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First published online 5 November 2008
doi: 10.1242/dev.025700
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Research Report |
1 NCCR, Frontiers in Genetics, University of Geneva, Department of Zoology and
Animal Biology, 30 Quai Ernest Ansermet, 1211 Geneva 11, Switzerland.
2 University of Geneva, Department of Zoology and Animal Biology, 30 Quai Ernest
Ansermet, 1211 Geneva 11, Switzerland.
3 Institute of Genetics, Biological Research Centre, PO Box 521, H-6701 Szeged,
Hungary.
* Author for correspondence (e-mail: francois.karch{at}unige.ch)
Accepted 9 October 2008
SUMMARY
Although the boundary elements of the Drosophila Bithorax complex (BX-C) have properties similar to chromatin insulators, genetic substitution experiments have demonstrated that these elements do more than simply insulate adjacent cis-regulatory domains. Many BX-C boundaries lie between enhancers and their target promoter, and must modulate their activity to allow distal enhancers to communicate with their target promoter. Given this complex function, it is surprising that the numerous BX-C boundaries share little sequence identity. To determine the extent of the similarity between these elements, we tested whether different BX-C boundary elements can functionally substitute for one another. Using gene conversion, we exchanged the Fab-7 and Fab-8 boundaries within the BX-C. Although the Fab-8 boundary can only partially substitute for the Fab-7 boundary, we find that the Fab-7 boundary can almost completely replace the Fab-8 boundary. Our results suggest that although boundary elements are not completely interchangeable, there is a commonality to the mechanism by which boundaries function. This commonality allows different DNA-binding proteins to create functional boundaries.
Key words: Bithorax, Chromatin, Boundaries, Insulator
INTRODUCTION
The large cis-regulatory region of the BX-C is divided into nine
parasegment-specific chromatin domains that control the expression of the
three BX-C homeotic genes along the anteroposterior (AP) axis (Ubx,
abd-A and Abd-B) (for reviews, see
Duncan, 1987
;
Maeda and Karch, 2006). The
precise parasegment-specific expression pattern of these genes determines the
segmental identity of each of the segments of the posterior two-thirds of the
fly. Each domain is kept separate and autonomous by specialized elements known
as domain boundaries (Barges et al.,
2000; Gyurkovics et al.,
1990; Karch et al.,
1994; Mihaly et al.,
1997). In transgenic constructs, these boundary elements behave as
insulators, blocking enhancer activity when placed between the enhancer and
its target promoter (Barges et al.,
2000; Gruzdeva et al.,
2005; Hagstrom et al.,
1996; Zhou et al.,
1996). However, within their native context, they are often found
between an enhancer and its target promoter. How BX-C enhancers bypass
intervening boundaries is still a topic of contention.
Boundary deletions indicate that these elements are required to provide
functional autonomy to the enhancers and silencers within the large
cis-regulatory region. The Fab-7 boundary element, for example, is
normally found separating the iab-6 and iab-7 cis-regulatory
domains (see Fig. 1A). The
iab-6 enhancer region controls the level of Abd-B expression
in parasegment 11 (PS11) and determines the identity of segment A6. The
iab-7 region, however, controls the level of Abd-B
expression in PS12 and determines the identity of segment A7
(Celniker et al., 1990;
Galloni et al., 1993;
Mihaly et al., 2006;
Sanchez-Herrero, 1991). When
Fab-7 is deleted, the iab-6 and iab-7 domains
become fused into a single domain, allowing both the iab-6 and
iab-7 enhancers or silencers to become active in PS11 and PS12. In
most cells in PS11, the iab-7 enhancers are activated by
iab-6 initiation elements, resulting in a homeotic transformation of
PS11/A6 into PS12/A7. However, in other cells of PS11, the iab-6
initiators fail to activate the fused domain before iab-7 Polycomb
Response Elements (PRE) silence the domain, causing these cells to take on a
PS10/A5 identity (Galloni et al.,
1993; Gyurkovics et al.,
1990; Mihaly et al.,
1997).
Previously, we have shown that insulators such as gypsy
(Geyer and Corces, 1992) or
scs (Kellum and Schedl,
1992) cannot substitute for Fab-7 within the
BX-C (Hogga et al.,
2001). Both of these insulators block interactions between the
distal Abd-B enhancers and the Abd-B promoter. To test
whether the boundaries of the BX-C can functionally replace each
other, we used gene conversion to exchange the Fab-7 and
Fab-8 boundaries within the BX-C. Although these two
boundaries perform similar functions, they share little sequence identity.
Surprisingly, we find that the Fab-7 boundary is almost completely
capable of replacing the Fab-8 boundary, indicating that there is a
similarity in the mechanism of boundary function that cannot be predicted
through modern sequence analysis.
MATERIALS AND METHODS
Fab-7 replacement by Fab-8
The Fab-8 boundary element is an AluI-MscI 659
bp fragment (3R:12745503-12744844) cloned into an NsiI site of a
P-CaSpER-based plasmid containing the genomic region surrounding
Fab72 (Hogga and
Karch, 1995). This construct was injected into
white1118 flies. Third chromosome inserts were recombined
with the bluetail insertion (Galloni et
al., 1993). Convertants were obtained and verified as described
previously (Hogga and Karch,
2001).
Fab-8 replacement by Fab-7
The genomic region surrounding the Fab-8305deletion
(3R:12745801-12744797) was generated by PCR using Pfu polymerase (Promega) and
the following primers: 5'-TCTAGAGCTCCACTTGCTCGGGGG-3' and
5'-CTCGAGTTCGGATTTCTGCTTTCTGAGC-3' for the proximal region, and
5'-TCTAGACATAAAGGGAAGCGGAGGC-3' and
5'-CTCGAGGTTCTTCATTATTGTGCCTTC-3' for the distal region. The
Fab-7 boundary (a 0.8 kb fragment) was generated by PCR using
5'-CTCGAGGCAGCAAAAATCGTAAAAAAG-3' and
5'-CTCGAGGCAGAAACAAAGGCCGACG-3', and was inserted between the two
break points of the Fab-8305 deletion. Transgenic flies
were made as above and recombined onto a chromosome carrying the
fs(3)5649 P-element insertion. In trans to this chromosome, we placed
the Df(3R)R59 chromosome carrying the 
2-3 transposase and a
Tp(3;1)bxd111 duplication to rescue the sterility of these dysgenic
males. Putative conversion events were screened as above.
|
), using
mouse monoclonal antibodies against Abd-B obtained from the Developmental
Studies Hybridoma Bank, developed under the auspices of the NICHD and
maintained by The University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242, USA.
Preparation of abdominal cuticles
Abdominal cuticles were prepared as described previously
(Mihaly et al., 1997).
RESULTS AND DISCUSSION
The Fab-7 boundary can substitute for the Fab-8 boundary
Three reasons dictated our choice in converting Fab-8 to
Fab-7 (F8
F7). First, Fab-7 and Fab-8 perform
similar functions yet share almost no sequence similarity. Second, we wanted
to test whether a BX-C cis-regulatory domain could interact with the
Abd-B promoter over a boundary element that it generally never
encounters. As Fab-7 is located on the promoter distal side of
iab-7, iab-7 enhancers are never faced with the challenge of
bypassing the Fab-7 boundary (see
Fig. 1A). And third, recent
data have suggested that BX-C boundaries are regulated along the AP
axis (Cléard et al.,
2006). From these data, it seems that boundaries interact with the
Abd-B promoter until the neighboring (probably more posterior) domain
becomes active (see also Maeda and Karch,
2007). If this regulated association is responsible for boundary
function and the association is controlled by the boundary element itself,
then a substitution of Fab-8 by Fab-7 should result in the
inactivation of the boundary one parasegment too anterior. The expected
phenotype resulting from such an event would be a homeotic transformation of
A7 to A8 (much like a boundary deletion).
In the gene conversion, the Fab-8 region was removed and replaced
by a minimal Fab-7 boundary element
(Chen et al., 2005), inserted,
in separate constructs, in each orientations. In order to completely remove
the Fab-8 boundary without removing potentially important
iab-7 or iab-8 sequences, we deleted the region around
Fab-8 that is removed in the Fab-8305 deletion.
The Fab-8305 deletion is the smallest characterized
Fab-8 deletion that displays a complete Fab-8 phenotype;
homozygous adult females are sterile and the A7 segment disappears due to an
A7 to A8 transformation (Fig.
2B). Both the iab-8PRE
(Barges et al., 2000) and most
of the promoter targeting sequence 7 (PTS7) element
(Zhou and Levine, 1999) are
left intact in the Fab-8305 deletion. As convertants for
both orientations display identical phenotypes, we will simply call them
F8F7.
Given the simple nature of the experiment, we expected one of three
outcomes: that the Fab-7 boundary would act as a simple insulator and
block iab-7 from interacting with the Abd-B promoter (like
an iab-7 deletion); that Fab-7 would not be functional in
replacing Fab-8 and behave as an Fab-8 deletion mutation; or
that Fab-7 would substitute for Fab-8. Scoring females
homozygous for either F8F7 conversion showed that Fab-7 can
almost completely substitute for Fab-8. Almost all F8F7 flies
are wild-type appearance and are fertile
(Fig. 2). In rare cases, we do
observe homozygous flies displaying evidence of slight Abd-B
misexpression. Patches of cells in A7 occasionally take on an A6 or A8
identity. To characterize this phenotype more carefully, we looked at
F8F7 hemizygous flies. F8F7/Df(3R)P9 flies have features
reminiscent of Fab-8 homozygotes
(Fig. 3), indicating that
although Fab-8 can mostly substitute for Fab-7, the boundary
system in F8F7 flies is less robust, occasionally allowing the iab-7
domain to be influenced by neighboring cis-regulatory domains. However, in a
non-sensitized background, this effect is quite mild, affecting <5% of the
flies scored.
Abd-B antibody staining confirms our results. In the embryonic CNS of
wild-type flies, Abd-B is expressed in a step gradient pattern that
noticeably increases parasegmentally from PS10 to PS13
(Fig. 2E). In Fab-8
deletion mutants, that pattern changes such that PS12 expression levels mimic
those found in PS13. Meanwhile in iab-7 mutants, PS12 expression
drops to the level of PS11. In F8F7 conversion lines, we observe a
staining pattern that is similar to that found in wild-type embryos.
This result was quite surprising. The fact that Fab-7 can
substitute for Fab-8 means that everything required to restore
Fab-8 function is present in the Fab-7 fragment inserted.
However, at the DNA sequence level, the Fab-7 and Fab-8
boundaries share almost no similarity. A detailed analysis of the two
sequences using dot-plot and Markov analysis found little in common between
the two elements other than GAGA factor-binding sites (six in Fab-7
and two in Fab-8). The GAGA factor binding sites have previously been
shown to be important for Fab-7 enhancer blocking activity in
transgenic contexts (Schweinsberg et al.,
2004). However, the role of the GAGA factor in Fab-8
enhancer blocking activity is still unknown. Thus far, the only factor shown
to be important for Fab-8 function is the dCTCF factor. Previously,
it has been shown that deleting the dCTCF-binding sites in Fab-8
impaired its insulator function in transgenic insulator assays
(Moon et al., 2005). Moreover,
dCTCF mutants display phenotypes reminiscent of Fab-8 mutants
(Mohan et al., 2007). As
Fab-7 was shown to be one of the few BX-C boundaries to which dCTCF
does not bind (Holohan et al.,
2007), our results show that dCTCF is not absolutely required for
Fab-8-like function.
|
|
). Given that each of the BX-C boundaries has been isolated as
a DNase hypersensitive site (Karch et al.,
1994; Barges et al.,
2000) and as a site of intense histone H3.3 replacement
(Mito et al., 2007), what
could be important for boundary function is the chromatin structure of the
locus. It is easy to imagine that different combinations of proteins might be
able to induce a similar chromatin structure, dCTCF and GAGA being two of
them.
The Fab-8 boundary cannot fully substitute for the Fab-7 boundary
In the F8F7 flies, the iab-7 enhancers are able to bypass
Fab-7 even if, in the wild-type situation, they are never faced with
the challenge of bypassing it. Because Fab-7 could substitute for
Fab-8, we wondered whether all BX-C boundaries are capable
of substituting for each other. We, therefore, decided to replace the
Fab-7 boundary with the Fab-8 boundary.
For this gene conversion, we replaced the Fab-7 boundary with a
minimal Fab-8 boundary element (in both orientations)
(Fig. 1C). To do this, we
removed a Fab-7 fragment identical to that deleted in the
Fab-72 deletion. The Fab-72 deletion
is the smallest characterized deletion that completely removes Fab-7
boundary function; Fab-72 homozygous adult flies primarily
show an A6 towards A7 transformation (Fig.
4) (Galloni et al.,
1993; Mihaly et al.,
1997). Previous genetic and molecular analysis indicates that the
nearby iab-7PRE (Mihaly et al.,
1997) and PTS6 element (Chen et
al., 2005) are left intact in the Fab-72
deletion. Again, we isolated conversants for each Fab-8 orientation.
Although the two conversions differ slightly in their intensity, for the most
part, they display similar phenotypes. Therefore, we will simply call the
mutants F7F8, indicating, when necessary, where the two orientations
differ.
Although Fab-8 can restore the autonomy of the iab-7
domain (freeing it from ectopic activation by iab-6), surprisingly, F7F8
homozygous flies show a transformation of A6 towards A5. This means that there
is a loss of Abd-B activation by iab-6
(Fig. 4). Abd-B antibody
staining confirms these results (Fig.
4E-G). Instead of the normal stepwise gradient seen in the
wild-type embryonic CNS, F7F8 embryos display PS10-like Abd-B expression
in PS11 (Fig. 4G). This
phenotype is reminiscent of the phenotype obtained by substituting a minimal
scs insulator for Fab-7
(Hogga et al., 2001). For that
substitution, it was believed that the loss of iab-6 function was due
to the blocking of iab-6 by the intervening insulator. A second
possible explanation for this phenotype is that iab-6 is somehow
being silenced in the F7F8 substitution by the nearby iab-7PRE.
This hypothesis is presented because Fab-7 functions, not only to
prevent the inappropriate activation of adjacent cis-regulatory domains, but
also to prevent the inappropriate silencing of adjacent domains. In
Fab-72 mutants, for example, one sees a clonal mixture of
both ectopic activation and ectopic silencing
(Mihaly et al., 1997). The
balance between these two clonal populations is sensitive to mutations in
Polycomb group genes. We, therefore, crossed F7F8 flies to the
Polycomb-group mutant, Pcl. Because the phenotype of F7F8 flies
does not change upon the introduction of a Pcl/+ mutation (data not
shown), we believe that Fab-8 is acting like a short-range insulator
at this locus, blocking iab-6 enhancers from interacting with the
Abd-B promoter.
As mentioned above, there is a slight orientation effect with the
F7F8 substitution. Lines with Fab-8 placed in the wild-type
orientation (F7F8+) (relative to the Abd-B gene)
display a slightly less-severe transformation than lines with Fab-8
placed in the opposite orientation (F7F8-). The difference in
phenotype can be seen by looking at the trichome pattern in the transformed A6
segment. In F7F8- flies, trichomes cover most of the
transformed segment (A5-like), whereas in F7F8+ flies,
trichomes primarily cover the ventral-anterior region of the transformed
segment (more A6-like) (see Fig. S1 in the supplementary material). In all
other assays, the two transformants behave identically
(Fig. 4).
|
F7 conversion, we found that the iab-7
cis-regulatory domain was capable of bypassing a boundary element that it
never has to bypass but in the case of the F7F8 conversion, we found
that iab-6 is partially blocked by a boundary element that it must
normally bypass (Fab-8 is located between iab-6 and the
Abd-B promoter). One possible explanation is discrepancy is that the
Fab-8 fragment inserted lacked a specific element required for
insulator bypass. Although this is a possibility, we do not believe this to be
the case. Both the Fab-7 and Fab-8 regions have been
extensively scanned for elements allowing insulator bypass. In these attempts,
elements called promoter-targeting sequences (PTSs) have been identified that
allow enhancers to bypass insulator elements on reporter transgenes
(Chen et al., 2005;
Zhou and Levine, 1999). In our
experiments, we replaced the smallest characterized boundary deletions with
the smallest characterized insulator fragments. In both cases, molecular data
suggest that the fragments we introduced were separated from any PTS-type
activity, but were capable, in transgenic contexts, of being bypassed by known
PTS elements. Conversely, the deletions we created were chosen to be clean
boundary deletions; as much as possible, all known nearby elements, including
PTS elements, were left intact. In the F7F8 substitution, for example,
the entire PTS-6 element that was capable of bypassing the identical
Fab-8 insulator fragment is still present.
Therefore, if no PTS-type elements were deleted, the main difference
between the cases tested is context. For example, in the wild-type situation,
Fab-8 is located between the iab-7 and iab-8
cis-regulatory domains, whereas in F7F8, Fab-8 is placed
between the iab-6 and iab-7 cis-regulatory domains. We have
recently found that the Fab-7 boundary seems to be regulated along AP
axis (Cléard et al.,
2006). If we assume that all boundaries behave in a similar
manner, then Fab-8 would also be regulated along the AP axis. As this
regulation does not seem to come from the boundary element itself (see above),
it must come through specific interactions with the nearby cis-regulatory
domains. Previous work has pointed to PTS elements as the mediators of this
function. However, based on our data and because PTS deletions have little
phenotype when deleted, we believe that there must be something more that
inactivates boundary elements (Mihaly et
al., 2006; Zhou and Levine,
1999). For now, the identity of these elements remains a
mystery.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/3983/DC1
ACKNOWLEDGMENTS
We thank Annick Mutero and Jean-Michel Gibert for critically reading this manuscript, and Eva Favre and Jorge Faustino for excellent technical assistance. C.I., F.C., R.K.M. and F.K. were supported by grants from the State of Geneva, the Swiss National Foundation and the Swiss National Center of Competence in Research. H.G. is supported by grants from OTKA and by the NIH as a subcontractor.
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