Summary
The mammalian Retinoblastoma (Rb) protein family, collectively called pocket proteins, regulate entry into and exit from the cell cycle [1-5]. Although pRb plays a dominant role, the Rb-like homologs p130 and p107 represent the ancestral proteins [6, 7] and functionally overlap with pRb to repress cell cycle gene expression during cellular quiescence (G0) [8-10]. Like pRb, p130 and p107 interact with an E2F-DP transcription factor heterodimer [11-13]. Unlike pRb, they also interact with the highly conserved 5-subunit MuvB complex, forming the DREAM (for Dp, Rb-like, E2F, and MuvB) complex, which mediates transcriptional repression through MuvB [8, 14-17]. To address how the Rb-like pocket protein contributes to MuvB-mediated gene repression, we disrupted the interaction between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit LIN-52 using CRISPR/Cas9 targeted mutagenesis. Disrupting the LIN-35-MuvB association did not affect DREAM chromatin occupancy but did cause a highly penetrant synthetic multivulval (SynMuv) phenotype, indicating that blocking DREAM assembly impairs MuvB function. Some DREAM target genes became derepressed, indicating that for those genes MuvB chromatin binding alone is not sufficient for gene repression and that direct LIN-35-MuvB association potentiates MuvB’s innate repressive activity. Our previous study [17] showed that in worms lacking LIN-35, E2F-DP and MuvB chromatin occupancy is reduced genome-wide. With LIN-35 present, this study demonstrates that the E2F-DP-LIN-35 interaction promotes E2F-DP’s chromatin localization, which we hypothesize supports MuvB chromatin occupancy indirectly through DNA. Altogether, this study highlights how the pocket protein family may recruit regulatory factors like MuvB to chromatin through E2F-DP to facilitate their transcriptional activity.
Results and Discussion
The pRb-like pocket proteins (in mammals, p130/p107) interact with repressive E2F-DPs (in mammals, E2F4/5-DP1/2) and a 5-subunit subcomplex called MuvB (in mammals, LIN9, LIN37, LIN52, LIN54, and RBAP48) to form the DREAM transcriptional repressor complex (Figure 1A) [8, 18-20]. E2F-DP and LIN54 direct site-specific chromatin localization [21-25], and the Rb-like pocket protein scaffold serves as a bridge between the 2 DNA-binding DREAM components [18]. The pocket protein-associated complex MuvB, isolated first in Drosophila melanogaster [14, 15] and Caenorhabditis elegans [16] before homologs were identified in mammals [8, 19, 20], mediates gene repression in the context of DREAM [17]. In C. elegans, LIN-35 represents the sole pocket protein, most closely resembling p130/p107 [26]. The C. elegans complex, called DRM, similarly regulates cell cycle genes [27], but also regulates cell fate specification by antagonizing Ras signaling during vulval development [16, 28, 29] and by protecting somatic cells from expressing germline genes [30, 31]. We previously reported how LIN-35 loss resulted in a genome-wide decrease in chromatin occupancy of both E2F-DP and MuvB, illustrating how DRM/DREAM disassembly likely proceeds during cell cycle progression [17]. However, our previous findings raised questions about how the pocket protein contributes to DRM/DREAM assembly and function.
Targeted mutagenesis to disrupt DREAM complex formation
Structural studies demonstrated that MuvB interacts with the pocket protein via the LIN52 subunit (Figure 1A) [18]. Using the self-excising cassette (SEC) method for C. elegans CRISPR/Cas9 [32], we generated a lin-52(KO) strain (lin52(bn133[lin-52p::TagRFP-T::3xFLAG]) by completely replacing the lin-52 gene with TagRFP-T coding sequence (Figure 1C). We observed that lin-52(KO) rendered worms sterile (Figure 1E), as previously observed in the lin-52(n3718) protein null strain [16, 33]. Loss of LIN-9, LIN-53 (C. elegans RBAP48), or LIN-54 in protein null strains also renders worms sterile and affects the levels of other MuvB subunits, suggesting that MuvB components require co-expression for assembly/stability of the complex [16]. Loss of LIN-37 does not cause sterility and does not affect assembly of the rest of MuvB in either C. elegans or mammalian cells [16, 34]. We next replaced the TagRFP-T coding sequence with lin-52 tagged with a C-terminal GFP-3xFLAG coding sequence, generating the lin-52(WT) strain (lin-52(bn139[lin-52::GFP::3xFLAG]), Figure 1C). We observed that lin-52(WT) completely rescued fertility (Figure 1E), indicating that the GFP tag does not disrupt LIN-52 function.
Since LIN-52 is essential for C. elegans fertility, we sought to disrupt the LIN-35-LIN-52 interaction without affecting protein integrity. The mammalian LIN52 protein interacts with the pocket protein LxCxE binding cleft via a suboptimal LxSxExL sequence which is rendered optimal by a nearby S28 phosphorylation site [18] (Figure 1B). S28 phosphorylation by DYRK1A kinase induces formation of mammalian DREAM [35]. In C. elegans, the conserved lin-52 gene encodes the optimal LxCxE sequence (Figure 1B). Additionally, since C. elegans lacks a DYRK1A homolog and its corresponding consensus motif RX(X)(S/T)P in LIN-52 (Figure 1B), C. elegans DREAM likely does not utilize a phospho-switch to induce DREAM formation [18, 35]. Importantly, the LxCxE binding motif mediates the high-affinity interaction that is employed by the human papillomavirus (HPV) viral oncoprotein E7 to disrupt association of LIN52 with mammalian pocket protein [18]. Therefore, we targeted the LIN-52 LxCxE sequence using CRISPR/Cas9-mediated precision mutagenesis. We generated 2 mutants of the LxCxE binding motif in lin-52(WT) using the dpy-10 co-CRISPR method of small oligo homology-directed repair [36]. We generated the lin-52(1A) single alanine mutation strain (lin-52(bn150[lin-52[C44A]::GFP::3xFLAG)) and the lin-52(3A) triple alanine mutation strain (lin-52(bn151[lin-52[L42A,C44A,E46A]::GFP::3xFLAG)) with the intent to completely disrupt LIN-52’s interaction with the C. elegans pocket protein LIN-35 (Figure 1D). Additional silent mutations were included in the oligo repair templates to generate new restriction enzyme cut sites to aid in genotyping (Figure 1D).
Full loss of C. elegans DREAM activity causes sterility, as observed in protein null mutants of worm E2F-DP (dpl-1 and efl-1) and worm MuvB (lin-9, lin-52, lin-53, and lin-54) [37-39]. Since the C-terminally GFP-tagged lin-52 coding sequence completely rescued lin-52(KO) sterility, we were able to test whether lin-52(1A) and lin-52(3A) disrupt DREAM function. We observed that neither the 1A nor 3A mutation in the LIN-52 LxCxE sequence caused a significant reduction in brood size (Figure 1E). Using western blot analysis of selected DREAM components from lin-52(WT) and mutant lysates, we observed that DREAM component protein levels were unaffected compared to N2 (Bristol) (Figure 1F, Figure S1). Similarly, using live image analysis of lin-52(WT), lin-52(1A), and lin-52(3A) L4 larvae, we observed that LIN-52 level and localization appeared normal in mutants (Figure 1C). Together, these results demonstrate that mutation of the LIN-52 LxCxE sequence does not cause a lin-52 null phenotype and does not alter the levels and tissue distribution of MuvB components.
Blocking DREAM complex formation recapitulates the classic SynMuv phenotype
C elegans DREAM components were initially identified in genetic screens for a Synthetic Multivulval (SynMuv) phenotype [16, 26, 33, 40]. All 8 components of DREAM were classified as SynMuv B genes; double mutant worms bearing a mutation in a SynMuv B gene along with a mutation in a SynMuv A gene have multiple vulvae along their ventral body instead of the usual single vulva [41]. We hypothesized that if DREAM function was affected by mutation of LIN-52’s LxCxE sequence, then pairing our 1A and 3A LIN-52 mutations with a SynMuv A mutation should generate a SynMuv phenotype. SynMuv A alleles lin-8(n2731) [42] or lin-15A(n767) [43] resulted in a SynMuv phenotype when paired with lin-52(3A) but not with lin-52(1A) or as expected with lin-52(WT) (Figure 2A). These results indicate that the triple alanine substitution of LxCxE affects DREAM function.
To test whether the triple alanine substitution in fact impaired pocket protein-MuvB association, we performed co-immunoprecipitations (co-IPs) from protein extracts prepared from lin-52(WT), lin-52(1A), and lin-52(3A) late embryos. We pulled down LIN-35 and tested for LIN-52 association using the GFP epitope, and we pulled down LIN-52 using either the GFP or FLAG epitope and tested for LIN-35 association (Figure 2B, Figure S2). In both co-IP experiments, we observed that LIN-52 association with LIN-35 was lost in lin-52(3A) extracts but not in lin-52(1A) extracts. These results demonstrate that the LIN-52 triple alanine substitution successfully severed the protein-protein association between LIN-52 and LIN-35, effectively blocking formation of an intact DREAM complex.
E2F-DP-LIN-35 and MuvB subcomplexes independently co-occupy chromatin sites
In the absence of LIN-35, E2F-DP and MuvB do not associate with one another and their chromatin occupancy is reduced genome-wide [17]. In our lin-52(3A) worm strain, LIN-35 is present, but its association with MuvB is severed. We tested the impact of this severing on the chromatin localization of DREAM components using chromatin immunoprecipitation (ChIP). We chose 4 genes, set-21, mis-12, polh-1, and air-1, as representative DREAM target genes; in lin-35 null embryos, the chromatin occupancy of DREAM components was greatly diminished at each of their gene promoters [17]. Importantly, DREAM component chromatin occupancy was undetectable at the air-1 promoter in the absence of LIN-35 [17]. We observed that all tested DREAM components remained similarly enriched at the 4 selected promoters in lin-52(3A) as compared to lin-52(WT) (Figure 3A). An additional 6 DREAM target gene promoters were tested and showed similar DREAM occupancy profiles (Figure S3A). This included C. elegans E2F-DP (DPL-1 and EFL-1) and LIN-35, suggesting that the chromatin association of the repressive E2F-DP transcription factor heterodimer is stabilized by its interaction with the pocket protein.
To test whether MuvB and E2F-DP-LIN-35 co-occupy DREAM target regions, we performed sequential ChIP analysis. We first ChIPed LIN-52 via its FLAG tag and then ChIPed LIN-35. We observed no significant difference in LIN-35 co-occupancy in lin-52(3A) extracts vs. lin-52(WT) extracts (Figure 3B). Our results indicate that, although the interaction of LIN-35 and MuvB is disrupted, DREAM components nevertheless co-localize at target promoters through their respective protein-DNA interactions. We previously observed that in the absence of LIN-35, E2F-DP and MuvB protein-DNA interactions were not sufficient for robust chromatin localization [17]. Importantly, in vitro analysis of heterodimeric mammalian E2F-DP complexes identified a distinct induction of DNA bending, especially in the case of the homologues of C. elegans EFL-1-DPL-1 (E2F-4/DP-1/2) [44]. Therefore, we propose that DREAM-associated E2F-DP heterodimers promote MuvB co-occupancy through a DNA bending-dependent mechanism. Together, our results suggest a model in which the LIN-35 pocket protein promotes E2F-DP chromatin occupancy, which in turn promotes MuvB chromatin occupancy.
Severing the LIN-35-MuvB connection impairs transcriptional repression of some but not all DREAM target genes
MuvB dissociation from E2F-DP-LIN-35 resulted in no observed loss in chromatin occupancy of DREAM at the 10 gene promoters tested (Figure 3A, Figure S3A). Each of the gene products targeted by the 4 selected promoter regions in Figure 3A was upregulated in the lin-35 null strain [17, 45]. We performed gene expression analysis of these 4 genes in lin-52(WT), lin-52(1A), and lin-52(3A) late embryos using RT-qPCR (Figure 3C). We observed that 2 genes, set-21 and polh-1, were significantly upregulated in both lin-52 mutant strains, while 2 genes, mis-12 and air-1, were not up-regulated. Transcript levels of each of the gene products targeted by the 6 selected promoter regions in Figure S3A were not affected (Figure S3B). Importantly, air-1 upregulation in the lin-35 null strain was accompanied by complete loss of MuvB promoter association [17]. Thus, MuvB chromatin occupancy is necessary but not sufficient for repression of DREAM target genes. Our findings reveal that the LIN-35-MuvB association potentiates MuvB-mediated transcriptional repression but is not required.
Outlook and Future Work
The trio of pocket proteins, pRb, p107, and p130, govern cell cycle exit and reentry through targeted transcriptional repression of cell cycle genes. We analyzed how the C. elegans Rb-like pocket protein LIN-35 contributes to the formation and function of the DREAM complex, which relies on the recruitment of the highly conserved and essential 5-subunit MuvB complex to direct target gene repression. Using CRISPR/Cas9-mediated targeted mutagenesis, we generated a mutant C. elegans strain in which MuvB’s LIN-35-interacting subunit LIN-52 was rendered incapable of interacting with LIN-35. This LIN-52 mutant recapitulated the classic Synthetic Multivulval phenotype observed in all C. elegans DREAM mutants that perturb its ability to repress genes. We determined that while LIN-35 and MuvB association was lost, the LIN-35 and E2F-DP occupancy on chromatin was unchanged. Additionally, even without direct protein-protein association, MuvB co-occupied sites with the heterotrimeric E2F-DP-LIN-35 complex. Our results highlight that the pocket protein stabilizes E2F-DP chromatin occupancy, which we hypothesize in turn supports MuvB occupancy potentially through local alteration of DNA shape.
Our results support an exciting model for how local E2F-DP-mediated alterations to DNA shape enhanced by their interaction with a pocket protein promote MuvB co-occupancy. Even with evolutionary divergence from the ancestral pocket protein, this model may also apply to pRb function. Many histone deacetylases and chromatin remodeling complexes associate with pRb through the LxCxE binding cleft, although many of these associations have only limited support thus far from structural/biochemical interaction studies [46]. Variation in pRb monophosphorylation events that can alter pRb structure and recognition of binding partners offered one explanation for how pRb can potentially interact with >300 individual protein partners [47, 48]. Our data provide an alternative, but not exclusive, mechanism for how direct and stable pRb association with these complexes may be unnecessary. Perhaps pRb association with E2F-DPs promotes localization of these complexes to genomic sites. Additional dissection of DREAM and pRb structure and function will shed light on how the pocket proteins mediate their essential cellular roles.
Author Contributions
Conceptualization, P.D.G and S.S. Methodology, P.D.G. Investigation, P.D.G. Writing – Original Draft, P.D.G. Writing – Review & Editing, P.D.G. and S.S. Funding Acquisition, P.D.G. and S.S. Resources, P.D.G. Supervision, S.S.
Declaration of Interests
The authors declare no competing interests.
Methods
Contact for Reagent and Resource Sharing
Requests for information, strains, and reagents should be directed to and will be fulfilled by Paul D. Goetsch (pdgoetsc{at}mtu.edu)
Experimental Model and Subject Details
Strains were cultured on Nematode Growth Medium (NGM) agarose plates with E. coli OP50 and incubated at 20°C. Experiments were performed on embryos, L4 larvae, and young adult hermaphrodites as indicated, with males used for genetic crosses. Genotyping of genome edited strains and progeny of subsequent genetic crosses was performed on single worm lysates using standard techniques with primers indicated in the Key Resources Table (Table 1).
Method Details
CRISPR/Cas9-mediated genome editing
To generate lin-52(KO), 2 Cas9 target sites were identified near the 5’ and 3’ ends of the gene using the MIT CRISPR design tool (http://crispr.mit.edu). The 20 nucleotide crDNA targeting sequences were cloned into the PU6::unc119_sgRNA vector (Addgene plasmid #46169) using the overlapping PCR fragment method described in [49]. The lin-52 KO homologous repair template was generated by amplifying homology arms containing the lin-52 promoter and lin-52 3’ UTR and cloned into the N-terminal tag digested pDD284 vector (Addgene plasmid #66825) using Glibson Assembly (New England Biolabs) [50], as described in [32]. The following CRISPR/Cas9 and co-injection marker [51] plasmid mix was microinjected into the germline of ∼50 N2 young adults: 50 ng / µL Cas9 expression plasmid (pDD162, Addgene #47549), 2.5 ng / µL Pmyo-2::mCherry::unc-54utr (pCJF90, Addgene #19327), 5 ng / µL Pmyo-3::mCherry::unc-54utr (pCFJ104, Addgene #19328), 10 ng / µL pRAB-3::mCherry::unc-54utr (pGH8, Addgene #19359), 50 ng / µL lin-52 5’ sgRNA (pPDG14), 50 ng / µL lin-52 3’ sgRNA (pPDG18), and 10 ng / µL lin-52p::TagRFP-T^SEC^3xflag::lin-52 3’ UTR (pPDG13). CRISPR/Cas9-positive progeny were treated with hygromycin and screened for the Roller phenotype and absence of fluorescent co-injection marker expression (the latter eliminates false-positive extrachromosomal arrays). Individuals from 1 positive selection plate were selected and balanced to create the strain SS1240 lin-52(bn132(lin-52p::TagRFP-T^SEC^3xflag::lin-52 3’ UTR)) III / hT2G [bli-4(e937) let-?(q782) qIs48] (I:III) [52]. The self-excising cassette (SEC) was removed by a 4-5 hour heat-shock of L1 larvae at 32°C. Non-Roller F1 progeny were isolated to create the strain SS1241 lin-52(bn133(lin-52p::TagRFP-T::3xflag::lin-52 3’ UTR)) III / / hT2G [bli-4(e937) let-?(q782) qIs48] (I:III).
To generate lin-52(WT), 2 Cas9 target sites were identified near the 5’ and 3’ ends of the TagRFP-T-3xFLAG coding sequence using the MIT CRISPR design tool. The 20 nucleotide crDNA targeting sequences were cloned into the pDD162 vector using the Q5 Site Directed Mutagenesis Kit (New England Biolabs), as described in [53]. The lin-52 WT homologous repair template was generated by amplifying homology arms containing the lin-52 promoter with the gene’s coding sequence and the lin-52 3’ UTR and cloned into the C-terminal tag digested pDD282 vector using Gibson Assembly, as described in [32]. The following CRISPR/Cas9 and co-injection marker plasmid mix was microinjected into the germline of ∼50 SS1241 young adults: 50 ng / µL TagRFP-T 5’ sgRNA-Cas9 vector (pPDG21), 50 ng / µL TagRFP-T 3’ sgRNA-Cas9 vector (pPDG22), 2.5 ng / µL pCJF90, 5 ng / µL pCFJ104, and 10 ng / µL lin-52p::lin-52 CDS-GFP^SEC^3xflag::lin-52 3’ UTR (pPDG17). CRISPR/Cas9-positive progeny were treated with hygromycin and screened for the Roller phenotype and absence of fluorescent co-injection marker expression. Individuals from 2 of 3 positive selection plates were selected and made homozygous to create strains SS1325 and SS1326 lin-52(bn138(lin-52::GFP^SEC^3xflag)) III. The SEC was removed by heat-shock, and non-Roller F1 progeny were isolated to create the strains SS1256 and SS1257 lin-52(bn139(lin-52::GFP::3xflag)) III. SS1256 was backcrossed 6 times to generate strain SS1272, which was used in downstream experiments.
To generate lin-52(1A) and lin-52(3A), 1 Cas9 target site was identified near the LxCxE coding sequence using the MIT CRISPR design tool and cloned into the pDD162 vector, as described above. Single strand DNA templates included at least 40 base pairs of homology flanking the LxCxE coding sequence and silent mutations to aid genotyping, as illustrated in Figure 1C. The following/Cas9 and co-injection marker plasmid mix was microinjected into the germline of 6 (for 1A) and 10 (for 3A) SS1256 young adults: 40 ng / µL lin-52 LxCxE sgRNA-Cas9 vector (pPDG59), 2.5 ng / µL pCJF90, 5 ng / µL pCFJ104, 20 ng / µL lin-52 mutagenesis ssDNA template (1A or 3A), 40 ng / µL dpy-10(cn64) sgRNA (pJA58, Addgene plasmid #59933), and dpy-10(cn64) ssDNA template. dpy-10(cn64) guide and ssDNA template were co-injected to select for positive CRISPR activity in injectant progeny, as described in [36]. Injected adults were cloned onto individual plates, and F1 progeny were screened for presence of a Roller (Rol) and/or Dumpy (Dpy) phenotype. Individual Rol and/or Dpy progeny were genotyped, resulting in 3 independent lin-52(1A) and 2 independent lin-52(3A) strains. Each strain was backcrossed 6 times to create SS1273-SS1275 lin-52(bn150(lin-52[C44A]::GFP::3xflag)) III, and SS1276 and SS1277 lin-52(bn151(lin-52[L42A,C44A,E46A]::GFP::3xflag)) III. SS1273 and SS1276 were used in downstream experiments.
Microscopy
L4 larvae were mounted on a 10% agarose pad and immobilized with a 1-2 µL suspension of 0.1 µm polystyrene beads (Polysciences), as described in [54]. Fluorescence images were acquired using a Solamere spinning-disk confocal system with µManager software [55]. The microscope setup was as follows: Yokogawa CSUX-1 spinning disk scanner, Nikon TE2000-E inverted stand, Hamamatsu ImageEM X2 camera, solid state 405-, 488-, and 561-nm laser lines, 435–485, 500–550, and 573–613 fluorescent filters, and Nikon Plan Fluor 40x air objective. Images were processed using Image J [56].
C. elegans phenotype scoring
For brood size analyses, L4 individuals were cloned to fresh plates every 24 hours and all progeny were counted. For SynMuv phenotype scoring, 3 replicate plates per strain were set up with 5-10 adults that were allowed to lay eggs for 6 hours. Progeny were incubated at 20°C for 3 days, then scored for the presence or absence of pseudovulvae. The percentages of multivulva worms in each replicate population were averaged, and the standard deviation was calculated.
Immunoblotting and co-immunoprecipitation (coIP)
For immunoblotting whole worm lysates, 200 adults from each strain were picked into SDS gel-loading buffer (50 mM pH 6.8 Tris-Cl, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 100 mM β-mercaptoethanol). For coIP, embryos collected after bleaching gravid worms were aged for 3.5 hours before freezing them in liquid nitrogen. Lysates were prepared by grinding frozen embryos using a mortar and pestle, resuspending in lysis buffer (25 mM HEPES pH 7.6, 150 mM NaCl, 1mM DTT, 1mM EDTA, 0.5 mM EGTA, 0.1% Nonidet P-40, 10% glycerol) with Complete EDTA-free Protease Inhibitors (Roche), and sonicating twice for 30 seconds. Lysates were clarified and precleared using a mix of Protein A and Protein G Dynabeads (ThermoFisher). Protein concentrations of coIP lysates were determined using a Qubit fluorometer (ThermoFisher). For each IP, 5 μg of anti-FLAG was crosslinked to Protein G Dynabeads and 2 μg of anti-GFP or anti-LIN-35 was crosslinked to Protein A Dynabeads using dimethyl pimelimidate in 0.2 M trimethylamine pH 8.2. Crosslinking was stopped using 0.1M Tris pH 8.0, and beads were washed with 0.1 M glycine pH 2.8 before being stored in phosphate buffered saline pH 7.2 with 0.05% Tween-20. For each IP, 8 mg of protein lysate was mixed with antibody-conjugated Dynabeads and incubated for 2 hours at 4°C. Each IP was washed with lysis buffer, and eluted with 50 μL 2x SDS gel-loading buffer for 5 minutes at 98°C
Proteins were separated by SDS/PAGE, and western blot analysis was performed using a 1:1,000-1:5000 dilution of primary antibody and 1:2,000 dilution of an appropriate HRP-conjugated secondary antibody. Serial western blot analysis was performed by stripping the blot with buffer containing 0.2M pH 2.2 glycine, 0.1% SDS, and 1% Tween-20 between antibody probings.
Chromatin immunoprecipitation (ChIP) and sequential ChIP
Embryos collected after bleaching gravid worms were aged for 3.5 hours before freezing them in liquid nitrogen. Lysates were prepared by grinding, crosslinking for 10 minutes in 1% formaldehyde, and sonicating to an average size of 250 base pairs in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl) using a Bioruptor (Diagenode) on the high setting with 60 rounds of 30 seconds on and 1 minute rest. Protein concentrations of lysates were determined using a Qubit fluorometer.
For ChIP, chromatin extracts were precleared with Protein A Dynabeads. ChIPs were performed with 2 mg of extract and 1 μg of antibody, with 2% of the extract set aside for an input reference control. ChIPs were incubated overnight at 4°C with 1% sarkosyl. Protein A Dynabeads equilibrated in 20 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs were washed with the following buffers: once with FA buffer containing 1 M NaCl, once with FA buffer containing 0.5 M NaCl, once with TEL buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). 2 elutions of 50 μL elution buffer containing TE plus 1% SDS and 250 mM NaCl were incubated at 55°C. Eluted ChIP and input samples were incubated with proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation using glycogen as a carrier. Quantitative PCR was performed using SYBR green reagents on an Applied Biosystems ViiA 7 Real-Time PCR System (ThermoFisher).
For sequential ChIP, chromatin extracts were precleared with Protein G Dynabeads and 4 parallel ChIPs per replicate were performed with 2.5 mg of extract and 2.5 μg of anti-FLAG antibody, with 2% of the extract set aside for an input reference control. ChIPs were incubated overnight at 4°C with 1% sarkosyl. Protein G Dynabeads equilibrated in 20 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs for each replicate were washed as described above and pooled. 2 elutions of 50 μL 0.1M NaHCO3plus 1% SDS were incubated at 55°C for 15 minutes. Elutions were divided, diluted with FA buffer with 1% sarkosyl, and incubated with anti-LIN-35 or IgG as a negative control, with 10% of the elution set aside as a reference control. The 2nd ChIP was incubated overnight at 4°C. Protein A Dynabeads equilibrated in 20 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs were washed and eluted twice with 50 μL elution buffer with incubation at 55°C. Eluted ChIP, reference, and input samples were incubated with proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation using glycogen as a carrier. Quantitative PCR was performed similarly to above.
Analysis of transcript levels by RT-qPCR
Embryos collected after bleaching gravid worms were aged for 3.5 hours before freezing them in Trizol for RNA isolation. A total of 1 μg RNA was treated with DNase and reverse transcribed using the High Capacity cDNA Kit (Applied Biosystems). qPCR was performed using SYBR green reagents on an Applied Biosystems ViiA 7 Real-Time PCR System (ThermoFisher). The relative quantity of experimental transcripts was calculated with act-2 as the control gene using the ΔCt method.
Quantification and statistical analysis
For brood size analysis, significance was determined using a Wilcoxon-Mann-Whitney test comparing CRISPR/Cas9-genome edited strains to N2 (Bristol). For ChIP-qPCR and transcript level analysis by RT-qPCR, significance was determined using a student’s T Test between lin-52(WT) and lin-52(1A) and lin-52(3A).
Acknowledgments
We thank Seth Rubin and members of the Rubin and Strome labs for helpful discussions. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by National Institutes of Health R01 grant GM34069 to S.S. and American Cancer Society Postdoctoral Fellowship PF-16-106-01-DDC to P.D.G.
Footnotes
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