Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
Accepted 16 July 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Transcription, Drosophila, Bithorax, Polycomb, Ultraabdominal
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Each regulatory region includes a variety of DNA elements which must work
together to establish and maintain correctly the proper segmental patterns for
the homeotic genes. Some DNA fragments, when tested in P element reporter
constructs, drive segmentally limited patterns in early embryos; we call these
`initiation elements'. A few initiation elements have been well defined, with
binding sites for gap and pair rule genes
(Qian et al., 1993;
Müller and Bienz, 1992
;
Shimell et al., 1994
). Other
DNA fragments mediate the repressive effects of the Polycomb Group of factors
(the PcG) to maintain the segmental limits of the initiation elements through
late embryonic and larval life. These are usually called `Polycomb response
elements' or `PREs'. The PREs have been more difficult to define, because most
Polycomb Group proteins are not DNA-binding proteins, and because PRE function
is best assayed in conjunction with initiation elements. The PRE in the middle
of the bxd region is the best studied
(Chan et al., 1994
;
Fritsch et al., 1999
;
Horard et al., 2000
). This PRE
does not have an intrinsic segmental address; it can maintain different
segmental boundaries when combined with different initiation elements
(Chiang et al., 1995
).
It is not known how initiation elements interact with PREs, or whether that
interaction is limited in distance or orientation. It is also unclear how
inappropriate influences between initiation elements in one segmental
regulatory region and PREs in another are prevented. The latter problem may be
solved by boundaries to the segmental regulatory regions, which block the
spread of activation or repression signals. Boundary elements between domains
were postulated to account for the phenotypes of several small deletions in
the BX-C. Removing a putative boundary gives a dominant gain-of-function
phenotype, a transformation of one segment to the character of the next more
posterior segment (Mihaly et al.,
1998; Barges et al.,
2000
). The best-studied boundaries, called Mcp, Fab7 and
Fab8, each have a PRE within or immediately adjacent to the boundary
(Busturia et al., 1997
;
Hagstrom et al., 1997
;
Barges et al., 2000
), although
there are reasonable arguments that the Fab-7 boundary is separable
from the PRE (Mihaly et al.,
1997
).
We describe here a series of P element mutations in the BX-C that disturb
the functions of PREs or boundaries. These give dominant gain-of-function
phenotypes that mimic those of boundary deletion mutations. The P elements
initiate transcripts that proceed through PREs and boundaries, and the
phenotypes depend on the production of these transcripts. The accompanying
paper (Hogga and Karch, 2002)
suggests that transcription across a different region of the BX-C can relieve
silencing, and another parallel study
(Rank et al., 2002
) also
correlates transcription with loss of silencing, using a BX-C fragment on a
transgene. These observations raise the possibility that non-coding RNAs in
wild-type animals may function to activate segmental regulatory regions.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Southern blotting
DNA was recovered from adult flies and digested with restriction enzymes as
described in Bender et al. (Bender et al.,
1983b). Fragments were separated on 0.7% agarose gels, transferred
to Magnacharge nylon membrane (Osmonics), and probed with radiolabeled probe
prepared by random priming.
RNA in situs
RNA probes for in situ hybridization to embryos were made by in vitro
transcription with digoxigenin-substituted UTP. Procedures for probe synthesis
and embryo treatments have been described
(Fitzgerald and Bender, 2001).
The genomic DNA fragments used for the probes are defined by restriction
enzyme sites (proximal end/distal end) as follows: A,
EcoRI/EcoRI; B, EcoRI/HindIII; C,
SalI/BamHI; D, BamHI/SalI; E,
SalI/EcoRI; F, EcoRI/BamHI; G,
EcoRI/EcoRI; H, EcoRI/EcoRI; I,
HindIII/HindIII; and J, EcoRI/BamHI.
Immunohistochemistry
Embryos were fixed and stained for ABD-A protein as described
(Karch et al., 1990), except
that the antibody was the 6A18.12 mouse monoclonal
(Kellerman et al., 1990
). The
UabHH1 chromosome was balanced over a version of TM3 with
a ftz/lacZ transgene, and embryos were stained simultaneously for
ABD-A and lacZ. Uab homozygous embryos (without lacZ
staining) were chosen for examination. Stained embryos were dissected and
flattened as described (Karch et al.,
1990
).
Abdominal cuticle preparation
Adults were preserved in a mixture of ethanol and glycerol (3:1). Abdomens
were separated from the thorax, and split mid-dorsally with a razor blade. The
abdomens were soaked in 10% KOH for 30 minutes, then rinsed in water. Internal
tissues were removed manually, and the cuticles were arranged on a slide in a
drop of Immumount (Shandon), and flattened with a cover slip.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Such Uab flies appeared with a surprisingly high frequency
(1/50). Most showed a mild phenotype as a heterozygote, but homozygotes
showed a fairly complete transformation of the dorsal first abdominal tergite
towards the character of the second abdominal segment (illustrated for the R1
allele, Fig. 2). The ventral
abdomens of such homozygotes showed a sternite on the first abdominal segment,
where wild-type animals show none (Fig.
2).
|
One line, a derivative of the HC184B P element called HH1, had a particularly strong Uab phenotype. HH1 heterozygotes showed transformations as complete as those in homozygotes of most of the other alleles, including R1. HH1 homozygotes usually died as pharate adults. In exceptional homozygotes that did eclose, the tergite on the first abdominal segment resembled that of the second, third or fourth abdominal segments in size, pigmentation and bristle morphology (Fig. 2). The first abdominal segment had a complete sternite, which resembled that of the third or fourth abdominal segment in the number and orientation of the bristles (Fig. 2). The sternite of the second abdominal segment was also transformed to the character of a more posterior segment. The HH1 homozygotes also show a thin band of cuticle between the thorax and abdomen. In rare individuals, this is expanded on one side into a recognizable half tergite (Fig. 2), similar to the normal tergites on the second through fourth abdominal segments, as judged by bristle morphology. This `extra' abdominal-like segment is most probably derived from the third thoracic segment, which normally does not contribute to dorsal cuticle in the adult. Homozygotes also had enlarged or missing halteres, they typically were not able to flatten their wing blades, and they often had malformed third legs. If the Uab mutation affects PS6, the transformation should include the posterior third thoracic segment. This would explain the emergence of the extra abdominal segment, as well as the haltere and leg phenotypes.
Molecular characterization of the new Uab alleles
When the Uab chromosomes were analyzed by Southern blotting, none
had deletions adjacent to the P elements, and all retained P element
sequences. The Southern blots revealed a variety of different rearrangements
of the starting P elements. Several independent lines appeared to have
generated inverted duplications of the starting P element, represented by the
G1 and L1 Uab alleles (Fig.
1). Several additional independent lines appeared to be precise
inversions of the starting P element, as in the M1 and R1 alleles
(Fig. 1). Others had more
complicated rearrangements, and were not analyzed further.
The UabHH1 line, with the particularly strong Uab phenotype, appeared to retain a small P element derivative. This was recovered by PCR and sequenced. The element was 690 bp in length, which included 656 bp from the 5' end of HC184B, and 15 bp from the 3' end, separated by 19 bp of AT-rich sequence with no obvious homology to the starting element. The element retained the P element promoter at the 5' end, and the first 65 bases of the ß-galactosidase sequence. This deleted P was inverted relative to HC184B, so that the P promoter pointed toward the distal end of the chromosome arm (Fig. 1).
Ectopic RNA transcripts on the Uab chromosomes
A comparison of all the P insertions suggests that the Uab
phenotypes are not caused by disruption of endogenous BX-C sequences or by
juxtaposition of particular P sequences with BX-C sequences. However, all the
Uab chromosomes had inversions of the P elements, resulting in
distally-directed promoters. Therefore, we looked for RNA products, downstream
of the P elements, coming from these promoters. The intact P elements (such as
the M1 and R1 alleles) have two poly(A) addition sites 3' to the
lacZ-coding region, one derived from the Hsp70 gene
(Mlodzik and Hiromi, 1992),
and the other from the 3' end of the P element. The latter poly(A) site
is known to be only
50% efficient
(Laski et al., 1986
); the
efficiency of the Hsp70 poly(A) site is not known. The strongest
allele, UabHH1 lacks both these terminators.
Distally directed transcripts from the Uab P elements were easy to detect. To probe for RNA products, we subcloned a variety of fragments from the region, as shown in Fig. 1. The fragments were used to prepare strand-specific RNA probes. These were hybridized to embryos from the various Uab strains. We first looked for transcripts 3' to the Uab P elements, using a probe made from fragment B to detect distally-directed transcripts. Wild-type embryos showed no detectable RNA with this probe. In UabHH1 embryos, weak staining was detectable as early as embryonic stage 5 (cellular blastoderm) in a narrow band from 25% to 35% egg length (from the posterior pole). During gastrulation, the zone of RNA expression spread forward to the anterior edge of PS6. By stage 10 (extended germ band), the RNA signal was at its highest level. Hybridization was widespread throughout the epidermis of PS6, with more narrow bands of hybridization in PS7-12 (Fig. 3A). The RNA signal gradually decreased in later embryonic stages, but it always showed a sharp boundary at the anterior edge of PS6 (Fig. 3B). Embryos from the UabR1 and UabM1 strains showed a very similar pattern with the same probe, but the signal intensities were much reduced (Fig. 3D). The G1, M1, L1 and R1 alleles all have intact lacZ-coding regions; all make ß-galactosidase in PS6-12 in a pattern similar to that of the RNA detected by probe B.
|
The transcripts from UabHH1 were surprisingly long.
Probes from fragments C through I (Fig.
1) all detected distally directed RNAs in the same pattern as that
seen with probe B, although the signal intensity declined with the more distal
probes. Wild-type embryos showed no distally directed transcripts with probes
from fragments A-I. Fig. 3C
shows UabHH1 embryos with probe I; the signal is quite
weak but still clearly in the same pattern as probe B
(Fig. 3A). Probe J detects the
`iab-4' RNA (Cumberledge et al.,
1990); this transcription unit most probably lies within the
iab-3 segmental domain (Bender and
Hudson, 2000
). The signal from probe J begins in PS8 in both
wild-type and UabHH1 embryos; there is no signal in PS6
like that seen with probes B-I. In summary, the UabHH1 P
element drives an RNA product that extends for about 50 kb, although the
majority of transcripts must terminate at shorter distances. The
UabR1 P element makes a similar transcript that is less
abundant, presumably because of partial termination within the P element.
RNA probes were also used to detect proximally directed transcripts. A
probe from fragment A detected a proximally directed transcript from the
HC184B starting P element, but nothing from wild type,
UabR1 or UabHH1. Three other
proximally directed RNAs were detected in both wild-type and
UabHH1 embryos. The first was the abd-A
transcript, revealed by probes from fragments C-G. The second RNA, detected
with the fragment I probe, was seen only in blastoderm and early gastrulating
embryos in a zone from 10% to 40% egg length (from the posterior pole). A
similar pattern of RNA expression was discovered
(Sánchez-Herrero and Akam,
1989
), using a double-stranded probe from the same region. The
third RNA appeared to be the `PS13-15' RNA
(Sánchez-Herrero and Akam,
1989
). It was seen most strongly with the fragment J probe,
beginning in early elongated germ band embryos, widespread in the epidermis of
PS13 and PS14. After germ band shortening, the RNA faded in the epidermis but
persisted in the posterior CNS. The same pattern was seen with probes from
fragments I and H, although weaker. With probes from fragments C-G, we did not
detect this posterior RNA in addition to the abd-A pattern, but a
probe from fragment B did show faint staining in the same pattern in the
posterior CNS of late embryos. This RNA product could represent a read-through
product initiated at one or more of the Abd-B promoters.
Alternatively, it might come from an `iab-8 promoter'
(Zhou et al., 1999
). In either
case, transcripts extending through fragment B would measure at least 125
kb.
Suppression of the Uab phenotype in P cytotype
If the Uab phenotypes are due to RNAs initiated at the P element
promoter, then P cytotype should revert the phenotype. P cytotype is conferred
by strains with multiple P element insertions, some of which produce a
truncated 66 kDa form of the P transposase, which is a repressor of P activity
(Misra and Rio, 1990). Crosses
to such strains repress P/lacZ fusion genes, regardless of the
direction of the cross (Lemaitre and Coen,
1991
). P cytotype did indeed revert the Uab phenotype.
UabHH1/MKRS flies were crossed to flies of the Harwich,
2, and
MR-h12 strains. UabHH1/Harwich heterozygotes appeared
virtually wild type (Fig. 4),
and the phenotype was the same with the Harwich chromosomes derived from
either the maternal or paternal lineage. Similar crosses to
2 gave partial
suppression of the Uab phenotype, although our MR-h12 stock gave no
suppression. UabHH1/Harwich males were crossed to
UabHH1/MKRS females to recover UabHH1
homozygotes with a
25% contribution of Harwich chromosomes. These
homozygotes were healthy, and had variable Uab phenotypes; some flies
were near wild type. These homozygotes all retained the
UabHH1 P element insertion, as determined by Southern
blots. The repression of P transcription was confirmed by RNA in situ
hybridization. UabHH1/Harwich embryos were stained for
distally directed RNA products homologous to probe B. Weak staining was seen
in blastoderm embryos, but transcripts in older embryos were nearly completely
suppressed.
|
Ectopic transcripts in Uab1 and
Uab5
Since our P element Uab alleles are associated with transcription
of the iab-2 region, we checked the preexisting Uab
rearrangement alleles for similar RNA products. Uab1 is
associated with an inversion within the bithorax complex between the
bxd (PS6) and iab-8 (PS13) regulatory regions
(Fig. 5A; between positions 225
kb and 22 kb in SEQ89E coordinates) (Karch
et al., 1985). Uab1 homozygous or heterozygous
embryos show RNA products with probe B starting in PS6, primarily in the
epidermis (Fig. 5B). The
staining is weaker in PS7-12, but strong again in PS14. The inversion
juxtaposes the iab-2 region of probe B with the iab-9
region. It seems likely that a promoter in the iab-9 region [most
likely the `class C' promoter of Abd-B
(Zavortink and Sakonju, 1989
)]
is influenced by both bxd and iab-9 regulatory sequences.
Such a transcript would extend at least 40kb to reach the position of fragment
B.
|
Uab5 is associated with a translocation to the X
chromosome (Lewis, 1978) which
breaks in the bithorax complex at a site nearly coincident with the HC184B
insertion site (Fig. 5A) (B.
Weiffenbach and W. B., unpublished). Probe B detects RNA in
Uab5 heterozygous embryos after stage 13 in the CNS and
lateral epidermal cells in all segments
(Fig. 5C). The posterior
transformation in Uab5 is limited to the first abdominal
segment (PS6). It is possible that ectopic expression of ABD-A in PS5 and in
more anterior segments has little effect because of high levels of the
Antennapedia protein in those segments.
ABD-A expression in UabHH1
In wild-type flies, the identities of the second through fourth abdominal
segments (PS7-9) are specified by the abd-A gene product. A posterior
transformation of the first abdominal segment (PS6) in Uab flies is
expected to be due to anterior misexpression of ABD-A. We found such ectopic
ABD-A in UabHH1 embryos, but it was surprisingly subtle
and late in onset. UabHH1 homozygotes looked like wild
type in ABD-A patterns prior to stage 13 (germ band retraction). About half of
the older homozygous embryos showed one or a few cells of the central nervous
system in PS6 expressing ABD-A, although there was no apparent misexpression
in the epidermis (Fig. 6).
Fig. 6 also shows reduced ABD-A
expression in the epidermis of PS7. This may be due to antisense effects of
the UabHH1 transcripts (see below). Embryos carrying the
Uab breakpoint alleles have near normal patterns of ABD-A expression
(Karch et al., 1990).
|
We also examined third instar larvae homozygous for the UabHH1 mutation, looking for evidence of segmental transformations. The denticle bands on the first abdominal segment of such larvae usually showed a subtle transformation towards the character of the wild-type second abdominal segment (Fig. 7). These larvae also showed a subtle transformation of the second abdominal denticle belt towards the first (Fig. 7). Again, this may be due to antisense effects (see below). In any case, the transformation of the larval epidermis in the first abdominal segment appeared much less severe than that of the adult (Fig. 2). It seems most likely that ABD-A misexpression in PS6 is most pronounced in the pupa, when the adult structures are forming.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 8 illustrates a simple model for the transcription effect. The iab-2 regulatory region is normally repressed in PS6 and in more anterior parasegments, and is active in PS7 and more posterior parasegments. Transcription across the iab-2 region in the cells of PS6 switches the region from repressed to active, so that the developmental program for the second abdominal segment (PS7) is expressed in the first abdominal segment (PS6). The strongest Uab mutation, UabHH1, drives transcription farther, through the iab-3 regulatory region, so that it, too, becomes active in PS6. The ectopic transcripts are also present in PS7 through PS12, and so the iab-3 region is also active in PS7. Thus in UabHH1 animals, both the first and the second abdominal segments (PS6 and PS7) assume the character of the third (PS8). In PS8-PS12, the transcripts have no apparent effect, because the transcribed regions are already active, according to the normal functioning of the BX-C.
|
Transcription might change the chromosome in several ways. The RNA
polymerase II complex involved in elongation includes a histone
acetyltransferase (Wittschieben et al.,
1999) that could modify nucleosomes across the transcribed region.
The act of transcription might remove bound complexes [such as the Polycomb
complex (Shao et al., 1999
)]
or prevent their spread along the chromosome. This mechanism was suggested by
Sandell et al., who showed that transcription of yeast telomeres relieved the
telomere position effect (Sandell et al.,
1994
). Transcription might also allow transient access to DNA
sequences near the RNA polymerase which might otherwise be covered with
nucleosomes or packaged in a `higher order' structure. A further possibility
is that the ectopic RNA product has a function in activation.
It is not clear what site or function is affected by the ectopic
transcripts. The boundary between the bxd and iab-2
regulatory regions most likely lies just distal to the
UabHH1 insertion site
(Bender and Hudson, 2000) (M.
McLaughlin and W. B., unpublished); perhaps the ectopic transcription disrupts
this boundary. Alternatively, the iab-2 region includes at least one
PRE (Chiang et al., 1995
;
Shimell et al., 2000
); perhaps
transcription across this site relieves the repression imposed by the Polycomb
Group. Unfortunately, there is no clear indication from the available BX-C
mutations what phenotype to expect from the loss of a PRE.
The ectopic RNAs appear not to affect the segmental regulation of the complex early in embryonic development, although the transcripts are abundant in PS6 from stage 10 (elongated germ band) onwards. Misexpression of ABD-A in PS6, which is presumably necessary for the observed segmental transformations, is not seen in embryos except in occasional cells in the central nervous system. Perhaps there is a critical time later in development when ectopic RNA matters, such as the time of abdominal histoblast proliferation in the pupa. Alternatively, continuous transcription might activate abd-A stochastically, so that over time the majority of PS6 cells switch to the active state.
The RNA transcripts from UabHH1 are antisense to the
normal transcripts of the abd-A gene in PS7-12, and one might expect
abd-A expression to be blocked. Indeed, the level of ABD-A protein in
UabHH1 embryos is reduced in the PS7 epidermis
(Fig. 6) relative to wild type
(Karch et al., 1990). ABD-A
expression appears normal in the developing central nervous system, and in the
epidermis of PS8-PS12, presumably because the UabHH1
transcripts in older embryos are primarily in the epidermis of PS6 and PS7
(Fig. 3B). In
UabHH1 larvae, there is also evidence of loss of
abd-A function in PS7; the second abdominal setal belt is weakly
transformed towards the first (Fig.
7). The UabHH1 adults don't show anterior
transformation (loss of abd-A function) in PS7
(Fig. 2), but any such effect
would be masked by the strong posterior transformation (gain of abd-A
function).
Other readthrough mutations
The discovery of ectopic transcripts in a variety of Uab alleles
prompts a reconsideration of other mutant classes. Mutations in the vestigial
locus have recently been reported
(Hodgetts and O'Keefe, 2001)
that are due to transcription from P elements, but these are recessive,
loss-of-function alleles. It is possible that loss-of-function P alleles in
the BX-C and elsewhere would be reverted by P cytotype; it has seldom been
checked. Other mobile element alleles could also do their damage with
readthrough transcripts.
It seems surprising that there are not more gain-of-function alleles in the
BX-C or elsewhere due to readthrough from P elements. However, most P element
transposons contain selectable marker genes downstream of the P promoter;
perhaps these sequences help to terminate transcripts initiated at the P
promoter. It is also likely that strong gain-of-function mutations would be
dominant lethals. There are a variety of gain-of-function mutations in the
BX-C associated with rearrangements, which could mediate their effects by
non-coding readthrough transcription from the juxtaposed DNA.
Contrabithorax alleles, like Cbx3 and
CbxTxt (Bender et al.,
1983a), are good candidates.
Role of transcripts in wild type
The dramatic effects of ectopic transcription hint at a function for
non-coding transcripts in the wild type BX-C. Non-coding transcripts have been
documented in the human ß-globin locus
(Ashe et al., 1997;
Plant et al., 2001
), and such
transcription has been correlated with changes in DNaseI sensitivity
(Gribnau et al., 2000
).
Several non-coding transcripts have been described in the BX-C, most notably
in the bxd and iab-3 regions
(Lipshitz et al., 1987
;
Cumberledge et al., 1990
).
These RNA products appear in blastoderm embryos, at or before the onset of
segment-specific expression of the homeotic proteins. Other early RNAs, not
associated with BX-C protein products, have been detected by RNA in situs in
early embryos (Sánchez-Herrero and
Akam, 1989
). The proximally directed RNA detected by probe I (see
above) represents one such transcript.
There is, so far, no evidence for a function of these RNAs. A deletion
(pbx1) that removes the promoter for the bxd RNA
has no effect on the embryonic expression pattern of UBX (W. B., unpublished),
although the UBX pattern in imaginal discs is changed
(White and Wilcox, 1985). The
latter effect of pbx1 may well be due to loss of imaginal
disc enhancers, but the bxd RNA could matter for the development of
the adult, just as our ectopic RNA does. A difference between embryos and
larvae has been reported (Poux et al.,
2001
) in their requirements for Polycomb Group repression. Perhaps
the later mode of Polycomb Group repression is sensitive to and regulated by
non-coding transcripts.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashe, H. L., Monks, J., Wijgerde, M., Fraser, P. and Proudfoot,
N. (1997). Intergenic transcription and transinduction of the
human ß-globin locus. Genes Dev.
11,2494
-2509.
Barges, S., Mihaly, J., Galoni, M., Hagstrom, K., Müller,
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.
Bender, W., Akam, M., Karch, F., Beachy, P. A., Peifer, M., Spierer, P., Lewis, E. B. and Hogness, D. S. (1983a). Molecular Genetics of the Bithorax Complex in Drosophila melanogaster.Science 221,23 -29.
Bender, W., Spierer, P. and Hogness, D. S. (1983b). Chromosomal walking and jumping to isolate DNA from the Rosy, Ace, and Bithorax loci in Drosophila melanogaster. J. Mol. Biol. 168,17 -33.[Medline]
Bender, W. and Hudson, A. (2000). P element
homing to the Drosophila bithorax complex.
Development 127,3981
-3992.
Busturia, A., Wightman, C. D. and Sakonju, S.
(1997). A silencer is required for maintenance of transcriptional
repression throughout Drosophila development.
Development 124,4343
-4350.
Chan, C. S., Rastelli, L. and Pirrotta, V. (1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J. 13,2553 -2564.[Abstract]
Chiang, A., O'Connor, M., Paro, R., Simon, J. and Bender, W.
(1995). Discrete Polycomb Binding Sites in each Parasegmental
Domain of the bithorax Complex. Development
121,1681
-1689.
Cumberledge, S., Zaratzian, A. and Sakonju, S. (1990). Characterization of two RNAs transcribed from the cis-regulatory region of the abd-A domain within the Drosophila bithorax complex. Proc. Natl. Acad. Sci. USA 87,3259 -3263.[Abstract]
Fitzgerald, D. P. and Bender, W. (2001).
Polycomb Group Repression Reduces DNA Accessibility. Mol. Cell.
Biol. 21,6585
-6597.
Fritsch, C., Brown, J. L., Kassis, J. A. and Müller, J.
(1999). The DNA-binding Polycomb group protein Pleiohomeotic
mediates silencing of a Drosophila homeotic gene.
Development 126,3905
-3913.
Gribnau, J., Diderich, K., Pruzina, S., Calzolari, R. and Fraser, P. (2000). Intergenic Transcription and Developmental Remodeling of Chromatin Subdomains in the Human ß-globin Locus. Mol. Cell 5,377 -386.[Medline]
Hagstrom, K., Muller, M. and Schedl, P. (1997).
A Polycomb and GAGA Dependent Silencer Adjoins the Fab-7 Boundary in the
Drosophila Bithorax Complex. Genetics
146,1365
-1380.
Hodgetts, R. B. and O'Keefe, S. L. (2001). The
mutant phenotype associated with P-element alleles of the
vestigial locus in Drosophila melanogaster may be caused by
a readthrough transcript initiated at the P-element promoter.
Genetics 157,1665
-1672.
Hogga, I. and Karch, F. (2002). Transcription
through the iab-7 cis-regulatory domain of the bithorax complex interferes
with Polycomb-mediated silencing. Development
129,4915
-4922.
Horard, B., Tatout, C., Poux, S. and Pirrotta, V.
(2000). Structure of a Polycomb Response Element and in vitro
binding of Polycomb Group Complexes containing GAGA factor. Mol.
Cell. Biol. 20,3187
-3197.
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]
Karch, F., Weiffenbach, B. and Bender, W. (1990). abdA expression in Drosophila embryos. Genes Dev. 4,1573 -1587.[Abstract]
Kellerman, K. A., Mattson, D. M. and Duncan, I. (1990). Mutations affecting the stability of the fushi tarazu protein of Drosophila. Genes Dev. 4,1936 -1950.[Abstract]
Laski, F. A., Rio, D. C. and Rubin, G. M. (1986). The tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell 44,7 -19.[Medline]
Lemaitre, B. and Coen, D. (1991). P regulatory products repress in vivo the P promoter activity in P-lacZ fusion genes. Proc. Nat. Acad. Sci. USA 88,4419 -4423.[Abstract]
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276:565 -570.[Medline]
Lewis, E. B. (1996). The Bithorax Complex: The First Fifty Years. In Les Prix Nobel 1995, pp. 235-260. Stockholm, Sweden: The Nobel Foundation.
Lipshitz, H. D., Peattie, D. A. and Hogness, D. S. (1987). Novel transcripts from the Ultrabithorax domain of the bithorax complex. Genes Dev. 1, 307-322.[Abstract]
Martin, C. H., Mayeda, C. A., Davis, C. A., Ericsson, C. L., Knafels, J. D., Mathog, D. R., Celniker, S. E., Lewis, E. B. and Palazzolo, M. J. (1995). Complete sequence of the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. USA 92,8398 -8402.[Abstract]
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., Misra, R. K., Hagstrom, K., Muller, M., Schedl, P., Sipos, L., Gausz, J., Gyurkovics, H. and Karch, F. (1998). Chromatin domain boundaries in the Bithorax complex. Cell Mol. Life Sci. 1, 60-70.
Misra, S. and Rio, D. C. (1990). Cytotype control of Drosophila P element transposition: the 66 kd protein is a repressor of transposase activity. Cell 62,269 -284.[Medline]
Mlodzik, M. and Hiromi, Y. (1992). Enhancer trap method in Drosophila: its application to neurobiology. Methods Neurosci. 9,397 -414.
Müller, J. and Bienz, M. (1992). Sharp anterior boundary of homeotic gene expression conferred by the fushi tarazu protein. EMBO J. 11,3653 -3661.[Abstract]
Poux, S., McCabe, D. and Pirrotta, V. (2001).
Recruitment of components of Polycomb Group chromatin complexes in
Drosophila. Development
128, 75-85.
Plant, K. E., Routledge, S. J. E. and Proudfoot, N. J.
(2001). Intergenic transcription in the human ß-globin gene
cluster. Mol. Cell. Biol.
21,6507
-6514.
Qian, S., Capovilla, M. and Pirrotta, V. (1993). Molecular mechanisms of pattern formation by the BRE enhancer of the Ubx gene. EMBO J. 12,3865 -3877.[Abstract]
Rank, G., Prestel, M. and Paro, R. (2002). Transcription through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigenetic switch. Mol. Cell. Biol. (in press).
Robertson, H. M., Preston, C. R., Phillis, R. W.,
Johnson-Schlitz, D. M., Benz, W. K. and Engels, W. R. (1988).
A stable genomic source of P element transposase in Drosophila
melanogaster. Genetics 118,461
-470.
Sánchez-Herrero, E. and Akam, M. (1989). Spatially ordered transcription of regulatory DNA in the bithorax complex of Drosophila. Development 107,321 -329.[Abstract]
Sandell, L. L., Gottschling, D. E. and Zakian, V. A.
(1994). Transcription of a yeast telomere alleviates telomere
position effect without affecting chromosome stability. Proc. Natl.
Acad. Sci. USA 91,12061
-12065.
Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C., Bender, W. and Kingston, R. E. (1999). Stabilization of Chromatin Structure by PRC1, a Polycomb Complex. Cell 98, 37-46.[Medline]
Shimell, M. J., Simon, J., Bender, W. and O'Connor, M. B. (1994). Enhancer point mutation results in a homeotic transformation in Drosophila. Science 264,968 -971.[Medline]
Shimell, M. J., Peterson, A. J., Burr, J., Simon, J. A. and O'Connor, M. B. (2000). Functional analysis of repressor binding sites in the iab-2 regulatory region of the abdominal-A homeotic gene. Dev. Biol. 218, 38-52.[CrossRef][Medline]
White, R. A. H. and Wilcox, M. (1985). Regulation of the distribution of Ultrabithorax proteins in Drosophila. Nature 318,563 -567.
Wittschieben, B. O., Otero, G., de Bizemont, T., Fellows, J., Erdjument-Bromage, H., Obba, R., Li, Y., Allis, C. D., Tempst, P. and Svejstrup, J. Q. (1999). A novel histone acetyltransfewrase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol. Cell 4,123 -128.[Medline]
Zavortink, M. and Sakonju, S. (1989). The morphogenetic and regulatory functions of the Drosophila Abdominal-B gene are encoded in overlapping RNAs transcribed from separate promoters. Genes Dev. 3,1969 -1981.[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.