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. 1997 Oct 15;11(20):2729-40.
doi: 10.1101/gad.11.20.2729.

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

P Morcillo  1 , C Rosen, M K Baylies, D Dorsett
Affiliations

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila

P Morcillo et al. Genes Dev. .

Abstract

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer-promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions.

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Figures

Figure 1
Figure 1
Model for regulation of cut by the remote wing margin enhancer and the sd, mam, and Chip genes. The model summarizes previous genetic and biochemical data (Morcillo et al. 1996a). The regulatory regions of cut genes (not to scale) are shown with the wing margin enhancer (wmE) to the left and the promoter (angled arrow) to the right. The sd protein (Sd) binds wing margin enhancer DNA and mam protein (Mam) is also postulated to be an enhancer-binding factor (see text). In females heterozygous for wild-type cut and an enhancer deletion allele (top pair), a heterozygous sd mutation (sd2/+) causes a partial cut wing phenotype (cut), while a heterozygous Chip mutation (Chipe5.5/+) displays wild-type wings (+). In contrast, when a female is heterozygous for wild-type cut and a gypsy insertion allele (bottom pair), which binds the enhancer-blocking su(Hw) protein (Suhw), a heterozygous sd mutation displays wild-type wings whereas a heterozygous Chip mutation causes a partial cut wing phenotype. These results, taken together, indicate that Suhw on one chromosome interferes with enhancer–promoter communication on both chromosomes and that Chip protein (Chip) facilitates enhancer–promoter communication.
Figure 2
Figure 2
The Chip gene and predicted Chip protein sequence. (A) Structure of the Chip gene. Shown is the restriction map of the 7-kb BglII–EcoRI genomic DNA fragment that rescues Chip homozygous mutant larval lethality. (Ba) BamHI; (Bg) BglII; (E) EcoRI; (H) HindIII; (O) EcoO109; (P) PstI. Locations of the l(2)k04405 P insertion, and the small deletions in Chipe5.5, Chipg371, and Chipg1 are indicated by arrows. The boxes below indicate the sequences present in the Chip cDNA clones, with the solid box indicating the ORF. The intron is indicated by a V. The Chip wild-type and mutant sequences have been deposited in GenBank (accession nos. AF010325–AF010328). (B) Sequence of Chip (D.m.) compared with the vertebrate homologs Xldb1 (X.l.) and Nli/Ldb1/Clim-2 (M.m.). Identities are silhouetted. The putative nuclear localization signal is underlined.
Figure 3
Figure 3
Interactions between Chip and the Ap LIM protein. (A) Genetic interaction between Chip and ap. The wing from a y*w*; Chipe5.5/ap56f male has gaps in the posterior margin. (B) Interaction between Chip and the Ap LIM domain in yeast. Yeast were transformed with a plasmid expressing the full length Chip ORF with the lexA DNA-binding protein fused to the amino terminus, and a second plasmid expressing the indicated protein containing the Gal4 activation domain (GAD, Gal4 activation domain alone; GAD–Chip, the Gal4 activation domain fused to the Chip ORF; GAD–APLIM, the Gal4 activation domain fused to the amino terminus of the Ap LIM domains). Interaction between Chip and the Ap LIM domains is indicated by the activation of the lexA operator–HIS3 reporter gene as detected by growth on histidine omission plates (his−), and expression of the lexA operator–lacZ fusion gene as detected by X-Gal staining (X-Gal). The GAD–APLIM protein does not activate the reporters when coexpressed with the LexA DNA-binding domain alone (not shown).
Figure 4
Figure 4
Expression of Chip transcripts during development. (top) An autoradiograph of a Northern blot showing the Chip 2.2-kb mRNA; (bottom) a photograph of the ethidium bromide-stained rRNA in the gel used for the Northern blot. Each lane contained 5 μg of total RNA isolated from Oregon-R wild-type flies at the indicated developmental stage [EE, 0–30 min after egg laying (AEL); LE, 30 min to 16 hr AEL; L1–L3, first to third instar larvae; P1–P4, first to fourth day of pupation; M, 0- to 1-day old male adults; F, 0- to 1-day old adult females]. Chip transcript was quantitated by PhosphorImager and normalized to the amount of rRNA as determined by densitometry. After normalization, the Chip transcript level in early embryos (EE) is ∼5-fold higher than in late embryos (LE) and ∼10-fold higher than in larval and pupal stages.
Figure 5
Figure 5
Expression of Chip protein. Embryos and larval tissues were stained with affinity-purified anti-Chip antibodies. All panels are wild-type Oregon-R and the same magnification unless indicated otherwise. Embryos are lateral views with anterior to the left and dorsal to the top. All larval tissues are from third instar larvae. (A) Cellular blastoderm; (B) mutant cellular blastoderm from Chipe5.5 germ-line clone; (C) gastrula; (D) wing imaginal disc; (E) eye–antennal imaginal disc; (F) leg imaginal discs; (G) larval fat body; (H) larval salivary gland; (I) salivary gland nucleus at 10-fold higher magnification than in panel H. The polytene chromosomes stain in a banded pattern.
Figure 6
Figure 6
Chipe5.5 germ-line clone embryonic cuticles. All panels are the same magnification. Anterior is to the left; dorsal is to the top. The wild-type cuticle is a lateral view and the mutant cuticles are slightly ventral aspects. (A) Wild-type Oregon-R embryo; (B) Chipe5.5 germ-line clone grown at 18°C. (C) Chipe5.5 germ-line clone grown at 25°C.
Figure 7
Figure 7
Expression of Eve in Chipe5.5 germ-line clone embryos. Embryos stained with anti-Eve antibodies. All are lateral views with anterior to the left and dorsal to the top. (A) Early wild-type cellular blastoderm. (B) Early Chipe5.5 germ-line clone cellular blastoderm. Eve is overexpressed relative to wild-type. (C) Wild-type cellular blastoderm with seven stripes of Eve expression. (D) Chipe5.5 germ-line clone cellular blastoderm showing weak Eve expression.
Figure 8
Figure 8
Expression of gap gene proteins in wild-type and Chipe5.5 germ-line clone embryos. Cellular blastoderm embryos were stained with antibodies against the indicated gap proteins (Hb, Kr, Kni, and Gt). All are lateral views with anterior to the left and dorsal to the top. (A,C,E,G) Wild type. (B,D,F,H) Mutant embryos from Chipe5.5 germ-line clones. Kr and Kni expression is lower in the mutants, and the domain of Gt expression is expanded.
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