DSP1, an HMG-like Protein, Is Involved in the Regulation of Homeotic Genes

Corresponding author: M. Decoville, CBM, CNRS, rue Charles Sadron, 45071 Orléans cedex 2, France., decovil{at}cnrs-orleans.fr (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Drosophila dsp1 gene, which encodes an HMG-like protein, was originally identified in a screen for corepressors of Dorsal. Here we report that loss of dsp1 function causes homeotic transformations resembling those associated with loss of function in the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Abdominal-B. The expression pattern of Scr is altered in dsp1 mutant imaginal discs, indicating that dsp1 is required for normal expression of this gene. Genetic interaction studies reveal that a null allele of dsp1 enhances trithorax-group gene (trx-G) mutations and partially suppresses Polycomb-group gene (Pc-G) mutations. On the contrary, overexpression of dsp1 induces an enhancement of the transformation of wings into halteres and of the extra sex comb phenotype of Pc. In addition, dsp1 male mutants exhibit a mild transformation of A4 into A5. Comparison of the chromatin structure at the Mcp locus in wild-type and dsp1 mutant embryos reveals that the 300-bp DNase I hypersensitive region is absent in a dsp1 mutant context. We propose that DSP1 protein is a chromatin remodeling factor, acting as a trx-G or a Pc-G protein depending on the considered function.

THE family of HMG-box proteins, originally defined by the presence of a common DNA-binding domain called the HMG box, includes diverse regulatory proteins (BIANCHI et al. 1992 ). The HMG box is a highly conserved basic motif, 70–80 amino acids in length, that adopts an L-shaped three-dimensional structure and is responsible for DNA-binding activity (READ et al. 1995 ). HMG-box proteins preferentially bind to curved microcircles or distorted DNA structures such as four-way junctions, cisplatin-modified DNA, and S-S tethered DNA. The HMG-box proteins are divided in two classes according to the sequence conservation and the number of their HMG boxes (GROSSCHEDL et al. 1994 ). Proteins belonging to the first class are generally transcription factors that bind to specific DNA sequences. They contain only one HMG box and are expressed in restricted cell types. They are exemplified by the human sex-determining factor SRY (SINCLAIR et al. 1990 ), the lymphoid enhancer binding factor Lef1 (TRAVIS et al. 1991 ), or the T-cell factor Tcf-1 (WATERMAN et al. 1991 ). The second class includes a larger number of nuclear proteins that contain two or more tandem HMG boxes and bind to DNA in a relatively sequence-aspecific manner. The archetype of this class are the mammalian HMG 1/2 proteins. In vitro studies have shown that these proteins are able to remodel chromatin and participate in DNA replication, nucleosome assembly, and transcription (BONNE et al. 1982 ; BONNE-ANDREA et al. 1984 ; TREMETHICK and MOLLOY 1988 ; SINGH and DIXON 1990 ). Recently, CALOGERO et al. 1999 have established that HMG1 is not essential for packaging DNA into chromosomes, or for embryonic and fetal development in mouse. Nevertheless, HMG1 is required for specific gene regulatory processes after birth. Despite intensive studies, the biological functions of these proteins still remain elusive.

LEHMING et al. 1994 isolated a new HMG1-like protein from Drosophila as a corepressor of Dorsal protein that was named DSP1 (Dorsal switch protein). This protein contains two HMG boxes, a small acidic tail, and two N-terminal glutamine-rich regions. DSP1 is expressed throughout embryogenesis, ubiquitously during the first stages (cellular blastoderm and germ band extension) and then exclusively in the central nervous system during the last stages (stages 15–16). In adults, the protein is detected only in ovaries and in brain (MOSRIN-HUAMAN et al. 1998 ). LEHMING et al. 1998 have proposed that DSP1 could be part of a repressing chromatin complex containing SP100 and HP1, a component of Drosophila heterochromatin involved in position effect variegation (PEV; EISSENBERG et al. 1990 , EISSENBERG et al. 1992 ). PEV occurs when a euchromatic gene is transposed adjacent to a segment of heterochromatin. Expression of the transposed gene is repressed in some cells and not in others, producing a mosaic phenotype. Many mutations that enhance or suppress PEV have been isolated (LOCKE et al. 1988 ; SINCLAIR et al. 1989 , SINCLAIR et al. 1992 ; WUSTMANN et al. 1989 ; DORN et al. 1993 ), and most of them identified genes that encode nonhistone chromatin proteins. Some of these proteins share a region of sequence similarity with other chromatin regulators such as Pc-G or trx-G proteins that control the expression of homeotic genes. For example, Pc shares a region of sequence similarity with Su(var)205, which encodes HP1 (EISSENBERG et al. 1990 ; PARO and HOGNESS 1991 ), and Su(var)309 shares a domain with Enhancer of zeste [E(z)], a Pc-G gene, and with trx (SET domain; TSIERSCH et al. 1994 ). Pc-G and trx-G genes may act by modifying chromatin structure. Several groups have found that some Pc-G or trx-G genes act as suppressors or enhancers of PEV. Examples are Enhancer of Polycomb [E(Pc); SINCLAIR et al. 1998a], cramped (YAMAMOTO et al. 1997 ), Asx (SINCLAIR et al. 1998B ), and Trl (FARKAS et al. 1994 ). These results have prompted us to investigate a possible function of dsp1 in homeotic gene regulation.

Homeotic genes encode transcriptional factors that specify the identities of body segments in Drosophila. They are clustered in two complexes, the Antennapedia and Bithorax complexes (ANT-C and BX-C; DUNCAN 1987 ; KAUFMAN et al. 1990 ). In early embryos, the initial boundaries of homeotic gene transcription are controlled by segmentation genes. Later in development, the pattern of homeotic gene transcription is maintained by two groups of regulatory proteins, the Polycomb-group of repressors (Pc-G) and the trithorax-group of activators (trx-G; reviewed in KENNISON 1995 ; SIMON 1995 ; PIROTTA 1997 ). Mutations in Pc-G genes cause homeotic transformations due to the ectopic expression of ANT-C and BX-C genes, resembling gain-of-function mutations of the BX-C and ANT-C. In contrast, mutations in trx-G genes cause homeotic transformations similar to loss-of-function mutations in BX-C and ANT-C, due to the failure to maintain expression of homeotic genes. It was proposed that Pc proteins package inactive homeotic genes into inaccessible complexes in the early embryo, therefore preventing their expression. Biochemical studies have demonstrated physical interactions between different members of Pc-G proteins. Genetic studies have suggested that transcription of homeotic genes is regulated by interaction between trx-G proteins. Three complexes containing trithorax group proteins have been identified (PAPOULAS et al. 1998 ). One of them, the BRM complex, is composed of at least seven major polypeptides, four of which are related to subunits of the yeast chromatin remodeling complexes SWI/SNF (DINGWALL et al. 1995 ) and RSC (CAIRNS et al. 1996 ).

Here, we report the phenotype of a loss-of-function mutant of dsp1 (named dsp1¹). We show that lack of dsp1 product causes homeotic transformations. Results of genetic interactions with BX-C and ANT-C mutants suggest that DSP1 is involved in the regulation of several homeotic genes. dsp1¹ mutation suppresses the homeotic transformations observed in Pc heterozygotes and on the contrary enhances the trx-G mutant phenotype. Overexpression of dsp1 results in enhancement of the Pc phenotype. Finally, analysis of the chromatin structure at the Mcp locus suggests that DSP1 could act as a chromatin remodeling factor. These results support the idea that dsp1 could function as an activator or repressor, depending on the considered function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In situ hybridization:
Scr expression was monitored by whole mount embryo in situ hybridization using digoxygenin-labeled riboprobes. Probes were prepared according to the manufacturer's directions (Boehringer Mannheim Biochemicals, Mannheim, Germany). Prehybridization and hybridization conditions were based on the protocol described by TAUTZ and PFEIFLE 1989 and conditions for embryos and imaginal discs were based on MASUCCI et al. 1990 . The Scr riboprobe was generated from pGEM3Zf(+) containing a 1011-bp DNA fragment [nucleotides (nt) 1833–2844] obtained by PCR.

Drosophila strains and crosses:
Flies were raised on standard medium at 22°. All mutations and chromosome aberrations are described in LINDSLEY and ZIMM 1992 unless otherwise noted. Isolation of the dsp1 null mutant was described previously (MOSRIN-HUAMAN et al. 1998 ). Ubx^bx-83kb/TM1, Pc¹¹/TM3, Ubx^bx34-e/TM1, Antp^D43/TM, and Df(3R)P9/Dp(3;3)P5 were obtained from the Umea Drosophila Stock Center. Df(1)19, f¹/C(1)RM, y¹ shi¹ f¹; Dp(1;Y)shi⁺³, y⁺, Scr⁴/TM3, Scr^s/TM3, trx¹/TM1, trx^E2/TM6, trx^E2brm²/TM6, ash2¹/TM6, and w[*]; were obtained from the Bloomington Fly Stock Center. ash2^x2/TM3, ash1^vv183/TM3, and w¹¹⁸ strains were kindly provided by A. Shearn and B. Limbourg-Bouchon, respectively. Oregon-R was used as wild-type reference strain.

Overexpression of dsp1:
A dsp1 transgenic strain was obtained by cloning a fragment of 1.3 kb spanning the whole dsp1 open reading frame and obtained by reverse transcriptase (RT)-PCR into pUAST vector (generous gift from B. Limbourg-Bouchon). P-element-mediated germ-line transformation was done using standard procedures (SPRADLING and RUBIN 1982A , SPRADLING and RUBIN 1982B ). The mini-white transformation marker in the pUAST transformation vector was designed to allow detection of transformants by the rescue of the white mutation in the recipient strain (w¹¹⁸). The transgenic strain was controlled by PCR and Southern assays. Chromosomal linkage of construct was determined by segregation with respect to the balancer chromosomes CyO and TM3. A homozygous transgenic strain was established and maintained at 22°.

Virgin homozygous dsp1 transgenic flies were mated to w[*]; males. w[*];P{w[+mC] = Gal4-HSP70.PB}2 virgin females were recovered and submitted to heat-shock treatment (3 heat shocks at 36° for 20 min, with equivalent recovery times at room temperature). Then, the females were crossed with Pc¹¹/TM3 males at 22°, and the progeny were recovered at different times after laying (0/24 hr, 24/40 hr, 40/48 hr, 48/72 hr, and >72 hr after heat shock). The same results were obtained for 0/24 hr and 24/40 hr.

Chromatin studies:
Nuclei were prepared from 0–12-hr mass-collected embryos as described (JOWETT 1986 ). The nuclei were incubated for 3 min at 25° with different concentrations of DNase I. The DNA was then purified by proteinase K treatment and phenol extraction and digested with EcoRI. After electrophoresis on an agarose gel, and blotting to nitrocellulose, the DNA was hybridized with a Mcp probe (PCR product corresponding to nt 8–2475).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

dsp1 mutant strain:
We have obtained by P mutagenesis a loss-of-function allele of dsp1. Molecular analysis has revealed a deletion of the dsp1 open reading frame that does not affect another transcription unit. This mutant does not produce detectable RNA or protein (MOSRIN-HUAMAN et al. 1998 ). We have named this allele dsp1¹ and we use this nomenclature hereafter. dsp1¹ was isogenized with wild-type Oregon-R chromosomes by recombination around the dsp1 locus and was maintained as a homozygous strain at 22°. REYNOLDS and TANOUYE 1998 have proposed that dsp1 is allelic to bang senseless (bss). bss mutants become paralyzed for several minutes following a vibration of the culture vial. Surprisingly, we do not observe this phenotype in dsp1¹ adults. To resolve this discrepancy we performed a complementation experiment between bss¹ and dsp1¹. Heterozygotes for bss¹ are distinguishable from homozygotes or hemizygotes by the length of time they remain paralyzed. The phenotype of bss¹/dsp1¹ heterozygotes and +/bss¹ was the same, indicating that bss¹ and dsp1¹ do complement. We observed the same result with other alleles of the bss gene. We concluded from this experiment that bss and dsp1 are not alleles. To avoid confusion between the two genes, we propose that dsp1 be named only dsp1 and not dsp1/bss.

dsp1¹ homozygotes or hemizygotes died prematurely and exhibited very low fertility. The same phenotypes were observed in dsp1¹/Df(1)19 flies bearing a deletion including dsp1. Inactivation of dsp1 also led to a reduction of the size of the sex comb in males. This phenotype was suppressed in dsp1¹/shi⁺ Y males bearing on the Y chromosome a translocation of 13F to 14F X region. To confirm that the phenotypes observed were a result of a lack of dsp1 function rather than an effect of other loci, a phenotype rescue test was performed by introducing an extra copy of the wild-type dsp1 gene into the dsp1¹ background. The wild-type copy of dsp1 rescued all phenotypes.

Lack of dsp1 function induces homeotic transformations:
Inspection of adults homozygous or hemizygous for the dsp1¹ allele has revealed various homeotic transformations. The first one corresponded to a T1 to T2 transformation. Adult males hemizygous for the dsp1¹ allele showed a reduced sex comb, with an average of 6 teeth instead of the 11 normally found in the wild type. The size of the sex comb in dsp1¹/Y males never exceeded 9 teeth and was always reduced whatever the mother (homozygous or heterozygous for dsp1¹), suggesting that this phenotype is the result of an absence of dsp1 function in the zygote. This phenotype mimics, to some extent, loss-of-function mutations in the homeotic gene Sex combs reduced (Scr). We have studied interactions of dsp1¹ with Scr⁴, a loss-of-function mutation (PATTATUCCI et al. 1991 ). dsp1¹/Y; Scr⁴/+ males showed an increase in the severity of the Scr phenotype; the size of the sex comb was greatly reduced and the average number of teeth was 4 in the double mutant vs. 6 in the Scr⁴ or dsp1¹ single mutants. We have also studied interactions of dsp1¹ with Scr^S, a gain-of-function mutation corresponding to a transposition that does not disrupt any of the identified Scr loci and is sensitive to mutation in Pc (PATTATUCCI et al. 1991 ). dsp1¹/Y; Scr^S/+ males exhibited a less severe phenotype than the Scr mutant alone (Table 1): the number of T3 legs with a sex comb was highly diminished (30 vs. 100%) and the size of the sex comb on the T2 legs was reduced (2.5 vs. 4.5 teeth). These results prompted us to study the expression of Scr in dsp1¹ embryos and imaginal discs of third instar larvae (Fig 1). No difference was observed in wild-type and dsp1¹ embryos (Fig 1A and Fig B; only stage 17 is shown). In contrast, Scr expression in dsp1¹ T1 imaginal discs was severely lowered (Fig 1C and Fig D). In particular, the strong expression of Scr in cells between the central knob and peripheral margin of the disc was not detected in dsp1¹ imaginal discs. These cells are the progenitors of the adult anterior tarsus and tibia, including the sex combs of adult males (BRYANT 1978 ). These data strongly suggest that dsp1 participates in activation of Scr in imaginal discs.

View larger version (43K): In this window In a new window Download PPT slide   Figure 1. Pattern of Scr expression in embryos and prothoracic imaginal discs. (A and C) Wild-type Oregon-R; (B and D) dsp11 mutant. (A and B) Ventral views of embryos at approximately stage 17 of development (the anterior is on the left). (C and D) Prothoracic (T1) imaginal discs.

The second homeotic transformation corresponded to a T3 to T2 transformation. Adults homozygous or hemizygous for the dsp1¹ allele showed partial homeotic transformations of metathoracic into mesothoracic structures, mainly in the anterior compartment. Generally, only one haltere was affected and the transformations included, to various extents, dorsal development of wing tissue in place of haltere or mesonotal tissue in place of metanotum. This phenotype resembles the one obtained for loss of function in the Ubx gene, especially with bx alleles. This led us to study the interactions of dsp1¹ with two bx alleles, bx^34e and bx^83kd. These two alleles correspond to insertions of transposable elements and show reductions of Ubx protein expression. In dsp1¹/Y; bx/+ males, the frequency of transformation of halteres into wings was enhanced by a factor >15 (Table 1). A similar result was obtained with each of the two bx alleles. The enhancement was dramatically reduced but not suppressed if females were heterozygous for dsp1¹, indicating that maternal DSP1 function is involved in a concentration-dependent manner.

The third homeotic transformation corresponded to an A6 into A5 transformation. About 25% of males hemizygous for the dsp1¹ allele showed bristles on the A6 sternite, some of them bearing more than six bristles (Fig 2). As this phenotype is reminiscent of mutations in the iab-6 regulatory region of AbdB, we studied the interaction between the dsp1¹ allele and Df(3R)P9, a deletion of BX-C. About 90% of dsp1¹/Y; Df(3R)P9/+ males exhibited bristles on the A6 sternite compared to 25% in dsp1¹ or Df(3R)P9 single mutants. This observation strongly suggests that DSP1 is involved in the regulation of the iab-6 locus. In addition to the A6 to A5 transformation, we observed in 50% of males patches of pigmentation on the A4 tergite, suggesting a partial transformation of A4 into A5 (Fig 2). This point is discussed later.

View larger version (81K): In this window In a new window Download PPT slide   Figure 2. Adult male phenotypes of hemizygous dsp11 mutation. Whole mounts of abdominal male cuticles of wild type (A) or dsp11 (B) are shown. Male abdomens were cut along the dorsal midline and flattened on a slide. The dorsal surface of each abdominal segment has a plate of hard cuticle called tergites. The ventral surface of abdominal segments is composed of pleura on the central midline of hard cuticle called sternites. In wild-type male (A), only the fifth and sixth tergites are pigmented. The sixth sternite is easily distinguished from the more anterior sternites by its different shape and by the absence of bristles. In dsp11 male (B), the fourth tergite shows patches of pigmentation, suggesting ectopic activation of iab-5 in A4 (white arrow). This activation probably does not take place in all cells. On the ventral side, bristles are found on the sixth sternite, indicating that iab-6 is inactive, at least in some cells, in segment A6 (black arrow). The number of bristles observed on the sixth sternite can vary from one to more than six.

To know whether dsp1 is involved in the expression of other homeotic genes, we looked at the interaction between dsp1¹ and Antp^D43, a gain-of-function mutation of Antennapedia (Table 1). Analysis of the dsp1¹ male progeny heterozygous for Antp^D43 revealed that the number of homeotic transformations of antennae into legs was strongly reduced (10 times). In addition, in the female progeny heterozygous for dsp1¹ and Antp^D43, the frequency of homeotic transformations was also reduced (2 times). On the contrary, when the Antp^D43 mutant was crossed with a Pc¹¹ mutant, 100% of the Antp^D43/Pc¹¹ progeny exhibited transformation of antennae into leg. This result suggests that dsp1 is also involved in expression of Antennapedia and acts in a concentration-dependent manner as it does for Ultrabithorax.

dsp1 genetically interacts with trithorax-group and Polycomb-group genes:
The results described above suggest that dsp1 is involved in the expression of several homeotic genes. Two groups of genes are known to control homeotic gene expression: the trx-G and the Pc-G genes. We studied genetic interactions between dsp1¹ and various mutations of trx-G or Pc-G genes (Table 2). Interaction with mutations in the trx gene was studied with two trx alleles: trx¹ and trx^E2. In both cases, we observed an increase in the number of transformations of halteres into wings. This enhancement was more pronounced with the trx^E2 allele (16%) than with the trx¹ allele (3%). This can be explained by the hypomorph nature of the trx¹ allele, which results from an insertion of 9 kb outside the coding sequences, and probably produces normal trx protein but at a reduced level (INGHAM and WHITTLE 1980 ; INGHAM 1985 ; BREEN and HARTE 1991 ). On the contrary, trx^E2 is an amorph allele (KENNISON and TAMKUN 1988 ). A strong enhancement in transformation frequency of halteres into wings (21%) was also observed in dsp1¹/Y; ash1^vv183/+ males. In contrast, interaction with mutations in other trx-G genes (ash2 and brm) did not affect significantly the rate of homeotic transformations. As with Ubx alleles, the enhancement of transformations was reduced when the mothers were heterozygous for dsp1¹.

Interaction with Pc showed a decrease of the extra sex comb phenotype of Pc (Table 2): the number of T2 legs with sex comb was reduced (only 37% of T2 legs showed a sex comb in dsp1¹/Y; Pc¹¹/+ flies vs. 54% in dsp1⁺/Y; Pc¹¹/+). The number of T3 legs with a sex comb was also lower, 19% in dsp1¹/Y; Pc¹¹/+ flies vs. 30% in dsp1⁺/Y; Pc¹¹/+. It is worth noting that the size of the sex comb on T1 legs in the double mutant dsp1¹/Y; Pc¹¹/+ was almost normal, as expected for two genes acting in an opposite manner in the same pathway.

Overexpression of dsp1 enhances a Polycomb mutation:
As loss of dsp1 function led to a reduced expression of Ubx and Scr, we expected a perturbation of homeotic gene expression by an overexpression of dsp1 and a subsequent ectopic expression of Ubx and Scr. To test it we used the Gal4/UAS system of induction to overexpress dsp1. As a driver we used Gal4-HSP, which is expressed after heat-shock treatments. Flies carrying the Gal4-HSP driver were crossed with those carrying the UAS-dsp1 construct. Virgin females were recovered, submitted to heat shocks, and crossed with Pc¹¹/TM3 males as described in MATERIALS AND METHODS. The Pc offspring were analyzed for transformations of wings into halteres and for the extent of the extra sex comb phenotype of Pc in males. In control experiments, a majority of Pc¹¹/+ flies showed normal wings and very few showed a mild transformation of the wing into haltere (Table 3). In contrast, when UAS-dsp1 mothers were submitted to heat shock, the majority of the Pc¹¹/+ progeny exhibited a mild transformation of wing into haltere and Pc¹¹/+ male offspring showed a considerable increase in their number of T3 legs with a sex comb (Table 3). These results strengthen the hypothesis that dsp1 is involved in the expression of different homeotic genes and could act as trx-G genes.

Absence of dsp1 modified the chromatin structure at the Mcp locus:
As already shown, dsp1¹ flies exhibited a partial transformation of A4 into A5, which could be the result of an activation of iab-5 in the A4 segment. The repression state of iab-5 in the A4 segment is controlled by Pc-G genes and by a boundary region, the Mcp region, which ensures that iab-4 and iab-5 are functionally autonomous and that the activation state of iab-4 does not spread into iab-5. The Mcp region is characterized by an unusual chromatin structure in embryos (KARCH et al. 1994 ). One prominent nuclease hypersensitive region of 300 bp has been identified. Deletion of this region leads to a transformation of A4 into A5. Thus it seemed to be of interest to determine whether the Mcp boundary region had the same chromatin structure in mutants lacking DSP1 protein. To examine the chromatin structure of the Mcp DNA segment, we prepared nuclei from 0–12-hr wild-type or dsp1¹ embryos and digested them with DNase I. In the experiment shown in Fig 3, EcoRI-restricted chromatin digests were probed with a 2.5-kb DNA fragment spanning almost all the Mcp region (Fig 3A). As illustrated in the autoradiogram in Fig 3B, the wild-type 6.0-kb EcoRI Mcp fragment contained a prominent hypersensitive region, as revealed by the decrease of the amount of the full-length Mcp DNA fragment and the appearance of specific DNase cleavage products around 4.3 kb and 1.7 kb (Fig 3B, lanes 1–4). These DNase cleavage products are chromatin-specific as they are not detected in control digests of naked DNA (Fig 3B, lane 9). Such a result is in agreement with the location of the hypersensitive sites of the Mcp region described by KARCH et al. 1994 . When the dsp1¹ 6.0-kb EcoRI Mcp fragment was DNase I digested, no specific DNase cleavage products appeared (Fig 3B, lanes 5–8). This result strongly suggests that the major DNase hypersensitive region of the Mcp boundary is absent in mutant dsp1¹ embryos and that DSP1 protein could act to remodel the chromatin structure at the Mcp locus.

View larger version (64K): In this window In a new window Download PPT slide   Figure 3. Absence of the DNase hypersensitive region in the Mcp boundary in a dsp11 mutant. (A) Schematic representation of the 6.0-kb EcoRI Mcp fragment. The map is shown in the proximal to distal orientation, with iab-4 to the left and iab-5 to the right. The solid square indicates the strong hypersensitive DNase region as described by KARCH et al. 1994 . The probe used in the experiment is indicated by an open rectangle below the map and the Mcp boundary is indicated by an arrow. E, EcoRI; P, PstI. (B) Nuclei prepared from wild-type (lanes 1–4) or dsp11 (lanes 5–8) embryos were digested with DNase I. After isolating the DNase I-digested DNA, the DNA samples were restricted with EcoRI and electrophoresed onto an agarose gel. After blotting to nitrocellulose filters, the DNA was hybridized with a probe encompassing the DNase hypersensitive region. If the hypersensitive region is present, several fragments are revealed at 4.3 and 1.7 kb; if it is absent, only one fragment is revealed at 6.0 kb. Lanes 1–4 and 5–8 correspond to different concentrations of DNase I (0, 1, 2, and 4 units/ml); lane 9 corresponds to naked DNA treated with DNase I (4 units/ml). Lanes containing a 1.0-kb Mr ladder were also included in the gels, but are not shown here.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

dsp1 is involved in homeotic gene expression:
Studies of the phenotype of a homozygous dsp1¹ mutant provide evidence that dsp1 is involved in the determination of body segment identity. We show that dsp1¹ mutants exhibit homeotic transformations typical of loss-of-function mutants for the two homeotic genes Ubx and Scr, with halteres transformed into wings and a sex comb reduced in size on the T1 leg. In the case of Scr, we have shown that Scr expression is diminished in T1 imaginal discs in homozygous dsp1¹ mutants. Hemizygous dsp1¹ males also show a moderate transformation of A6 into A5, resembling mutants at the iab-6 locus, and a mild transformation of A4 into A5, suggesting that iab-5 is ectopically activated in A4. Furthermore, by studying genetic interaction between dsp1¹ and a gain-of-function mutation of Antp, we show that the absence of DSP1 alters the function of Antp. All these results argue that dsp1 is implicated in the regulation of the function of homeotic genes.

dsp1 could be a remodeling chromatin factor acting as a trithorax- or a Polycomb-group gene:
Two groups of genes are known to control the expression of homeotic genes: the trx-G genes and the Pc-G genes. In view of some phenotypic traits observed in the mutant lacking DSP1, it appears that dsp1 could be classified as a trx-G gene. Studies of the genetic interaction between dsp1¹ and mutations of various trx-G genes show a strong enhancement of the haltere into wing homeotic transformation. On the contrary, interaction between dsp1¹ and a mutation in Pc reveals a partial suppression of the extra sex comb phenotype of Pc. Taken together, these findings suggest that DSP1 acts antagonistically to Pc to activate the transcription of Ubx, Scr, Antp, and iab-6. If this is the case, overexpression of dsp1 is expected to induce ectopic expression of these homeotic genes. This has been confirmed by studying overexpression of dsp1 in a Pc context. We observe an increase of transformations of wings toward halteres and an enhancement of the extra sex comb phenotype of Pc. Taken together, these results strongly support the idea that dsp1 acts as a member of trx-G. Interestingly, dsp1 function seems to be restricted to some particular loci. This is not unknown in flies since kismet, a suppressor of Pc, causes specific homeotic transformations when it is mutated (DAUBRESSE et al. 1999 ): transformation of the fifth abdominal segment into the fourth, with the other abdominal segments being not affected. The Kis protein seems to interact specifically with the iab-5 cis-regulatory element of AbdB and not with the other iab regions.

Surprisingly, one phenotypic trait of dsp1¹ seems to be characteristic of a mutation in Pc-G genes, the pigmentation of the A4 segment in adult males, corresponding to homeotic transformation of a segment into a more posterior one. However, analysis of the chromatin structure at the Mcp locus reveals that the DNase hypersensitive region is absent in dsp1¹. We propose that lack of DSP1 leads to remodeling of the chromatin structure at the Mcp locus, suppressing, at least in part, the boundary between iab-4 and iab-5, and then allowing the extension of the activation state of iab-4 to iab-5 in the A4 segment.

These results demonstrate that dsp1 could be a chromatin remodeling factor, acting as a trx-G or Pc-G gene depending on the considered function. These genes are involved in maintenance of an activation or repression state of homeotic genes. It has been proposed that they modify chromatin structure locally to maintain it in an "open" or "closed" configuration. DSP1 is an HMG1-like protein. It contains an HMG domain with two HMG boxes and a short acidic tail. HMG domains are known to interact with DNA, principally with bent DNA as four-way junctions or cisplatin-modified DNA. The interaction between an HMG box and DNA causes dramatic distortions on DNA structure and thus could participate with protein complexes in remodeling of the chromatin. In the case of the Brm complex, a protein, BAP111, containing an HMG domain has been identified. A counterpart for BAP111 has been identified in mammals as a member of a related SWI/SNF complex. This SWI/SNF complex is composed of BAF190, the human homologue of the Drosophila protein Brahma, and of BAF57, a high-mobility-group/kinesin-like protein (WANG et al. 1998 ). Considering the strong interaction between dsp1¹ and trx or ash1 mutations, DSP1 could be involved in a complex containing trx and ash1. Recently it has been shown that these two proteins interact with each other in vivo (ROZOVSKAIA et al. 1999 ). Such HMG proteins would not be obligatory members of the activating complexes, but could participate in the recognition of a higher-order chromatin structure and allow the interaction between chromatin and the activating complexes.

	FOOTNOTES

¹ Present address: Institut Curie, U.M.R. 144, 26 rue d'Ulm, 75248 Paris cedex 05, France.

	ACKNOWLEDGMENTS

We are grateful to M. J. Giraud-Panis for helpful discussions and useful suggestions and to A. Soulas and M. Martineau for excellent technical assistance. We are indebted to Dr. B. Limbourg-Bouchon for help in P-element-mediated transformation. We thank Bloomington Stock Center and Umeå Stock Center for supplying mutant strains used in this analysis. We especially thank A. Shearn for providing ash mutant strains. This work is supported in part by la Ligue contre le Cancer, la Fondation pour la Recherche Médicale, l'Association pour la Recherche contre le Cancer, and the E.U. (project ERB4061 PL97028).

Manuscript received February 18, 2000; Accepted for publication September 20, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BIANCHI, M. E., M. BELTRAME and L. FALCIOLA, 1992 The HMG box motif, pp. 112–128 in Nucleic Acids and Molecular Biology, Vol. 6, edited by F. ECKSTEIN and D. M. J. LILLEY. Springer-Verlag, Berlin.

BONNE, C., P. SAUTIERE, M. DUGUET, and A. M. DE RECONDO, 1982  Identification of a single-stranded DNA binding protein from rat liver with high mobility group protein 1. J. Biol. Chem. 257:2722-2725[Abstract/Free Full Text].

BONNE-ANDREA, C., F. HARPER, J. SOBCZAK, and A. M. DE RECONDO, 1984  Rat liver HMG1: a physiological nucleosome assembly factor. EMBO J. 5:1193-1199[Medline].

BREEN, T. R. and P. J. HARTE, 1991  Molecular characterization of the trithorax gene, a positive regulator of homeotic gene expression in Drosophila. Mech. Dev. 35:113-127[Medline].

BRYANT, P. J., 1978 Pattern formation in imaginal discs, pp. 229–335 in The Genetics of Drosophila, Vol. 2c, edited by M. ASHBURNER and T. R. F. WRIGHT. Academic Press, London.

CAIRNS, B. R., Y. LORCH, Y. LI, M. ZHANG, and L. LACOMIS et al., 1996  RSC, an essential, abundant chromatin-remodeling complex. Cell 87:1249-1260[Medline].

CALOGERO, S., F. GRASSI, A. AGUZZI, T. VOIGTLÄNDER, and P. FERRIER et al., 1999  The lack of chromosomal protein HMG1 does not disrupt cell growth, but causes lethal hypoglycaemia in newborn mice. Nat. Genet. 22:276-280[Medline].

DAUBRESSE, G., R. DEURING, L. MOORE, O. PAPOULAS, and I. ZAKRAJSEK et al., 1999  The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity. Development 126:1175-1187[Abstract].

DINGWALL, A. K., S. J. BEEK, C. M. MCCALLUM, J. W. TAMKUN, and G. V. KALPANA et al., 1995  The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex. Mol. Biol. Cell 6:777-791[Abstract].

DORN, R., J. SZIDONYA, G. KORGE, M. SEHNERT, and H. TAUBERT et al., 1993  P transposon-induced dominant enhancer mutations of position-effect variegation in Drosophila melanogaster.. Genetics 133:279-290[Abstract].

DUNCAN, I., 1987  The bithorax complex. Annu. Rev. Genet. 21:285-319[Medline].

EISSENBERG, J. C., T. C. JAMES, D. M. FOSTER-HARTNETT, T. HARTNETT, and V. NGAN et al., 1990  Mutation in a heterochromatin specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 87:9923-9927[Abstract/Free Full Text].

EISSENBERG, J. C., G. D. MORRIS, G. REUTER, and T. HARTNETT, 1992  The heterochromatin associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. Genetics 131:345-352[Abstract].

FARKAS, G., J. GAUSZ, M. GALLONI, G. REUTER, and H. GYURKOVICS et al., 1994  The Trithorax-like gene encodes the Drosophila GAGA factor. Nature 371:806-808[Medline].

GROSSCHEDL, R., K. GIESE, and J. PAGEL, 1994  HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. Trends Genet. 10:94-100[Medline].

INGHAM, P. W., 1985  A clonal analysis of the requirement for the trithorax gene in the diversification of segments in Drosophila. J. Embryol. Exp. Morphol. 89:349-365[Medline].

INGHAM, P. W. and R. WHITTLE, 1980  Trithorax: a new homeotic mutation of Drosophila melanogaster causing transformations of abdominal and thoracic imaginal segments. Mol. Gen. Genet. 179:607-614.

JOWETT, T., 1986 Preparation of nucleic acids, pp. 275–286 in Drosophila a Practical Approach, edited by D. B. ROBERTS. IRL Press, Oxford.

KARCH, F., M. GALLONI, L. SIPOS, J. GAUSZ, and H. GYURKOVICS et al., 1994  Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster.. Nucleic Acids Res. 22:3138-3146[Abstract/Free Full Text].

KAUFMAN, T. C., M. A. SEEGER, and G. OLSEN, 1990  Molecular and genetic organization of the Antennapedia gene complex of Drosophila melanogaster.. Adv. Genet. 27:309-362[Medline].

KENNISON, J. A., 1995  The Polycomb and trithorax group proteins of Drosophila: trans-regulators of homeotic gene function. Annu. Rev. Genet. 29:289-303[Medline].

KENNISON, J. A. and J. W. TAMKUN, 1988  Dosage-dependent modifiers of Polycomb and Antennapedia mutations in Drosophila.. Proc. Natl. Acad. Sci. USA 85:8136-8140[Abstract/Free Full Text].

LEHMING, N., D. THANOS, J. M. BRICKMAN, J. MA, and T. MANIATIS et al., 1994  An HMG-like protein that can switch a transcriptional activator to a repressor. Nature 371:175-179[Medline].

LEHMING, N., A. LE SAUX, J. SCHÜLLER, and M. PTASHNE, 1998  Chromatin components as part of a putative transcriptional repressing complex. Proc. Natl. Acad. Sci. USA 95:7322-7326[Abstract/Free Full Text].

LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego.

LOCKE, J., M. A. KOTARSKI, and K. D. TARTOF, 1988  Dosage-dependent modifiers of position effect variegation in Drosophila and a mass action model that explains their effect. Genetics 120:181-198[Abstract/Free Full Text].

MASUCCI, J. D., R. J. MILTENBERGER, and F. M. HOFFMANN, 1990  Pattern-specific expression of the Drosophila decapentaplegic gene in imaginal disks is regulated by 3' cis-regulatory elements. Genes Dev. 4:2011-2023[Abstract/Free Full Text].

MOSRIN-HUAMAN, C., L. CANAPLE, D. LOCKER, and M. DECOVILLE, 1998  DSP1 gene of Drosophila melanogaster encodes an HMG-domain protein that plays multiple roles in development. Dev. Genet. 23:324-334[Medline].

PAPOULAS, O., S. J. BEEK, S. L. MOSELEY, C. M. MCCALLUM, and M. SARTE et al., 1998  The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes. Development 125:3955-3966[Abstract].

PARO, R. and D. S. HOGNESS, 1991  The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila.. Proc. Natl. Acad. Sci. USA 88:263-267[Abstract/Free Full Text].

PATTATUCCI, A. M., D. C. OTTESON, and T. C. KAUFMAN, 1991  A functional and structural analysis of the sex comb reduced locus of Drosophila melanogaster.. Genetics 129:423-441[Abstract].

PIROTTA, V., 1997  Pc-G complexes and chromatin silencing. Curr. Opin. Genet. Dev. 7:249-258[Medline].

READ, C. M., P. D. CARY, S. CRANE-ROBINSON, P. C. DRISCOLL, M. O. M. CARILLO et al., 1995 The structure of the HMG box and its interaction with DNA, pp. 222–250 in Nucleic Acids and Molecular Biology, Vol. 9, edited by F. ECKSTEIN and D. M. J. LILLEY. Springer-Verlag, Berlin.

REYNOLDS, E. and M. TANOUYE, 1998  Cloning and characterization of bangsensitive mutants. A. Dros. Res. Conf. 39:588C.

ROZOVSKAIA, T., S. TILLIB, S. SMITH, Y. SEDKOV, and O. ROZENBLATT-ROSEN et al., 1999  Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter. Mol. Cell. Biol. 19:6441-6447[Abstract/Free Full Text].

SIMON, J., 1995  Locking in stable states of gene expression: transcriptional control during Drosophila development. Curr. Opin. Cell Biol. 7:376-385[Medline].

SINCLAIR, A. H., P. BERTA, M. S. PALMER, J. R. HAWKINS, and B. L. GRIFFITHS et al., 1990  A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346:240-244[Medline].

SINCLAIR, D. A., V. K. LLOYD, and T. A. GRIGLIATTI, 1989  Characterization of mutations that enhance position-effect variegation in Drosophila melanogaster.. Mol. Gen. Genet. 191:326-333.

SINCLAIR, D. A., A. A. RUDDELL, J. K. BROCK, N. J. CLEGG, and V. K. LLOYD et al., 1992  A cytogenetic and genetic characterization of a group of closely linked second chromosome mutations that suppress position-effect variegation in Drosophila melanogaster.. Genetics 130:333-344[Abstract].

SINCLAIR, D. A., N. J. CLEGG, J. ANTONCHUK, T. A. MILNE, and K. STANKUNAS et al., 1998a  Enhancer of polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.. Genetics 148:211-220[Abstract/Free Full Text].

SINCLAIR, D. A. R., T. A. MILNE, J. W. HODGSON, J. SHELLARD, and C. A. SALINAS et al., 1998b  The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique polycomb group sites on polytene chromosomes. Development 125:1207-1216[Abstract].

SINGH, J. and G. H. DIXON, 1990  High mobility group proteins 1 and 2 function as general class II transcription factors. Biochemistry 29:6295-6302[Medline].

SPRADLING, A. C. and G. M. RUBIN, 1982a  Transposition of cloned P-element into Drosophila germ lines chromosomes. Science 218:341-347[Abstract/Free Full Text].

SPRADLING, A. C. and G. M. RUBIN, 1982b  Genetic transformation of Drosophila with transposable element vectors. Science 218:348-353[Abstract/Free Full Text].

TAUTZ, D. and C. PFEIFLE, 1989  A non-radioactive in situ hybridization method for the localization of specific mRNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81-85[Medline].

TRAVIS, A., A. AMSTERDAM, C. BELANGER, and R. GROSSCHEDL, 1991  LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor alpha enhancer function. Genes Dev. 5:880-894[Abstract/Free Full Text].

TREMETHICK, D. J. and P. L. MOLLOY, 1988  Effects of high mobility group proteins 1 and 2 on initiation and elongation of specific transcription by RNA polymerase II in vitro. Nucleic Acids Res. 16:11105-11122.

TSIERSCH, B., A. HOFMANN, V. KRAUSS, R. DORN, and G. KORGE et al., 1994  The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13:3822-3831[Medline].

WANG, W., T. CHI, Y. XUE, S. ZHOU, and A. KUO et al., 1998  Architectural DNA binding by a high-mobility-group/kinesin-like subunit in mammalian SWI/SNF-related complexes. Proc. Natl. Acad. Sci. USA 95:492-498[Abstract/Free Full Text].

WATERMAN, M. L., W. H. FISHER, and K. A. JONES, 1991  A thymus-specific member of the HMG protein family regulates the human T cell receptor alpha enhancer. Genes Dev. 5:656-669[Abstract/Free Full Text].

WUSTMANN, G., J. SZIDONYA, H. TAUBERT, and G. REUTER, 1989  The genetics of position-effect variegation modifying loci in Drosophila melanogaster.. Mol. Gen. Genet. 217:520-527[Medline].

YAMAMOTO, Y., F. GIRARD, B. BELLO, M. AFFOLTER, and W. J. GEHRING, 1997  The cramped gene of Drosophila is a member of the Polycomb-group, and interacts with mus209, the gene encoding proliferating cell nuclear antigen. Development 124:3385-3394[Abstract].

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