HIPP1 stabilizes the interaction between CP190 and Su(Hw) in the Drosophila insulator complex

Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). Here, we performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720–892) and with CP190, but not with Mod(mdg4)-67.2. We have generated Hipp1 null mutants (HippΔ1) and found that the loss of Hipp1 does not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilized the interaction between CP190 and the Su(Hw)-dependent complex.

The proteins were blotted onto a PVDF membrane, which was then consecutively probed with antibodies to His (Amersham, designated αHis) or to GST (Amersham, designated αGST) used as loading control (at the bottom). The sample in the InPut lane contained 10% of His-fusion protein loaded onto Glutathione Sepharose together with GST-fusion proteins. The sample in the GST lane contained GST alone used as negative control. Numbers in brackets refer to amino acid residues. All results were reproduced in three independent experiments.
The immunoprecipitated complexes were washed with 150 mM KCl-containing buffers before loading onto SDS-PAGE for Western blot analysis. The PVDF membrane was consecutively probed with antibodies against the HIPP1 protein (designated αHIPP1) or FLAG epitope to visualize immunoprecipitated target proteins. Input is the input fraction (10% of lysate used for immunoprecipitation); Output, the supernatant after immunoprecipitation; IP, the immunoprecipitate; PI, immunoprecipitation with nonspecific IgG; Output PI, the supernatant after nonspecific IgG immunoprecipitation. The result of test for the specificity of HIPP1 antibodies by RNAi method is shown on the bottom panel. Anti-tubulin staining (αTub) was used as loading control. All experiments were repeated in triplicate.
To generate pGBT9Su(Hw) 1 To generate pGBT9CP190 308-470 and pGDACP190 308-470 , the fragment EcoRI-ApaI (filled in with Klenow) from CP190 cDNA was subcloned into pGBT cleaved with EcoRI and SalI (filled in with Klenow fragment). Then the EcoRI-SalI fragment from the resulting plasmid was subcloned into pGDA cleaved with the same enzymes.
To generate pGBT9HIPP1 1-778 and pGDAHIPP1 1-778 plasmids, HIPP1 cDNA was amplified with primers 5'-gaattcatggagcaggtgtcggata-3' (with introduced EcoRI site) and 5'ggatccaaaatcaacgtcgttaagatagc-3' (with introduced BamHI site) from cDNA library. The PCR product was cleaved with BamHI and EcoRI, and the 638-bp BamHI-EcoRI fragment was ligated into either pGBT9 or pGDA vector cleaved with the same enzymes. The remaining 2100bp EcoRI-EcoRI fragment of cDNA was then cloned into the EcoRI site of the resulting plasmid and checked for orientation.

Constructs for GST Pull Down assay
First, the pSKHIPP1 plasmid was generated. The pGBT9HIPP1 1-778 plasmid was cleaved with BamHI and EcoRI; the 638-bp BamHI-EcoRI fragment was ligated into pBluescript II SK(+) cleaved with the same enzymes, and the remaining 2100-bp EcoRI-EcoRI fragment of cDNA was then cloned into the EcoRI site of the resulting plasmid and checked for orientation.
To prepare pAc5.1CP190 1-1096 -FLAG plasmid, the HindIII-BstXI fragment containing full-length CP190 cDNA was cloned in frame with FLAG epitope into pAc5.1 cleaved with the same enzymes.

CRISPR/Cas9 genome editing
HIPP1 Δ1 mutants were generated by the homology-directed repair (HDR) method. For gRNA expression, the U6:3 promoter was used 7 , and target sites were designed so that they directed Cas9-mediated cleavage to the 5' end of the coding sequence, which targets the HIPP1 promoter: 5'-cctatcgatagatccacctgcgc-3' and 5'-gactctagaactaaaaacggcgg-3'. To reduce the risk of offtarget cleavage, target sites were chosen so as to have no highly homologous sites elsewhere in the genome. Off-target potential was assessed using CRISPR target finder Crispr fly design (http://www.crisprflydesign.org). Because a 5′ guanine is required for transcription from U6 promoters, target sites lacking this feature were extended in the 5′ direction by a single guanine.
pHD-DsRed-attP was used as a vector to generate dsDNA donor templates for HDR. This vector is designed for replacing a targeted locus with a 50-bp attP phage recombination site and is positively marked with a Cre recombinase-removable 3XP3-dsRed construct for screening. It has two multiple cloning sites (MCS) for inserting homology arms that immediately flank the targeted locus. The 5′ and 3′ homology arms were PCR-amplified from nos-Cas9 flies genomic DNA using primers 5'-acatggcaagccgtcgcttgat-3' and 5'-cgtgaaaatatcggaaaaagtggat-3' for the 5' arm and 5'-cgttagaaacactctcggaaag-3' and 5'-ggtgttggttccacagcaact-3' for the 3' arm. Two vectors expressing gRNAs and template for HDR vector were simultaneously injected into preblastoderm embryos.