CLAMP promotes gypsy insulator activity as an accessory factor

We found that CLAMP interacts physically with insulator proteins and acts as a positive factor required for gypsy insulator function. Ubiquitously expressed CLAMP promotes gypsy insulator activities in all tissues tested at various stages of development, suggesting a central and not necessarily regulatory role during development or within specific tissues. Although we observed physical interaction and partial co-localization between CLAMP and core gypsy insulator proteins, our ChIP-seq analysis showed that CLAMP co-localizes mainly with CP190 but does not overlap extensively with all three gypsy insulator proteins throughout the genome. This result is consistent with a low fraction of total insulator proteins observed in physical complex with CLAMP. Alternatively, low overlap with gypsy insulator sites could also reflect physical interaction of CLAMP and this particular class of insulator proteins separately from chromatin. Nevertheless, we did observe CLAMP chromatin association along with core insulator proteins at the Su(Hw)-binding site of the gypsy retrotransposon. Depletion of CLAMP resulted in a mild reduction of insulator proteins at these sites, suggesting that CLAMP may affect accessibility of binding.

We observed altered nuclear localization of gypsy insulator bodies in CLAMP-depleted flies, indicating that CLAMP affects the overall distribution of insulator complexes. CLAMP contains a glutamine-rich N-terminal domain ( Larschan et al., 2012 ), and proteins harboring this domain are able to aggregate nuclear factors ( Kato et al., 2012 ; Wilkins and Lis, 1999 ). However, it should be pointed out that the functional significance of insulator bodies is still debated. Assigning function to a visually defined structure remains a general challenge of modern cell biology. Nevertheless, CLAMP could be involved in promoting higher order gypsy insulator complex formation. Interestingly, both CLAMP and CP190 are associated with TAD borders ( Ramirez et al., 2015 ; Sexton et al., 2012 ), but further study is required to determine whether either CLAMP or CP190 plays a role in overall TAD formation or maintenance.

CLAMP but not CP190 is a component of LBC

Recent work showing that CLAMP associates with a non- gypsy class of insulator may provide insights into how CLAMP promotes gypsy insulator function. CLAMP was identified as a component of LBC, which binds the Fab - 7 insulator of the Abd - B homeobox gene during embryonic development ( Wolle et al., 2015 ). The LBC is estimated to be a 1 MDa complex that also contains GAF and at least one isoform of Mod(mdg4) that does not correspond to Mod(mdg4)67.2, but CP190 was not detected in this complex ( Kaye et al., 2017 ). Both CLAMP and GAF bind similar GA-rich sequences ( Kuzu et al., 2016 ), which are also present in Fab - 7 . Interestingly, CLAMP and GAF association with Fab - 7 is interdependent ( Kaye et al., 2017 ) and both can directly compete with each other for a binding site ( Kaye et al., 2018 ). GAF-binding sites are known to be required for Fab - 7 function ( Wolle et al., 2015 ). However, neither clamp nor trithorax - like (encoding GAF) have yet been tested genetically for this activity. Since both factors have been shown to promote binding of the MSL complex to multiple chromosome entry sites ( Kaye et al., 2017 ), it is possible that the function of CLAMP is to load LBC onto Fab - 7 DNA.

CLAMP affects CP190 recruitment to chromatin at a handful of sites

CP190 is a component of multiple insulator complexes but does not appear to bind DNA directly, suggesting the need for interaction with DNA-binding factors such as CLAMP to be recruited to chromatin. In the case of the gypsy insulator, Su(Hw) provides the specificity of binding ( Parkhurst et al., 1988 ), and CTCF recruits CP190 to the Fab - 8 insulator ( Moshkovich et al., 2011 ). Furthermore, Ibf1 and Ibf2 have been shown to recruit CP190 to specific binding sites that do not overlap with either Su(Hw) or CTCF ( Cuartero et al., 2014 ). Recent studies have shown that zinc finger-containing proteins Pita and ZIPIC co-localize with CP190 across the genome and play a role in targeting CP190 at least to specific sites ( Maksimenko et al., 2015 ). Pita and ZIPIC binding is mostly distinct from Su(Hw), Mod(mdg4)67.2 ( Maksimenko et al., 2015 ), as well as Ibf1 and Ibf2 binding sites throughout the genome. Likewise, we found that CLAMP does not co-localize extensively with any of these factors; therefore, CLAMP may function independently of these proteins to recruit CP190 to shared CLAMP-CP190 sites. In fact, we found that co-occupied CLAMP-CP190 sites resemble GA-rich CLAMP genome-wide consensus sites ( Kuzu et al., 2016 ; data not shown), but when we depleted CLAMP in Kc cells, we did not observe extensive changes in overall CP190 chromatin association by ChIP-seq except for at a minority of sites, including the gypsy retrotransposon. Although this result could suggest that CLAMP is not generally required for CP190 recruitment, CLAMP could act redundantly with another factor to recruit CP190 to chromatin. In fact, knockdown of the CP190-associated DNA-binding insulator protein BEAF-32 also has no effect on CP190 recruitment ( Lim et al., 2013 ; Schwartz et al., 2012 ).

CLAMP chromatin binding is dependent on CP190

Based on both differential ChIP-seq results and staining of polytene chromosomes, we find that CLAMP chromatin association at many sites in the genome is dependent on CP190. Since CLAMP and CP190 physically associate within the nucleus, CP190 may directly recruit CLAMP to some shared sites. However, we also found that CLAMP chromatin association is dependent on CP190 at some additional sites where CP190 is not observed to be stably associated. A similar phenomenon was observed for AGO2 chromatin recruitment, which is fully CP190-dependent despite AGO2 being present at additional sites ( Moshkovich et al., 2011 ). Therefore, we speculate that recruitment of CLAMP to additional sites might be achieved by CP190-dependent chromatin looping ( Moshkovich et al., 2011 ; Ong and Corces, 2014 ; Wood et al., 2011 ). The dependence of CLAMP chromatin association on CP190 supports a functional relationship between CLAMP and the gypsy insulator complex.

Antibodies

For western blotting, guinea pig serum against CP190 ( Matzat et al., 2012 ) was used at 1:10,000, guinea pig serum against Mod(mdg4)67.2 ( Moshkovich and Lei, 2010 ) was used at 1:1000, guinea pig serum against Su(Hw) ( Moshkovich and Lei, 2010 ) was used at 1:7500, mouse monoclonal antibody against Tubulin (Cat. no. T6074-100UL, Sigma-Aldrich) was used at 1:10,000, rabbit antibody against CLAMP (Cat. no. 49880002, Novus/SDIX) was used at 1:1000, rabbit serum against Pc was used at 1:1000 ( Moshkovich et al., 2011 ) and purified mouse serum against Pep ( Amero et al., 1991 ) was used at 1:1000. For immunofluorescence, rabbit serum against CP190 ( Pai et al., 2004 ) was used at 1:10,000. For polytene immunostaining rabbit antibody against CLAMP was used at 1:500 (Cat. no. 49880002, Novus/SDIX), rabbit antibody against CLAMP was used at 1:500 (custom antibody generated through a contract to Abcam) ( Rieder et al., 2017 ), guinea pig serum against CP190 was used at 1:200, guinea pig serum against Mod(mdg4)67.2 was used at 1:200, guinea pig serum against Su(Hw) was used at 1:200, and mouse serum against Pep ( Amero et al., 1991 ) was used at 1:100. For ChIP, 3 μl (1:333 dilution) rabbit serum against Mod(mdg4)67.2 ( Van Bortle et al., 2014 ), 3 μl (1:333 dilution) guinea pig serum against Su(Hw), 3 μl (1:333 dilution) rabbit serum against CP190 ( Pai et al., 2004 ), 5 μl (1:200 dilution) rabbit antibody against CLAMP ( Rieder et al., 2017 ) and 5 μl (1:200 dilution) rabbit antibody against CLAMP (Cat. no. 49880002 Novus/SDIX) were used.

Luciferase insulator barrier activity assay

Luciferase insulator barrier activity assay was carried out as described previously ( Matzat et al., 2012 ). Luciferase signal was quantified using a Spectramax II Gemini EM plate reader (Molecular Devices). Luciferase levels were measured for twelve individual whole third instar male and female larvae for all genotypes indicated in a single panel simultaneously. Luciferase activity was normalized to total protein of each larva measured by BCA reagent (Thermo Scientific). Then the relative luciferase activity of a population of a single genotype was aggregated into a box and whisker plot. Populations were compared with one-way ANOVA followed by a Tukey HSD post-hoc test to obtain P -values for each pairwise comparison. The P -values for pairwise comparisons between the control and RNAi lines within the insulated group are listed in Tables S1 and S2 .

Immunostaining of imaginal discs

Imaginal discs and brains were dissected from at least six larvae of each genotype and subjected to whole-mount staining as previously described ( Moshkovich et al., 2011 ) using ProLong Gold (Life Technologies) mounting media.

Polytene staining

Polytene chromosomes were prepared from the salivary glands of third instar y ² larvae as described previously ( Lei and Corces, 2006 ). Three pairs of salivary glands were processed for each slide. We co-stained polytene chromosomes with rabbit anti-CLAMP (Novus/SDIX, 1:500) and either with guinea pig anti-Su(Hw), guinea pig anti-Mod(mdg4)67.2 and guinea pig anti-CP190 primary antibodies. We also co-stained with anti-CLAMP (1:500) ( Rieder et al., 2017 ) with mouse anti-Pep (1:100) ( Amero et al., 1991 ) antibodies. For detection, we used anti-rabbit Alexa Fluor 488 (Cat. no. A11008, Life Technologies), anti-guinea pig Alexa Fluor 594 (Cat. no. A11076, Life Technologies) and anti-mouse Alexa Fluor 594 (Cat. no. A11032, Life Technologies) secondary antibodies at a concentration of 1:1000. Chromosomes were imaged using a Leica DM5000B epifluorescent microscope using a 40× objective and captured using OpenLab software (Improvision).

Co-immunoprecipitation

Drosophila embryonic nuclear extract was prepared from 20 g of mixed stage (0–24 h) embryos as described previously ( Lei and Corces, 2006 ). Nuclei were lysed with 5 ml of nuclear lysis buffer (60 mM HEPES pH 7.5, 10 mM MgCl ₂ , 100 mM KCl, 0.1% Triton X-100, 10% glycerol, Roche cOmplete protease inhibitor) and sonicated for 8 cycles with 10 s on and 50 s off. The soluble fraction of extracts was collected by centrifugation. First, two sets of 20 µl of Protein G Sepharose beads (GE Healthcare) were washed three times with nuclear lysis buffer for immunoprecipitation. Rabbit anti-CLAMP antibody (3 µg; Cat. no. 49880002 Novus/SDIX) and rabbit-IgG antibody (1.2 µg; Cat. no. sc-2027, Santa Cruz Biotechnology) were incubated with Protein G Sepharose beads for 1 h at 4°C, and unbound antibodies were removed by means of centrifugation at 1500 g for 1 min. Beads were washed three times with 0.2 M of sodium borate, pH 9, followed by crosslinking with 20 mM DMP in sodium borate for 30 min at room temperature ( Harlow and Lane, 1988 ). Beads were collected by means of centrifugation and washed once with ethanolamine and three times with lysis buffer. After crosslinking, 500 µg of nuclear extract was used for each immunoprecipitation and incubated with antibody-bound beads overnight at 4°C. Next, beads were collected by means of centrifugation and washed three times with nuclear lysis buffer. Then samples were eluted with SDS sample buffer through boiling, separated using SDS-PAGE, transferred to nitrocellulose membrane in 10 mM CAPS, pH 11, and detected using western blotting.

For reverse-immunoprecipitation, 20 µl of Protein A Sepharose beads (GE Healthcare) for each immunoprecipitation with identical amounts (3 µl) of guinea pig pre-immune serum (Covance Research Products) or anti-serum against Su(Hw) (guinea pig) were used and followed the same method. The bound protein was eluted in sample buffer by means of boiling and detected using western blotting. The co-immunoprecipitation efficiency was calculated for each immunoprecipitated protein based on the percentage of total input protein.

Cell lines

Kc167 cells were grown in CCM3 media (Thermo Scientific HyClone, Logan, UT) and maintained in monolayer at 25°C.

Knockdown of clamp and Cp190 in Kc167 cells

1×10 ⁷ Kc167 cells were transfected with either 5 µg of dsRNA (29935, DRSC/TRiP Functional Genomics Resources) against clamp , 12 µg of dsRNA ( Moshkovich et al., 2011 ) against Cp190 or no RNA (mock) using Amaxa cell line Nucleofector kit V (Lonza) and electroporated using G-30 program. Cells were incubated for 5 d or 6 d at 25°C to obtain efficient depletion of CLAMP or CP190 protein, respectively.

ChIP and ChIP-seq library preparation

2–3×10 ⁷ cells were fixed by adding 1% formaldehyde directly to cells in culture medium for 10 min at room temperature with gentle agitation. Then formaldehyde was quenched with 0.125 M glycine with gentle agitation for 5 min at room temperature. Cells were pelleted by means of centrifugation at 2000 g and washed twice with ice-cold PBS. Pellets were resuspended in 0.8 ml ice-cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40, supplemented with Roche cOmplete protease inhibitor), incubated on ice for 10 min, then pelleted by means of centrifugation at 2000 g for 5 min at 4°C. Next, the supernatant was removed and pellet was resuspended in 1 ml nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, supplemented with Roche cOmplete protease inhibitor) and incubated for 10 min at 4°C on a rotator. Afterwards, 0.5 ml of IP dilution buffer (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS, supplemented with Roche cOmplete protease inhibitor) and 300 mg of acid-washed 212–300 μm glass beads (Sigma-Aldrich) were added to the lysate, and chromatin was fragmented to an average size range of 200–500 bp using a Bioruptor (Diagenode) using 10 cycles of 30 s on and 30 s off, maximum output. Samples were centrifuged at max speed for 10 min at 4°C, and the supernatant (sheared chromatin) was saved at −80°C. Chromatin was diluted to 1:5 with IP dilution buffer and added to 50 µl prewashed Protein A Sepharose beads (GE Healthcare) and 5 µg of respective antibody and rotated overnight at 4°C. The next day, beads were washed with the following wash buffers: three times with low-salt wash buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), three times with high-salt wash buffer (20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), and two times with LiCl wash buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholate). Chromatin was eluted twice with 200 μl of elution buffer (0.1 M NaHCO ₃ , 1% SDS) for 30 min at 65°C each in a thermomixer at 800 rpm. De-crosslinking solution (20 μl of 5 M NaCl, 8 μl of 0.5 M EDTA, 10 μl of 1 M Tris-HCl pH 8) was added to the eluates and further incubated overnight at 65°C. After de-crosslinking, samples were treated with 4 µl of proteinase K (20 mg/ml) for 2 h at 50°C and then purified using phenol-chloroform followed by ethanol precipitation with 0.1 vol of 3 M NaOAc pH 5.2 and 2.5 vol of 100% ethanol supplemented with 2 μl of Glycoblue (Ambion). After incubating overnight at −80°C, samples were centrifuged 20 min at 10,000 g at 4°C. Pellets were washed with 70% ethanol. Pellets were air-dried at room temperature prior to resuspension in 10 μl of nuclease-free water. Libraries were constructed by pooling two immunoprecipitation (IP) samples using TruSeq adapters (Illumina) according to the TruSeq Illumina ChIP-seq sample preparation protocol with the following modifications: after adaptor ligation and PCR amplification, samples were purified by using AMPure XP Beads (sample:bead ratio 1:0.8) according to manufacturer's protocol. All samples were sequenced with HiSeq2500 (Illumina) through 50 bp single-end sequencing.

ChIP-seq data are available at Gene Expression Omnibus (GSE118700; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE118700 ).

ChIP-seq data analysis

FASTQ files of sequenced single-end 50 bp reads were trimmed using cutadapt v1.8.1 ( Martin, 2011 ) with arguments ‘--quality-cutoff 20’, ‘-a AGATCGGAAGAGC’, ‘--minimum-length 25’ and ‘--overlap 10’. Trimmed reads were mapped with bowtie2 v2.2.9 ( Langmead and Salzberg, 2012 ) with default arguments to the dm6 assembly. Multimapping reads were removed from mapped reads with samtools v1.2 ( Li et al., 2009 ) view command using the argument -q 20. Duplicates were removed from mapped, uniquely mapping reads with picard MarkDuplicates v2.9.2 ( http://broadinstitute.github.io/picard/index.html ). MACS v2.1.0.20150731 ( Zhang et al., 2008 ) ( https://github.com/taoliu/MACS ) was used to call peaks by providing replicate IPs and inputs as multiple BAMs, effectively calling peaks on pooled samples and using additional arguments ‘-f BAM’, ‘--gsize=dm’, ‘--mfold 3 100’ (the latter to include a larger set of preliminary peaks for fragment size estimation).

Binary heatmaps were created using pybedtools ( Dale et al., 2011 ; Quinlan and Hall, 2010 ). Since peaks for one protein can potentially overlap multiple peaks for other proteins, the output represents unique genomic regions as determined by bedtools multiinter with the -cluster argument. As a result, when considering the multi-way overlap with other proteins, the sum of unique genomic regions for a protein is not guaranteed to sum to the total number of called peaks for that protein.

FlyBase release 6.16 annotations were used to annotate peaks as follows. Exons were defined as any exon from any transcript of any gene. Introns were defined as the space between exons calculated in a per-transcript manner using the gffutils ( https://github.com/daler/gffutils ) method FeatureDB.create_introns(). Promoters were defined as the TSS of each transcript plus 1500 bp upstream. Intergenic regions were defined as all regions between gene bodies. Unique or shared peaks were determined using pybedtools BedTool.intersect with the v=True or u=True argument, respectively. Each set of peaks was intersected with the annotations. Using the hierarchy ‘promoter>exon>intron>intergenic’, a peak was classified according to the highest priority feature it intersected. Thus, a peak simultaneously intersecting a promoter of one isoform and an intron of a different isoform would be classified as ‘promoter’. To compare percentages across annotated peaks in different types of peaks (CLAMP alone, CP190+CLAMP, etc), the number of peaks in each class was divided by the total number of peaks of that type.

Pairwise co-localization was assessed using the BEDTools ‘jaccard’ command ( Dale et al., 2011 ; Quinlan and Hall, 2010 ). The heatmap was clustered with the scipy.cluster.hierarchy module, using ‘euclidean’ as the distance metric and ‘Ward’ as the clustering method. Since the Jaccard statistic is independent of the order of comparison and therefore symmetric across the diagonal, only the upper triangle is shown.

Differential ChIP-seq

For detecting differential ChIP-seq binding, we used the Diffbind v1.16.3 (CLAMP ChIP-seq) or v2.6.6 (CP190 ChIP-seq) Bioconductor/R package ( Ross-Innes et al., 2012 ) using the config object ‘data.frame(RunParallel=TRUE,DataType=DBA_DATA_FRAME, AnalysisMethod=DBA_EDGER, bCorPlot=FALSE, bUsePval=FALSE, fragmentSize=300)’ and otherwise used defaults. Input files consisted of the final peak calls described above, and the IP and input BAM files for each replicate as described above with multimappers and duplicates removed. The final results were exported with the dba.report function with parameters ‘th=1, bCalled=TRUE, bNormalized=TRUE, bCounts=TRUE’ and final differentially gained or lost peaks were those that had a log ₂ fold change of >0 or <0, respectively, and an FDR<0.05.

We thank Erica N. Larschan for w ¹¹⁸ ; clamp ² /CyO - GFP null mutant flies, rabbit anti-CLAMP antibody, discussions and comments on the manuscript. We also thank Ann L. Beyer for anti-Pep, Patrick H. O'Farrell for anti-Pc antibodies, and members of the Lei laboratory for critical reading of the manuscript.