NBT/BCIP solution (Tautz and Pfeifle, 1989). Processed embryos were mounted on glass slides.

(A) Staining for w expression. Control constructs with a spacer inserted between lacZ and the 3' located NEE. The HI enhancer activates transcription in the anterior region of the embryo, while NEE activates w in the lateral region (arrows).

(B) Staining for lacZ expression. Same construct as in panel (A).

(C) w expression is activated by HI, but not, or minimally, by NEE when the 0.8 kb Fab-7 is inserted at the 3' position between lacZ and NEE.

(D) Similar to w, lacZ is activated by HI but not NEE. Staining represents most embryos, which show no NEE activity.

(E) In the line shown, w is activated by HI only.

(F) Instead of blocking NEE, the 3.7 kb DNA from Fab-7 boundary region (include Fab-7) actually facilitates the NEE–lacZ interaction. Compare with panel (B).

P-element transformation and in situ hybridization

P-element transformation vectors containing lacZ and white reporter genes were introduced into the Drosophila germline by injecting yw⁶⁷ embryos as described previously (Rubin and Spradling, 1982). Between 15 and 35 independent transformant insertions were obtained for each of the recombinant P-elements shown. In situ hybridization was performed essentially as described in previous reports (Tautz and Pfeifle, 1989 and Zhou and Levine, 1999).

Fly strains and crosses

Transgenic flies expressing the Flip recombinase were kindly provided by Gary Struhl and Steve Small (Wu et al., 1998). To recombine different FRT-flanked DNA elements away from the transgene, females carrying the transgene were mated with males that express the Flp recombinase under the control of a sperm-specific tubulin promoter (Wu et al., 1998). In F₁ males, the recombinase binds the FRT sites and deletes the intervening DNA. These male flies were collected and mated to yw virgin females to establish stocks that were subsequently analyzed by RNA in situ hybridization.

Results:

Previous studies have demonstrated that the PTS overcomes the enhancer blocking activity of an insulator and selectively targets and facilitates a distal enhancer to one of two transgenic promoters (Lin et al., 2003 and Zhou and Levine, 1999). In addition, the promoter targeting function is strain-specific: when a collection of individual strains is examined, the enhancer only activates the proximal promoter in a portion of the strains (Type I strains). In other strains, this enhancer only activates the distal promoter (Type II strains). In the remaining strains, the enhancer is blocked by the insulator, and neither promoter is activated (Type III strains) (Lin et al., 2004).

Identification of a new promoter targeting activity from the 3' Abd-B

Domain boundary regions of the Abd-B locus appear to have multiple cis-regulatory elements with similar organizations. For example, both Fab-7 and Fab-8 are comprised of an insulator located 3' of a PRE element (Mihaly et al., 1998). It is possible that other cis elements, such as the PTS, are also similarly arranged. Because the original PTS is located just 3' to the Fab-8 insulator, the best chance of locating a new PTS element will be the region 3' to the Fab-7 insulator. For this reason, we tested genomic DNA near Fab-7 for potential anti-insulator activity. To detect this activity, we analyzed a 3.7 kb HindIII fragment encompassing the Fab-7 region, in a transgenic P-element, shown in Fig. 2. This region contains the 0.8 kb Fab-7 element and a nearby 5' PRE (Hagstrom et al., 1996, Hagstrom et al., 1997, Mihaly et al., 1997 and Zhou et al., 1996). We reasoned that, if the 3.7 kb DNA contains a PTS, it should overcome Fab-7 and target a distal rhomboid neuroectoderm enhancer (NEE) (Ip et al., 1992) to one of the 5' promoters.

As shown in Figs. 2A and B, when placed at 1.6 kb away from the 3' end of lacZ, the NEE enhancer directs transcriptional activation of both w and lacZ genes, producing ventral lateral stripes along the anterior–posterior axis of the embryos. When the 0.8 kb Fab-7 was inserted between the 3' end of lacZ and the more distal enhancer, NEE activity is either severely attenuated or totally abolished (Figs. 2C, D). This result is consistent with our earlier observation (Zhou et al., 1996). However, when the 3.7 kb DNA from the Fab-7 genomic region was inserted in this location, the NEE enhancer is not always blocked in a number of transgenic lines examined. Instead, NEE selectively activates either the lacZ promoter (Figs. 2E, F) or the w promoter (not shown) in a strain-specific manner. A total of 19 transgenic strains were analyzed, and four exhibited selective lacZ activation, while three showed w-specific activation by NEE. The remaining lines showed no activation of either w or lacZ by NEE. The 5' hairy stripe one enhancer (HI) (Riddihough and Ish-Horowicz, 1991) is not affected by Fab-7 or the PTS in most transgenic strains and is used as an internal control for enhancer strength. The pattern of promoter activation by NEE among different strains is similar to Type I, Type II, and Type III strains obtained when the PTS and Fab-8 were included in a similarly constructed transgene. Thus, this result suggests that the 3.7 kb DNA may contain a new PTS element.

To definitively test the 3.7 kb region for promoter targeting activity, we tested this region against a heterologous insulator, suHw, in the #125 P-transformation vector described earlier (see Fig. 3) (Lin et al., 2003). This vector contains the 1.6 kb IAB8 enhancer located 3' of the lacZ gene and a 360 bp suHw insulator (Cai and Levine, 1995) inserted between the lacZ and IAB8. The IAB8 enhancer alone at the 3' of lacZ activates both w and lacZ producing moderate, but clearly detectable, transgene expression (Figs. 3A, B). When w and lacZ expression from embryos carrying W263FLP (same as #125) was analyzed, no detectable IAB8 activity could be seen (Figs. 3C, D). We then deleted the 0.8 kb Fab-7 insulator from the 3.7 kb DNA and inserted the rest between the suHw insulator and IAB8. As shown in Figs. 3E through H, this 3.7DFab-7 is able to target the IAB8 enhancer to the lacZ promoter, overcoming the intervening suHw insulator. A total of 13 transgenic lines were analyzed, two of which targeted lacZ (type I strains, Figs. 3E, F), one targeted w (type II strains, Figs. 3G, H), while the remaining nine showed no promoter targeting: neither of the promoters is activated (data not shown, type III). To prove that the IAB8 enhancer activity is due to promoter targeting rather than position effects associated with differential insertion sites, the two Type II transgenic strains were analyzed by FLP-induced recombination, which removes the intervening 3.7DFab-7 DNA flanked by the direct repeat of FRT sites (black arrows). Deleting the 3.7DFab-7 DNA from these transgenic strains leads to the total loss of IAB8 activity (Figs. 3C, D), suggesting that a PTS element exists within the 2.9 kb 3.7DFab-7.

Fig. 3. Characterization of the promoter targeting activity from the 3.7 kb Fab-7 region.

Fig. 3. Characterization of the promoter targeting activity from the 3.7 kb Fab-7 region.

(A, B) The IAB8 enhancer located 3' of lacZ in embryos carrying the control transgene W76 activates both w and lacZ in low but detectable levels.

(C, D) When the suHw insulator is present, IAB8 is blocked, and no transcription can be detected for either w or lacZ. This strain (W263FLP) was obtained from W263 (see below) after the 3.7kDFab-7 is recombined away from the transgene.

(E, F) When both suHw and 3.7kDFab-7 are present, IAB8 activates robust lacZ expression but no w activation.

(G, H) In a different strain, carrying W263, IAB8 selectively activates w instead of lacZ, leading strong staining in the posterior region of the embryo.

To map the minimal PTS, the 3.7 kb DNA was cut into three overlapping pieces, F3.7a, F3.7b, and F3.7c (Fig. 4A), and tested in transgenic vector #125. The result was summarized in Table 1. Approximately one third of the transgenic strains from F3.7a and a quarter from F3.7b exhibit promoter targeting. None of the transgenic line carrying F3.7c showed any strong (typical of promoter targeting) activation of either w or lacZ (Table 1), suggesting that the promoter targeting activity resides within F3.7a and F3.7b. Since there is only a 400 bp overlap between these two fragments, we tested the 200 bp DNA from the 3' end of F3.7a and found that it is sufficient to mediate anti-insulator and promoter targeting activity (Table 1). This new 200 bp PTS is located just 230 bp 3' of the Fab-7 insulator, a similar position where the original PTS is located relative to Fab-8. To distinguish between the two PTS elements, we refer the newly discovered PTS as PTS-6 to reflect the fact that it is located in the iab-6 domain. Similarly, we name the previously identified PTS as PTS-7.

Fig. 4. Mapping the PTS-6 element from the 3.7kb Fab-7 region.

Fig. 4. Mapping the PTS-6 element from the 3.7kb Fab-7 region.

Diagram showing the different regions tested from the 3.7 kb Fab-7 region. Top diagram shows the structure of the Abd-B locus. The promoter (arrow) and coding regions (black bars) are located to the far right. The iab-9 region located upstream is not shown. The 3' regulatory regions, iab-5 through iab-8, are shown. Red ovals represent domain boundaries. Blue squares indicate Promoter Targeting Sequences.

These DNA sequences were inserted between the IAB8 enhancer and the suHw insulator located at the 3' of lacZ. Transformants were classified into three types according which promoter is activated by IAB8 (Lin et al., 2004). Briefly, in Type I, IAB8 activates lacZ, not w. In Type II, it activates w but not lacZ, while, in Type III, the IAB8 enhancer activates neither w nor lacZ. The selective activation of a single promoter is an indication of promoter targeting activity. Asterisks indicate that several strains obtained exhibit excessive enhancer trap or background staining that prevents them from being characterized as one of the three categories.

During our analysis, we noticed that, when NEE is targeted by PTS-6 or PTS-7, it is only moderately facilitated (see Fig. 2, compare B with F). However, when IAB8 is targeted, a much greater degree of facilitation is observed (see Fig. 3, compare B with F and G). This result suggests that PTS elements may be more efficient in facilitating Abd-B enhancers than heterologous enhancers. Supporting this notion, we found that PTS-7 provides very little augmentation to the evenskipped stripe 3 enhancer (Small et al., 1993) (data not shown), while it greatly enhanced the IAB5 enhancer (Lin et al., 2003).

PTS elements overcome multiple insulators

Unlike enhancers from the iab-7 domain, which are separated from the Abd-B promoter by a single insulator, Fab-8, enhancers from iab-6 must overcome two insulators, Fab-7 and Fab-8. To test whether PTS-6 could overcome both of these insulators, we inserted the Fab-7, Fab-8, and PTS-6 between the 3' of lacZ and the IAB8 enhancer (see W270 in Fig. 5). After transgenic strains were analyzed by in situ hybridization, we detected all three types of transgenic strains: 5 of the 21 strains target lacZ, while three selectively activate w. A representative line showing lacZ targeting is shown in Figs. 5C and D. In the control experiment, the 0.8 kb Fab-7 and the 0.7 kb Fab-8 were inserted between the 3' of lacZ and the more 3' IAB8. No w or lacZ activation could be detected after analyzing 6 transgenic strains carrying this construct, suggesting that IAB8 is blocked (Figs. 5A, B). These results suggest that, in principle, PTS-6 may be able to target an enhancer over both Fab-7 and Fab-8 insulators in the endogenous Abd-B locus.

Fig. 5. The PTS element overcomes multiple insulators from different positions.

Fig. 5. The PTS element overcomes multiple insulators from different positions.

(A, B). The combination of the 0.8 kb Fab-7 and 690 bp Fab-8 insulators totally blocks the IAB8 enhancer. No transgene expression can be detected.

(C, D) The 200 bp PTS-6 can overcome Fab-7 plus Fab-8 insulator combination and selectively activates lacZ (in the strain shown). From 21 strains analyzed, three strains selectively activated w, five selectively activated lacZ, and the rest did not show activation of either promoters.

(E, F) The PTS-7 element overcomes a combination of suHw and Fab-8 and selectively activates w in 4 of 32 lines or lacZ in 7 of 32 lines.

(G, H) When PTS-7 is placed between suHw and Fab-8, it targets IAB8 to w in 3 of 29 strains and to lacZ in 8 of 29 lines analyzed.

To test whether overcoming multiple insulators is a general property of PTS elements, we also challenged PTS-7 with two insulators, Fab-8 and suHw. This experiment was done by inserting the 1.7 kb region (Zhou and Levine, 1999) from Fab-8 (containing both the Fab-8 insulator and the 625 PTS-7) into #125 (see construct W114 in Fig. 5, under E, F). Similar to PTS-6, PTS-7 overcomes a combination of two insulators, suHw and Fab-8, since IAB8 strongly activates lacZ in seven of 32 strains tested, and it activates w in four of these lines. An example of lacZ-targeted strain is shown in Figs. 5E, F, where IAB8 activates only lacZ. There is no detectable level of w expression. Similar results were obtained when the 3.7 kb DNA containing both PTS-6 and Fab-7 was inserted between suHw insulator and IAB8 enhancer in transgenic vector #125 (data not shown). These data suggest that there is no difference between PTS-6 and PTS-7 in terms of bypassing which insulator or insulator combination, thus overcoming that multiple insulators is a general property of the PTS.

Previous genetic analyses suggested that, when both PTS-7 and PTS-6 are deleted from Abd-B locus, none of the enhancers from iab-5, iab-6, or iab-7 region could activate Abd-B (Zhou and Levine, 1999). This implies that PTS-6 and PTS-7 might play a role in targeting enhancers from the iab-5 region. To test if this may be possible, we constructed a transgenic vector that partly mimics the arrangement of different cis elements in the iab-5 and iab-6 region relative to the Abd-B promoter. As illustrated in W115 of Fig. 5, PTS-7 is located at the 3' end of lacZ, flanked by a pair of insulators, suHw and Fab-8. The IAB8 enhancer is placed at the most 3' position. This layout of different cis elements in the transgene is similar to the endogenous arrangement of cis elements in Abd-B in that there are insulators both between the PTS and the promoter and between the PTS and the enhancer. When transgenic strains carrying this construct were analyzed, we could readily recover Type I (8/29), Type II (3/29), and Type III (18/29) strains, indicating that the PTS medicates promoter targeting in this transgene. One example of Type I strains is shown in Figs. 5G, H. Here, IAB8 strongly activates lacZ, while the neighboring w promoter is not activated. This result suggests that the PTS is able to mediate promoter targeting from various positions relative to the insulator, thus in the endogenous location PTS-6 (as well as PTS-7) could potentially regulate iab-5 enhancers.

Discussion:

Here, we described the identification and characterization of a new Promoter Targeting Sequence, PTS-6, from the iab-6 domain of the Abd-B 3' regulatory region. PTS-6 is located just next to the Fab-7 boundary. It overcomes both the Fab-7 insulator and the heterologous suHw insulator and selectively targets enhancers to a single promoter in transgenic embryos. PTS-6 preferentially facilitates the IAB8 enhancer from Abd-B to bypass the heterologous NEE enhancer. More importantly, PTS elements could overcome a combination of two insulators including Fab-7 plus Fab-8 and could function from a number of positions relative to the insulators. These findings suggest that PTS elements could target distal enhancers in Abd-B over several insulator elements to activate the promoter. These results strongly support the promoter targeting model of long-range enhancer–promoter interactions in the Abd-B locus.

The function of the PTS-6 is consistent with our previous genetic study (Zhou and Levine, 1999), where a P-element insertion into the PTS-7 region produced loss of function of Abd-B only in the 7th abdominal segment. However, a deletion in PTS-7 in combination with the deletion of 3.7 kb in Fab-7 ^{1 R73} (which removes both the Fab-7 insulator and the PTS-6) produced a much stronger loss of function phenotype, which is evident as almost a complete loss of Abd-B function in A5, A6, and A7 segments (Zhou and Levine, 1999). Although the deletion of PTS-7 alone is not available, this result, nonetheless, suggests that the deletion of PTS-6 (plus Fab-7) enhances the loss of function phenotype of PTS-7 mutation. Since the Fab-7¹ mutation alone does not have loss of Abd-B function phenotype in A5 and A6 abdominal segments (Gyurkovics et al., 1990 and Mihaly et al., 1997), PTS-6 and PTS-7 may have redundant functions.

Our study provides support to the model that the iab regulatory regions in Abd-B have modular organizations near the domain boundary, i.e. PTS, insulator, and PRE elements could be found near the boundary region. Although the iab-5/iab-6 boundary has not been studied, this is apparently the case for both iab-6/iab-7 and iab-7/iab-8 boundaries. In both cases, these elements are arranged in the same order: PTS–insulator–PRE. It is not clear why the PTS elements are located so close to the insulators in both cases (less than 300 bp). In transgenic embryos, separating the PTS from the insulator by 3 kb l insertion (Lin et al., 2003) and a 6 kb l insertion (Zhou J., unpublished results) did not affect promoter targeting. However, in the endogenous location, this arrangement could be of functional significance. For example, the close proximity between the insulator and the PTS may be necessary for the protection of the “enhancer–promoter complex” against the PRE element located just opposite of the insulator when an iab domain is looped to the promoter.

In this study, PTS-6 overcomes both the homologous Fab-7 insulator and the heterologous suHw insulator. In addition, it overcomes a combination of both suHw and Fab-7. These results are in apparent disagreement with a previous study where the replacement of the endogenous Fab-7 insulator with suHw led to the loss of Abd-B function in the 5th and 6th abdominal segments (Hogga et al., 2001). One of the interpretations was that the suHw is a stronger insulator than Fab-7, consequently, enhancers located in iab-5 and iab-6 are blocked and could not activate sufficient Abd-B expression. Results from our current study suggest that the strength of an insulator does not seem to affect the promoter targeting function of the PTS: the PTS facilitates the IAB8 enhancer equally well, regardless of which insulator, or insulators, are in its way. In light of our findings, we favor the alternative interpretation of the insulator replacement study, which emphasizes the qualitative differences between Fab-7 and other heterologous insulators. For example, Fab-7 has been shown to be developmentally regulated, while suHw is not (Schweinsberg and Schedl, 2004).

Previous observations with the suHw insulator suggested that two copies of the insulator could interact and cancel the enhancer blocking function (Cai and Shen, 2001 and Muravyova et al., 2001). In our study, we also tested several combinations of insulators: Fab-7/Fab-8, suHw/Fab-8, and Fab-7/suHw (Chen Q. and Zhou J., unpublished results). In all three combinations, the double insulators exert stronger enhancer blocking than a single one. Thus, these insulators do not appear to interact with each other to cancel enhancer-blocking function as do two suHw insulators.

Acknowledgments:

This work is supported by NIH Grant GM65391, the March of Dimes Birth Defect Foundation, the Edward Mallinckrodt Jr. Foundation, the Concern Foundation, and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of health to J.Z.

References:

Barges et al., 2000 S. Barges, J. Mihaly, M. Galloni, K. Hagstrom, M. Muller, G. Shanower, P. Schedl, H. Gyurkovics and F. Karch, The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain, Development 127 (2000), pp. 779–790.

Bell et al., 1999 A.C. Bell, A.G. West and G. Felsenfeld, The protein CTCF is required for the enhancer blocking activity of vertebrate insulators, Cell 98 (1999), pp. 387–396.

Boulet et al., 1991 A.M. Boulet, A. Lloyd and S. Sakonju, Molecular definition of the morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila Abd-B homeotic gene, Development 111 (1991), pp. 393–405.

Busturia and Bienz, 1993 A. Busturia and M. Bienz, Silencers in abdominal-B, a homeotic Drosophila gene, EMBO J. 12 (1993), pp. 1415–1425.

Busturia et al., 1997 A. Busturia, C.D. Wightman and S. Sakonju, A silencer is required for maintenance of transcriptional repression throughout Drosophila development, Development 124 (1997), pp. 4343–4350.

Cai and Levine, 1995 H. Cai and M. Levine, Modulation of enhancer–promoter interactions by insulators in the Drosophila embryo, Nature 376 (1995), pp. 533–536.

Cai and Shen, 2001 H.N. Cai and P. Shen, Effects of cis arrangement of chromatin insulators on enhancer-blocking activity, Science 291 (2001), pp. 493–495.

Celniker et al., 1989 S.E. Celniker, D.J. Keelan and E.B. Lewis, The molecular genetics of the bithorax complex of Drosophila: characterization of the products of the Abdominal-B domain, Genes Dev. 3 (1989), pp. 1424–1436.

Chan et al., 1994 C.S. Chan, L. Rastelli and V. Pirrotta, A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression, EMBO J. 13 (1994), pp. 2553–2564.

Chung et al., 1993 J.H. Chung, M. Whiteley and G. Felsenfeld, A 5' element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila, Cell 74 (1993), pp. 505–514.

Dorsett, 1993 D. Dorsett, Distance-independent inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding protein of Drosophila, Genetics 134 (1993), pp. 1135–1144.

Duncan, 1987 I. Duncan, The bithorax complex, Annu. Rev. Genet. 21 (1987), pp. 285–319.

Gaszner et al., 1999 M. Gaszner, J. Vazquez and P. Schedl, The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer–promoter interaction, Genes Dev. 13 (1999), pp. 2098–2107.

Geyer and Corces, 1992 P.K. Geyer and V.G. Corces, DNA position-specific repression of transcription by a Drosophila zinc finger protein, Genes Dev. 6 (1992), pp. 1865–1873.

Gyurkovics et al., 1990 H. Gyurkovics, J. Gausz, J. Kummer and F. Karch, A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation, EMBO J. 9 (1990), pp. 2579–2585.

Hagstrom et al., 1996 K. Hagstrom, M. Muller and P. Schedl, Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex, Genes Dev. 10 (1996), pp. 3202–3215.

Hagstrom et al., 1997 K. Hagstrom, M. Muller and P. Schedl, A Polycomb and GAGA dependent silencer adjoins the Fab-7 boundary in the Drosophila bithorax complex, Genetics 146 (1997), pp. 1365–1380.

Hark et al., 2000 A.T. Hark, C.J. Schoenherr, D.J. Katz, R.S. Ingram, J.M. Levorse and S.M. Tilghman, CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus, Nature 405 (2000), pp. 486–489.

Hogga et al., 2001 I. Hogga, J. Mihaly, S. Barges and F. Karch, Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex, Mol. Cells 8 (2001), pp. 1145–1151.

Ip et al., 1992 Y.T. Ip, R.E. Park, D. Kosman, E. Bier and M. Levine, The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo, Genes Dev. 6 (1992), pp. 1728–1739.

Kanduri et al., 2000 C. Kanduri, V. Pant, D. Loukinov, E. Pugacheva, C.F. Qi, A. Wolffe, R. Ohlsson and V.V. Lobanenkov, Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive, Curr. Biol. 10 (2000), pp. 853–856.

Karch et al., 1985 F. Karch, B. Weiffenbach, M. Peifer, W. Bender, I. Duncan, S. Celniker, M. Crosby and E.B. Lewis, The abdominal region of the bithorax complex, Cell 43 (1985), pp. 81–96.

Karch et al., 1994 F. Karch, M. Galloni, L. Sipos, J. Gausz, H. Gyurkovics and P. Schedl, Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster, Nucleic Acids Res. 22 (1994), pp. 3138–3146.

Kellum and Schedl, 1992 R. Kellum and P. Schedl, A group of scs elements function as domain boundaries in an enhancer-blocking assay, Mol. Cell. Biol. 12 (1992), pp. 2424–2431.

Lewis, 1978 E.B. Lewis, A gene complex controlling segmentation in Drosophila, Nature 276 (1978), pp. 565–570.

Lin et al., 2003 Q. Lin, D. Wu and J. Zhou, The promoter targeting sequence facilitates and restricts a distant enhancer to a single promoter in the Drosophila embryo, Development 130 (2003), pp. 519–526.

Lin et al., 2004 Q. Lin, Q. Chen, L. Lin and J. Zhou, The promoter targeting sequence mediates epigenetically heritable transcription memory, Genes Dev. 18 (2004), pp. 2639–2651.

Martin et al., 1995 C.H. Martin, C.A. Mayeda, C.A. Davis, C.L. Ericsson, J.D. Knafels, D.R. Mathog, S.E. Celniker, E.B. Lewis and M.J. Palazzolo, Complete sequence of the bithorax complex of Drosophila, Proc. Natl. Acad. Sci. U. S. A. 92 (1995), pp. 8398–8402.

McGinnis and Krumlauf, 1992 W. McGinnis and R. Krumlauf, Homeobox genes and axial patterning, Cell 68 (1992), pp. 283–302.

Mihaly et al., 1997 J. Mihaly, I. Hogga, J. Gausz, H. Gyurkovics and F. Karch, In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb-response element, Development 124 (1997), pp. 1809–1820.

Mihaly et al., 1998 J. Mihaly, I. Hogga, S. Barges, M. Galloni, R.K. Mishra, K. Hagstrom, M. Muller, P. Schedl, L. Sipos, J. Gausz, H. Gyurkovics and F. Karch, Chromatin domain boundaries in the Bithorax complex, Cell. Mol. Life Sci. 54 (1998), pp. 60–70.

Morata et al., 1986 G. Morata, E. Sanchez-Herrero and J. Casanova, The bithorax complex of Drosophila: an overview, Cell Differ. 18 (1986), pp. 67–78.

Muller et al., 1999 M. Muller, K. Hagstrom, H. Gyurkovics, V. Pirrotta and P. Schedl, The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions, Genetics 153 (1999), pp. 1333–1356.

Muravyova et al., 2001 E. Muravyova, A. Golovnin, E. Gracheva, A. Parshikov, T. Belenkaya, V. Pirrotta and P. Georgiev, Loss of insulator activity by paired Su(Hw) chromatin insulators, Science 291 (2001), pp. 495–498.

Paro et al., 1998 R. Paro, H. Strutt and G. Cavalli, Heritable chromatin states induced by the Polycomb and trithorax group genes, Novartis Found. Symp. 214 (1998), pp. 51–61.

Pirrotta, 1998 V. Pirrotta, Polycombing the genome: PcG, trxG, and chromatin silencing, Cell 93 (1998), pp. 333–336.

Pirrotta et al., 2003 V. Pirrotta, S. Poux, R. Melfi and M. Pilyugin, Assembly of Polycomb complexes and silencing mechanisms, Genetica 117 (2003), pp. 191–197.

Riddihough and Ish-Horowicz, 1991 G. Riddihough and D. Ish-Horowicz, Individual stripe regulatory elements in the Drosophila hairy promoter respond to maternal, gap, and pair-rule genes, Genes Dev. 5 (1991), pp. 840–854.

Rubin and Spradling, 1982 G.M. Rubin and A.C. Spradling, Genetic transformation of Drosophila with transposable element vectors, Science 218 (1982), pp. 348–353.

Sanchez-Herrero et al., 1985 E. Sanchez-Herrero, J. Casanova, S. Kerridge and G. Morata, Anatomy and function of the bithorax complex of Drosophila, Cold Spring Harbor Symp. Quant. Biol. 50 (1985), pp. 165–172.

Schweinsberg and Schedl, 2004 S.E. Schweinsberg and P. Schedl, Developmental modulation of Fab-7 boundary function, Development 131 (2004), pp. 4743–4749.

Small et al., 1993 S. Small, D.N. Arnosti and M. Levine, Spacing ensures autonomous expression of different stripe enhancers in the even-skipped promoter, Development 119 (1993), pp. 762–772.

Tautz and Pfeifle, 1989 D. Tautz and C. Pfeifle, A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback, Chromosoma 98 (1989), pp. 81–85.

Udvardy et al., 1985 A. Udvardy, E. Maine and P. Schedl, The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains, J. Mol. Biol. 185 (1985), pp. 341–358.

Vazquez et al., 1993 J. Vazquez, G. Farkas, M. Gaszner, A. Udvardy, M. Muller, K. Hagstrom, H. Gyurkovics, L. Sipos, J. Gausz and M. Galloni, Genetic and molecular analysis of chromatin domains, Cold Spring Harbor Symp. Quant. Biol. 58 (1993), pp. 45–54.

Wu et al., 1998 X. Wu, R. Vakani and S. Small, Two distinct mechanisms for differential positioning of gene expression borders involving the Drosophila gap protein giant, Development 125 (1998), pp. 3765–3774.

Zhao et al., 1995 K. Zhao, C.M. Hart and U.K. Laemmli, Visualization of chromosomal domains with boundary element-associated factor BEAF-32, Cell 81 (1995), pp. 879–889.

Zhou and Levine, 1999 J. Zhou and M. Levine, A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo, Cell 99 (1999), pp. 567–575.

Zhou et al., 1996 J. Zhou, S. Barolo, P. Szymanski and M. Levine, The Fab-7 element of the bithorax complex attenuates enhancer–promoter interactions in the Drosophila embryo, Genes Dev. 10 (1996), pp. 3195–3201.

Zhou et al., 1999 J. Zhou, H. Ashe, C. Burks and M. Levine, Characterization of the transvection mediating region of the abdominal-B locus in Drosophila, Development 126 (1999), pp. 3057–3065.

Enhancer gene loci activate promoter sites for gene transcription. Recent data (Ling J, et al, 2005) indicate that such activation is mediated by the syntheis of enhancer noncoding RNA species, which then travel to specific promoter sites and there activate the synthesis of specific coding RNAs. This new study by Qi Chen, Lan Lin, Sheryl Smith, Qing Lin and Jumin Zhou demonstrates that such enhancer elements are capable of by-passing intervening insulator elements on their journey to the promoter sites.

2. Song X, Sun Y, and Garen A, "Roles of PSF protein and VL30 RNA in reversible gene regulation".

3. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble", RNA2005", p. 279, The RNA Society, Bethesda, MD 20814-3998, (2005).

4. Frenster JH, "Mechanisms of Repression and De-Repression within Interphase Chromatin", In Vitro, vol. 1, pp. 78-101 (1965).

5. Frenster JH, "Correlation of the Binding to DNA Loops or to DNA Helices with the Effect on RNA Synthesis", Nature vol. 208, no. 5015, p. 1093 (December 11, 1965).

6. Herstein PR, and Frenster JH, "Mated Models of Gene Regulation in Eukaryotes", in: "Embryonic and Fetal Antigens in Cancer", vol. 2, pp. 5-7, Oak Ridge National Lab., Oak Ridge, Tenn., 1972.

7. Jabri E, "Non-Coding RNA: Small, but in Control", Nature Reviews Molecular Cell Biology, vol.6, no. 5, 361 (May, 2005).

8. Kuwabara T, Hsieh J, Nakashima K, Taira K, and Gage FH, "A Small Modulatory dsRNA Specifies the Fate of Adult Neural Stem Cells".

9. De Carvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo".

Links to RNA and Biological Causality:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).