Hairy transcriptional repression targets and cofactor recruitment in Drosophila

Abstract

Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s) surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP), and Drosophila silent information regulator 2 (dSir2) as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. We have used the chromatin profiling method DamID to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. We also adapted DamID profiling such that we could use tightly gated collections of embryos (2-6 h) and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. We have identified 59 putative transcriptional targets of Hairy. In addition to finding putative targets for Hairy in segmentation, we find groups of targets suggesting roles for Hairy in cell cycle, cell growth, and morphogenesis, processes that must be coordinately regulated with pattern formation. Examining the recruitment of Hairy's three characterized cofactors to their putative target genes revealed that cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, we find here that it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique provides a systematic means of identifying transcriptional target genes and of obtaining a global view of cofactor recruitment requirements during development.

Figure 1. Hairy Binds to a Specific Set of Transcriptional Targets

(A and B) Comparison of DamID-identified targets for Hairy with the Drosophila Myc and Mad/Mnt family proteins. Venn diagram comparing DamID-identified Hairy downstream targets in Kc cells compared to the transcriptional activator dMyc (A) and the transcriptional repressor dMnt (B). (C) Venn diagram comparing DamID-identified Hairy targets from Kc cells and embryos.

Figure 2. Expression of Hairy Target Genes Is Disrupted in hairy Mutant Embryos

Whole mount in situ hybridization on wild-type (A, C, E, G, I, K, and M) or hairy^7H mutant (B, D, F, H, J, L, and N) embryos with probes recognizing prd (A and B), stg (C and D), ImpL2 (E and F), mae (G and H), egh (I and J), kayak (K and L), or Idgf2 (M and N). Anterior is to the left. Dorsal is up, except in (M) and (N), which are dorsal views.

Figure 3. hairy Affects stg-lacZ Reporter Expression

(A–F) β-galactosidase expression from the stg-lacZ reporter lines pstg β-E4.9 (A and B), pstg β-E6.4 (C and D), and pstg β-E6.7 (E and F) in wild-type (A, C, and E) and hairy mutant (B, D, and F) embryos. Note the expanded (de-repressed) lacZ expression in the hairy mutant background compared to wild-type for the E4.9 and E6.4 lines (compare [B] to [A] and [D] to [C], respectively). (G) β-galactosidase expression from the stg-lacZ reporter line pstg β-E4.9^ΔHairy (same as the reporter construct shown in [A], but with a Hairy binding site mutation) in a wild-type background. Note the expanded (de-repressed) lacZ expression (compare with [A]). Anterior is to the left. Dorsal is up in (A–D) and (G), whereas the ventral surface is shown in (E–F).

Figure 4. Binding of Hairy to Class C (C-Box) Sites in Putative Targets In Vitro

(A) Schematic diagram (not to scale) of C-boxes within putative Hairy targets. C-boxes (Hairy binding sites) are denoted by white boxes, black arrows indicate transcription start sites (Ra, Rb, and Rc), ATG denotes the initiating methionine, and capital letters indicate bases matching with the Hairy consensus C-box. The distances in kilobases of the C-boxes from transcription start sites are noted in gray. (B) EMSA with either GST or GST–Hairy and the ac h/E-1 oligonucleotide. Lane 1, probe alone; lane 2, binding to probe by GST; lanes 3–5, binding to probe by GST–Hairy. In lanes 4 and 5, binding to probe by GST–Hairy was in the presence of competitor unlabeled oligos. An arrow indicates the Hairy–DNA complex; comp_wt and comp_mut indicate wild-type and mutated cold probes, respectively. (C) EMSA with either GST or GST–Hairy to the C-boxes within the stg and prd genes. Lanes 1–5, GST and GST–Hairy binding to the stg C-box (location: 25072658); lanes 6–10, GST and GST–Hairy binding to the prd C-box. (location: 12074032). Lane order and annotations are as in Figure 4B. (D) EMSA with GST–Hairy to the same C-box within the stg 4.9-kb genomic fragment is not competed by the presence of mutant competitor unlabeled oligo. Lane 1, probe alone; lane 2, binding to GST; lane 3, binding to probe by GST–Hairy; lanes 4 and 5, binding to probe by GST–Hairy in the presence of wild-type and mutant competitor unlabeled oligos, respectively. (E) Differential binding to C-boxes within the egh gene. EMSA with either GST or GST–Hairy to C-boxes within the egh promoter and transcribed region. Binding to three putative C-box sites is shown: egh1 (location: 2341609), egh2 (location: 2350367), and egh3 (location: 2352168). Lanes 1, 5, and 9: probe alone; lanes 2, 6, and 10: binding to probes by GST; lanes 3, 7, and 11: binding to probes with GST–Hairy. Lanes 4, 8, and 12: binding with GST–Hairy in the presence of unlabeled wild-type competitor. C-box locations and promoter information generated using Apollo (Berkeley Drosophila Genome Project).

Figure 5. Hairy Binds to Specific Loci on Polytene Chromosomes

(A and B) Hairy staining (green) on third instar larval salivary gland polytene chromosome sets counterstained with DAPI (blue) to visualize the chromosomes. (C and D) Higher magnification of chromosome arms X, 3R (C) and 2L, 2R (D).

Figure 6. Hairy Binds to Putative Target Loci on Polytene Chromosomes

(A) Hairy binds to polytene region 1A, the location of the Hairy target, ac. (B) Hairy is not found at 84A, the cytological location for ftz. (C–F) Hairy also binds to polytene region 99A, the location of stg (C); polytene region 3A, the location of egh (D); polytene region 33C, the location of prd (E); and polytene region 82A, the location of hkb (F). (G–I) Hairy is recruited to the insertion site for the pstg βE-4.9 reporter construct (arrow in [H] and [I]). Compare to the equivalent region of wild-type X chromosomes marked by brackets in (A), (D), and (G). (J) In situ hybridization to polytene chromosomes from pstg βE-4.9 larvae showing that this line has two insertions on the X chromosome at 1F and 6C. The probe also recognizes sequences to the endogenous white locus (asterisk).

Figure 7. Hairy Overlaps with Cofactors Differentially

(A–C) Venn diagram showing the overlap between Hairy targets and those loci also binding to the cofactors Groucho (A), dCtBP (B), and dSir2 (C). (D) Venn diagram showing combined overlaps of Hairy with its three known cofactors.

Figure 8. Hairy Target Gene Expression Is Disrupted in the Mutant Background of the Cofactors Associated with a Particular Target

Whole mount in situ hybridization on wild-type (A, E, and I), groucho germline clone (B, F, and J), dCtBP germline clone (C, G, and K), and dSir2 mutant (D, H, and L) embryos with probes recognizing stg (A–D), kayak (E–H), or prd (I–L). Anterior is to the left. Dorsal is up.

Figure 9. Hairy Shows Context-Dependent Association with Its Cofactors

(A) Sites of Hairy binding and Hairy cofactor recruitment based on DamID. The gray lines depict the relative position on the chromosomes of the approximately 6200 cDNAs on the microarray chip. The blue dots below the line represent Hairy binding sites while the green (Groucho), red (dCtBP), and yellow (dSir2) dots represent the positions of cofactor recruitment. (B–D) Cofactor recruitment visualized on third instar larval salivary gland chromosomes. Polytene chromosome sets stained (green) with antibodies to Groucho (B), dCtBP (C), and dSir2 (D). All chromosomes were counterstained with DAPI (blue) to visualize the DNA. (E) Higher magnification view of chromosome arms 2L and 2R costained with Groucho (red) and Hairy (green), and the merged image. (F) Higher magnification view of chromosome arm 2L costained with dCtBP (red) and Hairy (green), and the merged image, compared to the predicted DamID map. Note that both the DamID projected map and polytene chromosomes have more dCtBP recruitment sites to the left of the dashed line than to the right of the dashed line. (G) Chromosome arm 3R stained with dSir2 (green), highlighting regional specificity of dSir2 recruitment. (H and I) Higher magnification view of the distal ends of chromosome arms 2R (H) and 3L (I) from (D), stained with dSir2 (green), showing regional specificity and lack of dSir2 recruitment, respectively.