Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax complex

Abstract

Boundaries in the Bithorax complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including Fab-7 and Mcp To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6↔iab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7, we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7: it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.

Keywords: Architectural proteins; Chromatin organization; Insulator; Protein-protein interactions; Zinc-finger transcription factors.

Fig. 1.

Boundaries of the BX-C coincide with dCTCF and Pita. (A) Distribution of ChIP signals for Pita and dCTCF across the BX-C. The BX-C is presented as a sequence coordinate line. Transcripts of Ubx, abd-A and Abd-B are marked by horizontal arrows. The mapped insulator elements are indicated under the coordinate map as vertical black bars. Red ovals denote sequence motifs for Pita found in the BX-C. Tracks show the binding profiles for Pita and dCTCF according to the ChIP-Seq data. (B) Binding of Pita at the boundaries of BX-C. Chromatin was isolated from pupae and treated with antibodies against Pita. Nonspecific IgG was used as a negative control (not shown, see Fig. S1 for comparison). The results of ChIPs are presented as a percentage of input DNA. The γTub37C (tub)-coding region (devoid of binding sites for Pita) was used as a negative control. Error bars indicate standard deviations of the triplicate PCR measurements from two independent biological samples of chromatin. (C) EMSA of recombinant zinc-finger domains of Pita with the DNA fragments from BX-C containing Pita-binding sites (Fub-2, Fub, Mcp, Fab-7 and AB-E). Zinc-finger domains of Pita fused with MBP were incubated with the Cy5-labeled DNA fragments. Specificity of the interaction was demonstrated by incubation of the DNA fragments with varying amounts of Pita protein, presented as a series of twofold dilutions. In parallel, the FAM-labeled DNA fragments with no binding sites for Pita were used as a negative control (data not shown).

Fig. 2.

The phenotypic effects of Fab-7 replacement by multimerized Pita-binding sites. (A) A schematic presentation of the Fab-7 boundary. Hypersensitive sites are shown as gray boxes. The proximal and distal deficiency endpoints of the F7^attP50 deletions are shown. GAF- and Pita-binding sites are designated as blue and red ovals, respectively. In this experiment, HS*, HS1 and HS2 are replaced by five Pita sites, whereas HS3 (PRE^iab-7) is retained. Bold black half-arrows (marked i7) indicate primers that were used for ChIP experiments. (B) Binding of Pita and GAF at the Pita^×5 fragment in Pita^×5 pupae. Nonspecific IgG was used as a negative control. The results of ChIP are presented as a percentage of input DNA. The Fab-8 (F8) region was used as a positive control for GAF binding, the 100C region was used as a positive control for Pita binding. The γTub37C-coding region (tub), devoid of binding sites for Pita and GAF, was used as a negative control. Error bars indicate standard deviations of triplicate PCR measurements from two independent biological samples of chromatin. (C) EMSA of the recombinant zinc-finger domains of Pita with a Pita^×5 DNA fragment. Zinc-finger domains of Pita fused with MBP were incubated with a Cy5-labeled DNA fragment. In parallel, the FAM-labeled DNA fragment with no binding site for Pita was used as negative control (data not shown). (D) Morphology of the 4th to 6th male abdominal segments (numbered) is determined by the Abd-B cis-regulatory regions. F7^attP50 males have the classic gain-of-function transformation of A6 (PS11) into A7 (PS12) seen in mutations that remove both the Fab-7 boundary and the PRE^iab-7, HS3. Pita^×5 demonstrates a strong loss-of-function phenotype: A6 sternite in males is quadrilateral and covered in bristles, just like the sternite in A5; whole A6 tergite is covered with trichomes, similar to the A5 tergite. Wild-type (wt) males have pigmented A5 and A6 tergites. The A6 sternite is recognizable by the absence of bristles and a specific form; A7 does not contribute to any visible cuticle structures. Trichomes are visible in the dark-field images (bottom row) and cover all the surface of A5 tergite, and only a thin stripe along the anterior and ventral edges of the A6 tergite.

Fig. 3.

Role of Pita-binding sites in Fab-7 boundary function. (A) A schematic of the Fab-7 boundary replacement constructs at the F7^attP50 site. Hypersensitive sites are shown as gray boxes. The proximal and distal deficiency endpoints of the F7^attP50 deletions are shown. GAF- and Pita-binding sites are designated as blue and red ovals, respectively. Bold black half-arrows (marked i7) indicate primers that were used for ChIP experiments. (B) Binding of Pita and GAF at the HS1 and HS2 regions in pupae of tested transgenic lines (HS1+2+3, HS1+2^ΔPita +3, HS1+2 and HS1+2^ΔPita). The results of ChIP are presented as a percentage of the input DNA. The Mcp region was used as a positive control for Pita binding, the Hsp70 region was used as a positive control for GAF binding and the RpL32-coding region was used as a negative control for binding. Error bars indicate standard deviations of triplicate PCR measurements from two independent biological samples of chromatin. (C) All abdominal segments in HS1+2+3 and HS1+2^ΔPita +3 males (first row, bright field; second row, dark field) and females (third row, bright field; fourth row, dark-field images) have essentially a wild-type identity. HS1+2 males have a mixed wild-type and gain-of-function phenotype: an almost complete A6 tergite in combination with an absence of the A6 sternite. HS1+2^ΔPita males and females have a strong gain-of-function phenotype with small spots of cells with loss-of-function features.

Fig. 4.

Role of Pita- and CTCF-binding sites in Mcp insulator function. (A) Schematic of the Mcp boundary and replacement fragments used for the insertion at F7^attP50. The dCTCF-binding site is shown as magenta ovals. Bold black half-arrows (marked i7) below the map indicate primers that were used for ChIP experiments. Hypersensitive sites are shown as gray boxes. GAF- and Pita-binding sites are designated as blue and red ovals, respectively. (B) Binding of Pita, dCTCF and GAF at the PRE^iab-7 region in pupae of tested transgenic lines (M340, M^340ΔPita and M^340ΔCTCF). The results of ChIPs are presented as percentages of the input DNA. The 100C region was used as a positive control for Pita and hsp70 for GAF binding, the F8 region was used as a positive control for dCTCF and GAF binding, and the γTub37C-coding region was used as a negative control for binding. Error bars indicate standard deviations of triplicate PCR measurements from two independent biological samples of chromatin. (C) Morphology of the abdominal segments of the M³⁴⁰ mutant males (bright field, top row; dark field, bottom row). In M³⁴⁰ homozygous males, A6 is completely transformed into A5. The phenotypic effects are the same as in the case of Pita^×5. M^340ΔPita and M^340ΔCTCF males demonstrate a mixed gain- and loss-of-function transformation of A6 segment. Phenotypes of M³⁴⁰ mutant females can be seen in Fig. S3.