Molecular Genetics Division, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
* Author for correspondence (e-mail: zhouj{at}wistar.upenn.edu)
Accepted 21 October 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Abdominal-B, Insulator, PTS, Fab-8, suHw, IAB5
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent genetic studies in the homeotic selector gene Abdominal-B
from the Drosophila bithorax complex (BX-C) have identified a
cis-regulatory region, termed transvection mediating region
(tmr), that may facilitate long-range enhancer-promoter interactions
(Hendrickson and Sakonju,
1995; Hopmann et al.,
1995
; Sipos et al.,
1998
). Abd-B contains an extended 3' regulatory
region that is functionally subdivided into distinct enhancer domains called
infra-abdominal (iab)-5, iab-6, iab-7 and
iab-8 by insulators, or boundary elements such as
Frontabdominal (Fab)-7 and Fab-8
(Barges et al., 2000
;
Hagstrom et al., 1996
;
Karch et al., 1985
;
Mihaly et al., 1998
;
Mihaly et al., 1997
;
Zhou et al., 1999
;
Zhou et al., 1996
)
(Fig. 1A,B). Enhancer elements
from the 3' regulatory region can activate the Abd-B promoter
over the intervening insulators and long distances. These enhancers continue
to activate Abd-B when the 3' regulatory region is translocated
to different chromosomal locations, or even to a different chromosome (from
the third to the Y chromosome)
(Hendrickson and Sakonju,
1995
; Hopmann et al.,
1995
; Sipos et al.,
1998
). This strong regulatory interaction depends on the 9.5 kb
tmr (Hopmann et al.,
1995
). These observations suggest that an enhancer-facilitating
mechanism exists in Abd-B, possibly within the tmr.
Consequently, a novel cis element, the promoter targeting sequence
(PTS) (Zhou and Levine, 1999
)
has been identified from the tmr. The PTS has a distinctive
anti-insulator activity, allowing an enhancer to activate its promoter over
the intervening insulator in transgenic embryos. In the presence of an
insulator, the PTS also appears to have a promoter targeting activity,
allowing an enhancer to activate only one promoter when two are present in the
transgene. We report that the PTS facilitates long-range enhancer-promoter
interactions in transgenic embryos, and that it mediates the promoter
targeting function by restricting enhancer activities to a single promoter. We
also show that the PTS functions only when itself and an insulator are located
between an enhancer and a promoter.
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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 30 and 60 independent transformants were obtained for each of the
recombinant P-element shown. In situ hybridization was performed essentially
as described in previous reports (Tautz
and Pfeifle, 1989
; Zhou et
al., 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 element 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 are subsequently analyzed
by RNA in situ hybridization.
Quantitative analysis of the activity of PTS
Embryos from different transgenic strains were collected and stained with
anti white or lacZ RNA probes in parallel. Enhancer strength
was quantified by measuring the differential absorption of transmitted light
(EV) between spot a (unstained region) and b (stained
region) in the embryos in Fig.
5A using a digital spot light meter (Sekonic L608). The reading
reflects the relative intensity (2
EV-1) of the staining that
is linear with staining reaction time during a 1 hour incubation (see
Fig. 5B). It is assumed that
alkaline phosphatase (AP) activity represents enhancer strength, which can be
expressed as: enhancer strength=constant x
(2
EV-1)/time. To compare enhancer strength, heterozygous
embryos from different samples are fixed and stained in parallel for 45
minutes. The
EV values of approximately 30 embryos are measured.
Enhancer strengths are plotted as bar graphs in
Fig. 5C,D.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The IAB8 enhancer directs a narrow band of transcription in the posterior
region of the embryo (see arrow in Fig.
2A). Similar to other early Drosophila enhancers that
have been tested (Ohtsuki et al.,
1998; Zhou et al.,
1996
), its activity attenuates as its distance from the promoter
increases (compare Fig. 2A with
2B). This 5.3 kb tmr was placed in both the forward
(5'
3' when IAB8 is between the promoters and
Fab-8/PTS, see construct #17 in
Fig. 2) and the reverse
orientation (construct #28 in Fig.
2). To monitor transcriptional activity of the IAB8 enhancer,
embryos from individual transgenic strains were collected and subjected to
whole-mount RNA in situ hybridization for the white (w) or
lacZ genes.
In the forward orientation, IAB8 weakly stimulated both the divergently
transcribed w and Tp-lacZ genes
(Fig. 2C;
Table 1). Its activity on the
Tp promoter is very similar to that of the 1.6 kb IAB8 alone at the
same location (Fig. 2B),
suggesting that the distally located PTS and Fab-8 do not affect the
communication between IAB8 and the transgenic promoters. In the reverse
orientation, however, IAB8 activates the transgenic promoters despite the
intervening Fab-8 insulator. In any particular strain, IAB8 exhibits
the selective activation of either the Tp-lacZ
(Fig. 2D) or the w
(Fig. 2E) gene. This effect is
seen in about 50% of the transgenic lines
(Table 1). In the remaining
strains, IAB8 does not activate any of the transgenic promoters due to the
enhancer blocking effect of the Fab-8 insulator (data not shown).
These results are similar to that of the previous study
(Zhou and Levine, 1999),
suggesting that the 290 bp PTS exhibits the anti-insulator and promoter
targeting activities. This is confirmed by transgene W14
(Table 1), where PTS could
overcome a heterologous suHw insulator and target IAB8 to the
w or Tp promoters. It should be noted that in the forward
orientation (Fig. 2C), IAB8 is
located 5.5 kb away from the w promoter and 4.9 kb away from the
lacZ promoter, whereas in the reverse orientation
(Fig. 2D,E), it is 8.2 kb away
from w and 7.5 kb away from lacZ. Rather than reducing the
activity, the greater distance between IAB8 and the promoters caused by
inserting Fab-8 and PTS resulted in an increase of IAB8 enhancer
activity. This is seen by comparing the activities of IAB8 on Tp-lacZ
in Fig. 2B versus 2D, or
w in Fig. 2C versus
2E. In either case, the enhancer activity is much stronger when
both PTS and Fab-8 are located between IAB8 and the promoters. These
results indicate that PTS may facilitate the long distance interactions
between IAB8 and the w or the Tp promoter.
|
The PTS, in combination with an insulator, facilitates a distant
enhancer and restricts the transcription activation to only one of the two
available promoters
To confirm the enhancer facilitating activity and to eliminate position
effects due to differential chromosomal insertion sites of the transgene, we
used the Flp-FRT system, which causes the removal of FRT-flanked DNA sequences
from the transgene after introducing the Flp recombinase by genetic cross
(Golic and Lindquist, 1989).
This technique permits the analysis of the transgene in the same chromosomal
context before and after the test DNA is removed. We flanked a 2.0 kb
PstI-HindIII fragment from the tmr region (see
Fig. 1B) that contains both the
minimal 290 bp PTS and the 580 bp Fab-8 insulator with the FRT sites
and placed this group of elements between the 3' end of lacZ
and 2.7 kb EcoRI fragment from the same region
(Fig. 1B) that contains the 1.6
kb IAB8 enhancer (W32 in Fig.
3). When transgenic embryos were analyzed, results similar to
those without FRT sites were obtained. In about half of the transgenic strains
IAB8 selectively activates one of the two divergently transcribed w
and the Tp-lacZ genes (Table
1). In the remaining strains, IAB8 does not activate any of the
transgenic promoters. An example of a PTS-mediated IAB8-w interaction
is shown in Fig. 3A,B. Here,
IAB8 strongly activates w but not the closely positioned Tp
promoter (compare Fig. 3A with
3B). However, after the removal of PTS and Fab-8 by
Flp-mediated recombination, the intense and selective IAB8-w
interaction disappeared. Instead, IAB8 activates both w and
Tp-lacZ, but with greatly reduced activity, despite the fact that the
enhancer is now 2.0 kb closer to the promoters
(Fig. 3C,D). Similar result was
obtained when we place the Fab-8 and PTS elements between the
3' end of lacZ and the heterologous Neural Ectoderm Enhancer
(NEE) from the rhomboid gene (Ip
et al., 1992
) (data not shown). These results strongly suggest
that the PTS, in combination of the Fab-8 insulator, facilitates
enhancer-promoter interaction, and that it restricts the enhancer activity to
a single promoter.
|
To confirm that the enhancer-facilitating and single promoter activating
effects were due to the PTS but not a possible synergy between DNA sequences
located within the tmr, we tested the PTS in the absence of
Fab-8 and other tmr sequences. The PTS, the heterologous
suHw insulator (Cai and Levine,
1995; Dorsett,
1993
; Geyer and Corces,
1992
), and the IAB5 enhancer
(Busturia and Bienz, 1993
) were
placed at the 3' of lacZ in the order given (see construct W81
under Fig. 3E,F). PTS and
suHw were flanked by a direct repeat of FRT sites so that both
elements could be removed by recombination. In about one third of all
transgenic strains, IAB5 selectively activated only one of the two (w
or Tp) promoters (see Fig.
3E,F for selective IAB5-lacZ interaction), suggesting
that the PTS mediates promoter targeting in these transgenic lines
(Table 1). The activities of
IAB5 on the targeted promoters are consistently strong, with slight variations
among different strains (Fig.
5D). In the remaining lines, IAB5 does not activate either of the
promoters. Presumably, PTS does not function in these lines, and the IAB5
enhancer is blocked by the intervening suHw insulator. The
enhancer-facilitating activity was confirmed by Flp-FRT analysis
(Fig. 3G,H). Similar to
Fig. 3A-D, the simultaneous
removal of both PTS and suHw dramatically reduced IAB5-lacZ
interaction (over 10-fold reduction, see
Fig. 5D). By contrast,
IAB5-w interaction, which was undetectable before the recombination,
could now be detected (compare w activity in
Fig. 3G with 3E).
To confirm that the PTS could indeed help IAB5 overcome the suHw insulator, we also constructed transgenes W78 and W79 that are similar to W81, but contain an FRT-flanked PTS (Table 1). In the former, PTS was placed between the 3' end of lacZ and the suHw insulator, while in the latter, PTS was interposed between suHw and IAB5. Transgenic embryos carrying either of these constructs exhibit a selective activation of w or lacZ in any given strain (see Fig. 4A,B for selective activation of lacZ by IAB5 in transgene W78). Removal of the PTS by Flp-mediated recombination caused the loss of IAB5 activated transcription, as IAB5 became blocked by the suHw insulator (compare Fig. 4B with 4D). These experiments indicate that the PTS exhibits the anti-insulator and promoter targeting activities when it is either upstream or downstream of an insulator.
|
In summary, these results clearly demonstrate that the enhancer facilitating function depends on the PTS element, not other unknown elements located within the tmr. This result also indicates that the promoter targeting activity is not a result of random positional effect, or preferential insertion of the transgene into specific chromosomal locations that may silence one of the promoters present in the transgenic vector. It is due to a PTS-dependent, active restriction of the enhancer activity to only one of the two available promoters (Fig. 3G,H). The enhancer-facilitating and single promoter activating effects are genetically stable in that they are memorized in up to 60 generations without a loss or change of promoter targeting.
Quantitative analysis of the enhancer facilitating and single
promoter activating effects
We also conducted semi-quantitative analyses of PTS-mediated enhancer
facilitating and single promoter activating activities by quantifying RNA in
situ hybridization. The staining intensity (as measured by optical absorption
of stained Drosophila embryos,
Fig. 5A) shows linear
relationship with staining time 60 minutes after the addition of substrates
for alkaline phosphatase (AP) (Fig.
5B). Assuming that the AP activity directly reflects enhancer
strength and that the AP activity remains constant during an 1 hour
incubation, we can compare the activity of the same enhancer in different
transgenes if the embryos are fixed and stained in parallel. Using this
method, we found that IAB8 is two- to fivefold stronger when it is located
less than 100 bp from the Tp promoter than when located 5.5 kb
downstream of Tp-lacZ (Fig.
2A,B, and Fig. 5C).
We then compared IAB8 and IAB5 activities with or without facilitation by the
PTS. We found that PTS facilitates IAB8 about eightfold and IAB5 ten- to
17-fold, respectively (Fig.
5C,D). In transgenic lines (W81) showing specific
IAB5-lacZ interaction, no w expression could be detected,
but after the Flp-mediated removal of PTS and suHw, w became
activated by IAB5 (Fig. 5D). In
two of the four lines (W81-2 and W81-4) w expression was detectable
within 45 minutes, whereas in the remaining two (W81-1 and W81-3), w
expression could be detected only after extended incubation (2 hours,
data not shown). The strength of IAB5-w or IAB5-lacZ
interactions after the removal of suHw/PTS is similar to that of IAB5
alone originally cloned at the same location (data not shown). These results
provided quantitative evidence for the enhancer-facilitating and single
promoter-activating activities of the PTS.
The PTS and an insulator must be located between an enhancer and a
promoter
The results shown in Fig. 2
also suggest that PTS must be interposed between an enhancer and a promoter.
To test whether this is true, the PTS and suHw DNA were placed distal
to the IAB5 enhancer, downstream of lacZ (W82 in
Table 1). From over 40
transgenic strains isolated and examined, none displayed enhancer facilitating
and the distinctive single promoter-activating effects
(Table 1). In these lines, IAB5
activated both the w and Tp promoters, with activities
similar to that of IAB5 alone in the same location (data not shown). This
result, together with the data from Fig.
2 suggests that PTS activity is location dependent, in that it
only functions when itself (and an insulator) is located between the enhancer
and the promoter.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the Abd-B locus, the enhancer-facilitating property of the PTS
could help distal enhancers such as IAB7 overcome the long distances and
direct robust transcription activation of Abd-B. This notion is
consistent with the genetic functions of the tmr and the
loss-of-function phenotype of PTS mutants
(Castrillon et al., 1993;
Gyurkovics et al., 1990
;
Zhou and Levine, 1999
). The
single promoter-activating function could ensure that enhancers in the BX-C
activate the cognate Abd-B promoter only. In this study, we have also
shown that promoter targeting only occurred when PTS/insulator was placed
between an enhancer and a promoter. This location-dependent characteristic of
PTS suggests that strategic placement of PTS within Abd-B can
facilitate specific enhancers. For example, in the Abd-B locus, PTS
and Fab-8 are located between IAB7 (but not IAB8) and Abd-B
promoter (Fig. 1A). In this
arrangement, PTS may facilitate only the distal IAB7 but not the proximal IAB8
to the Abd-B promoter. In fact, this appears to be the case, as
deletion of PTS causes a loss-of-function transformation of the seventh but
not the eighth abdominal segment
(Castrillon et al., 1993
;
Gyurkovics et al., 1990
;
Zhou and Levine, 1999
). On
this note, it is possible that multiple PTS-like elements exist in the BX-C to
mediate long-distance regulatory interactions.
It can be seen from Fig. 3
that the PTS-facilitated single enhancer-promoter interaction is stronger than
the sum of enhancer-w and enhancer-Tp interactions when the
IAB5 or IAB8 enhancers are placed alone at the 3' of lacZ.
These results suggest that the PTS-mediated enhancer facilitation is not just
the consequence of restricting an enhancer to a single promoter, it must also
actively promote long distance enhancer-promoter communications. It is
possible that the PTS functions by establishing an insulator-insensitive,
stable chromatin structure between the enhancer and a promoter, e.g. forming a
`stable loop' and bringing the enhancer closer to the promoter
(Fig. 6). Similar `loop'
hypothesis has been proposed based on genetic analysis of the
enhancer-promoter interactions in the Abd-B locus
(Galloni et al., 1993). This
model can not only explain the anti-insulator activity but can also account
for the enhancer-facilitating and the single promoter activating activities.
Such a stable association would ensure that enhancer-interacting activators
are constantly present at the promoter, which would result in efficient
promoter activation and, at the same time, prevent the enhancer from
activating other promoters.
|
Our study also suggests that PTS functions as a generic element in
transgenic embryos as it can target and facilitate a heterologous
neuroectoderm enhancer NEE (Ip et al.,
1992). It is possible that in other large genetic loci such as the
odorant receptor gene complex (Mombaerts,
1999
) and the neural cadherin-like adhesion gene complex
(Wu and Maniatis, 1999
), where
only one among several dozen promoters is activated in any given cell,
PTS-like elements may contribute to the promoter-selective transcriptional
activation.
In transgenic embryos, the PTS does not appear to exhibit promoter-specific activity as it can target either the w or the Tp promoter present in the transgene. It is not known what determines which of the two promoters to select. One possibility is that the decision is made by the interaction between the PTS and local chromatin structure. Alternatively, the selection could be a stochastic process. In the endogenous BX-C, however, the PTS must target the Abd-B promoter. Additional mechanism(s), therefore, must be in place to ensure enhancer-promoter specificity in the Abd-B locus.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barges, S., Mihaly, J., Galloni, M., Hagstrom, K., Muller, M.,
Shanower, G., Schedl, P., Gyurkovics, H. and Karch, F.
(2000). 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
127,779
-790.
Bulger, M. and Groudine, M. (1999). Looping
versus linking: toward a model for long-distance gene activation.
Genes Dev. 13,2465
-2477.
Busturia, A. and Bienz, M. (1993). Silencers in abdominal-B, a homeotic Drosophila gene. EMBO J. 12,1415 -1425.[Abstract]
Cai, H. and Levine, M. (1995). Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo. Nature 376,533 -536.[CrossRef][Medline]
Castrillon, D. H., Gonczy, P., Alexander, S., Rawson, R.,
Eberhart, C. G., Viswanathan, S., DiNardo, S. and Wasserman, S. A.
(1993). Toward a molecular genetic analysis of spermatogenesis in
Drosophila melanogaster: characterization of male-sterile mutants generated by
single P element mutagenesis. Genetics
135,489
-505.
Dorsett, D. (1993). Distance-independent
inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding
protein of Drosophila. Genetics
134,1135
-1144.
Dorsett, D. (1999). Distant liaisons: long-range enhancer-promoter interactions in Drosophila. Curr. Opin. Genet. Dev. 9,505 -514.[CrossRef][Medline]
Fernandez, L. A., Winkler, M., Forrester, W., Jenuwein, T. and Grosschedl, R. (1998). Nuclear matrix attachment regions confer long-range function upon the immunoglobulin mu enhancer. Cold Spring Harb. Symp. Quant. Biol. 63,515 -524.[Medline]
Galloni, M., Gyurkovics, H., Schedl, P. and Karch, F. (1993). The bluetail transposon: evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J. 12,1087 -1097.[Abstract]
Geyer, P. K. and Corces, V. G. (1992). DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 6,1865 -1873.[Abstract]
Golic, K. G. and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59,499 -509.[Medline]
Grosveld, F., Antoniou, M., Berry, M., de Boer, E., Dillon, N., Ellis, J., Fraser, P., Hanscombe, O., Hurst, J., Imam, A. et al. (1993). The regulation of human globin gene switching. Philos. Trans. R. Soc. Lond. B Biol. Sci. 339,183 -191.[Medline]
Gyurkovics, H., Gausz, J., Kummer, J. and Karch, F. (1990). A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation. EMBO J. 9,2579 -2585.[Abstract]
Hagstrom, K., Muller, M. and Schedl, P. (1996). Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev. 10,3202 -3215.[Abstract]
Hendrickson, J. E. and Sakonju, S. (1995). Cis
and trans interactions between the iab regulatory regions and abdominal-A and
abdominal-B in Drosophila melanogaster. Genetics
139,835
-848.
Hopmann, R., Duncan, D. and Duncan, I. (1995).
Transvection in the iab-5,6,7 region of the bithorax complex of Drosophila:
homology independent interactions in trans. Genetics
139,815
-833.
Ip, Y. T., Park, R. E., Kosman, D., Bier, E. and Levine, M. (1992). The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev. 6,1728 -1739.[Abstract]
Karch, F., Weiffenbach, B., Peifer, M., Bender, W., Duncan, I., Celniker, S., Crosby, M. and Lewis, E. B. (1985). The abdominal region of the bithorax complex. Cell 43, 81-96.[Medline]
Li, X. G., Liu, D. P. and Liang, C. C. (2001). Beyond the locus control region: new light on beta-globin locus regulation. Int. J. Biochem. Cell Biol 33,914 -923.[CrossRef][Medline]
Mihaly, J., Hogga, I., Gausz, J., Gyurkovics, H. and Karch,
F. (1997). In situ dissection of the Fab-7 region of the
bithorax complex into a chromatin domain boundary and a Polycomb-response
element. Development
124,1809
-1820.
Mihaly, J., Hogga, I., Barges, S., Galloni, M., Mishra, R. K., Hagstrom, K., Muller, M., Schedl, P., Sipos, L., Gausz, J. et al. (1998). Chromatin domain boundaries in the Bithorax complex. Cell Mol. Life Sci. 54,60 -70.[CrossRef][Medline]
Mombaerts, P. (1999). Molecular biology of odorant receptors in vertebrates. Annu. Rev. Neurosci. 22,487 -509.[CrossRef][Medline]
Ohtsuki, S., Levine, M. and Cai, H. N. (1998).
Different core promoters possess distinct regulatory activities in the
Drosophila embryo. Genes Dev.
12,547
-556.
Rollins, R. A., Morcillo, P. and Dorsett, D.
(1999). Nipped-B, a Drosophila homologue of chromosomal adherins,
participates in activation by remote enhancers in the cut and Ultrabithorax
genes. Genetics 152,577
-593.
Rubin, G. M. and Spradling, A. C. (1982). Genetic transformation of Drosophila with transposable element vectors. Science 218,348 -353.[Medline]
Sipos, L., Mihaly, J., Karch, F., Schedl, P., Gausz, J. and
Gyurkovics, H. (1998). Transvection in the Drosophila Abd-B
domain: extensive upstream sequences are involved in anchoring distant
cis-regulatory regions to the promoter. Genetics
149,1031
-1050.
Tautz, D. and Pfeifle, C. (1989). 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, 81-85.[Medline]
Torigoi, E., Bennani-Baiti, I. M., Rosen, C., Gonzalez, K.,
Morcillo, P., Ptashne, M. and Dorsett, D. (2000). Chip
interacts with diverse homeodomain proteins and potentiates bicoid activity in
vivo. Proc. Natl. Acad. Sci. USA
97,2686
-2691.
Wu, Q. and Maniatis, T. (1999). A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97,779 -790.[Medline]
Wu, X., Vakani, R. and Small, S. (1998). Two
distinct mechanisms for differential positioning of gene expression borders
involving the Drosophila gap protein giant.
Development 125,3765
-3774.
Zhou, J. and Levine, M. (1999). A novel cis-regulatory element, the PTS, mediates an anti-insulator activity in the Drosophila embryo. Cell 99,567 -575.[Medline]
Zhou, J., Barolo, S., Szymanski, P. and Levine, M. (1996). The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo. Genes Dev. 10,3195 -3201.[Abstract]
Zhou, J., Ashe, H., Burks, C. and Levine, M.
(1999). Characterization of the transvection mediating region of
the abdominal- B locus in Drosophila. Development
126,3057
-3065.