METHOD DETAILS

In order to map the binding sites for Treslin-MTBP in the human genome, we set out to attach a 3X-FLAG tag onto the endogenous MTBP by using the CRISPR-Cas9 system. Our previous studies have indicated that Treslin and MTBP associate quantitatively with one another and also mutually depend upon each other for stability (Kumagai and Dunphy, 2017). Accordingly, the localization of MTBP would reflect the presence of the Treslin-MTBP complex. As a consideration for this approach, we also wished to verify that cells could replicate their DNA normally in the presence of only a FLAG-tagged version of MTBP. We also wanted to establish a system in which we could examine mutants of MTBP. We used a multi-step strategy to address these issues. First, we tagged the endogenous copies of MTBP in a human cell line with the mini auxin-inducible degron (mAID). We initially considered using HCT-116 colon cancer cells for these experiments due to the fact that use of the AID system has been well characterized in these cells (Natsume et al., 2016). However, HCT-116 cells have three copies of the gene encoding MTBP due to a chromosomal duplication, which complicated the use of CRISPR-Cas9 technology (Tym et al., 2016). Accordingly, we chose to use human DLD-1 cells, a similar colorectal adenocarcinoma cell line. Other advantages of DLD-1 cells are that they are pseudo-diploid and maintain relatively normal chromosomes.

To introduce OsTIR1 into the safe-harbor AAVS1 locus, we transfected DLD-1 cells (2 mL culture) with 1 μg AAVS1 T2 CRISPR in pX330 (Addgene #72833) and 1μg pMK232 (CMV-OsTIR1-PURO) (Addgene #72834) by using 8 μl Lipofectamine 2000. After 48 hr, cells were diluted and plated in DMEM containing 2 μg/ml puromycin. After 12 days, clones were picked and grown on 24-well plates. Genotyping was performed for each clone by PCR with LongAmp Taq DNA polymerase.

CRISPR/Cas9 vectors were constructed according to a standard protocol (Ran et al., 2013) using pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem(1/110) (Gutschner et al., 2016) with guide oligonucleotides for Exons 19 and 22 of MTBP to yield pX330-Cas9-MTBP-Exon19 and pX330-Cas9-MTBP-Exon22, respectively. Donor template arms for Exons 19 and 22 of MTBP (1,600 bp each) were amplified from genomic DNA of DLD-1 cells using Q5 DNA polymerase and cloned into pBluescript to yield pBlue-script-MTBP-Exon19 and pBluescript-MTBP-Exon22, respectively. DNA fragments corresponding to mAID-Neo, mAID-Hygro, and 3X-FLAG-Hygro were derived from pMK286 (Addgene plasmid # 72824), pMK287 (Addgene plasmid # 72825), and pMK284 (Addgene plasmid # 72800), respectively. These fragments were cloned into pBluescript-MTBP-Exon22 to make an in-frame fusion of the MTBP C-terminal end and these tags (pBluescript-MTBP-mAID-Neo, pBluescript-MTBP-mAID-Hygro, and pBluescript-MTBP-WT-FLAG-Hygro). In addition, the sequence 3X-FLAG-Hygro from pMK284 was cloned into pBlue-script-MTBP-Exon19 to make an in-frame fusion after amino acid 817 in order to delete 87 amino acids from the C-terminal end of MTBP (pBluescript-MTBP-ΔC-FLAG-Hygro). The appropriate silent mutations were introduced into the gRNA target sites of all the donor templates.

For preparation of MTBP-mAID/MTBP-mAID and MTBP-mAID/MTBP-WT-FLAG cell lines, DLD-1 cells harboring CMV-OsTIR1 were transfected with pX330-Cas9-MTBP-Exon 22 and combinations of either pBluescript-MTBP-mAID-Neo and pBluescript-MTBP-mAID-Hygro or pBluescript-MTBP-mAID-Neo and pBluescript-MTBP-WT-FLAG-Hygro. Cells were selected in the presence of both 300 μg/ml Hygromycin and 800 μg/ml G418. Drug-resistant clones were selected for genotyping.

For preparation of the MTBP-mAID/MTBP-ΔC-FLAG cell line, DLD-1 cells harboring CMV-OsTIR1 were transfected with pX330-Cas9-MTBP-Exon22 and pBluescript-MTBP-mAID-Neo. Cells were selected in the presence of 800 μg/ml G418. Drug-resistant clones were picked for genotyping to identify cell lines in which both MTBP alleles were tagged with mAID at the C-terminal end. These cells were transfected with pX330-Cas9-MTBP-Exon19 and pBluescript-MTBP-ΔC-FLAG-Hygro. Clones were selected in the presence of both 300 μg/ml Hygromycin and 800 μg/ml G418. Drug-resistant clones were picked for genotyping to identify cell lines with one allele of MTBP-mAID and one allele of MTBP-ΔC-FLAG.

Cells were arrested in G1 in the presence of 1 μM Cdk4/6i for 20 hr. Cells were labeled with 10 μM EdU for 20 min either at the end of this arrest or at various times after release from arrest and incubation in the absence or presence of 500 μM auxin. After fixation in 2% formaldehyde for 10 min, cells were permeabilized in phosphate-buffered saline (PBS) containing 0.5% Triton X-100. Click reactions were performed in Tris-buffered saline (TBS) containing 4 μM CuSO₄, 4 mM Alexa Fluor 488 azide, and 10 mM sodium ascorbate. Nuclei were stained with 0.5 μg/ml DAPI for 10 min. Images were obtained with a Zeiss LSM 800 microscope with a 20 × Plan-Apochromat objective in wide-field mode. Incorporation of EdU and staining with DAPI in individual nuclei were quantified with CellProfiler. More than 1,500 nuclei were counted for each time point. For determination of replication-timing patterns, images were obtained with a Zeiss LSM 800 laser scanning confocal microscope using a 63 × Plan-Apochromat objective.

For depletion of the MTBP-mAID protein, asynchronous cells were incubated in the presence of auxin for 16 hr prior to harvesting. CUT&RUN reactions were performed on 500,000 cells bound to BioMag Plus Concanavalin A beads (Bangs Laboratory) according to the original protocol (version 1) (Skene et al., 2018; Skene and Henikoff, 2017). Cells were incubated with mouse monoclonal anti-FLAG M2 antibody (Sigma) at 10 μg/ml in 200 μl digitonin-wash buffer containing 0.04% digitonin (Millipore #300410). Next, cells were incubated successively with rabbit anti-mouse IgG and protein A-micrococcal nuclease (pA-MNase). Thereafter, CaCl₂ was added to initiate digestion with pA-MNase, and DNA fragments were subsequently released by incubation at 37°C for 10 min in Stop buffer containing yeast spike-in DNA. The use of yeast spike-in DNA allowed a quantitative comparison between samples. After removal of magnetic beads containing undigested chromatin, released DNA fragments were purified using the Monarch PCR & DNA Cleanup Kit (NEB). Purified fragments were end-repaired, adaptor-ligated, and PCR-amplified with NEBNext Multiplex Oligos using the NEBNext Ultra II DNA Library Prep Kit. Adapters and PCR primers were removed with AMPure beads (Beckman Coulter). Libraries were sequenced in the paired-end mode (150 bp) at a commercial facility (Novogene) on an Illumina HiSeq platform. For CUT&RUN analysis of H3K4me2, we incubated auxin-treated MTBP-mAID/MTBP-WT-FLAG cells with anti-H3K4me2 rabbit antibodies or control rabbit antibodies and thereafter with pA-MNase.

CUT&RUN sequence data was processed using Trim Galore (version 0.5.0) to remove adaptor and low-quality reads (q < 20). Next, reads were mapped to the human hg38 genome assembly (GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index) as described (Skene et al., 2018) using Bowtie2 (version 2.3.5) with options:–local–very-sensitive-local–no-unal–no-mixed–no-discordant–phred33 -I 10. Reads that mapped to mitochondrial DNA and random contigs were removed. Also, reads were mapped to the yeast genome assembly in order to quantify yeast spike-in DNA. MACS2 (version 2.1.2) was used to call peaks on the reads for each of three replicates for the MTBP-WT and MTBP-ΔC CUT&RUN experiments using options:–keep-dup 1 -f BAMPE–nolambda -q 0.01. Peaks identified in at least two replicates were selected as high-confidence peaks. For H3K4me2 CUT&RUN, we performed the experiments twice and identified peaks that overlapped in the replicates. Peaks were called again on pooled replicated reads in MACS2 and reproducible peaks were identified. These reproducible peaks were used for analyses unless indicated otherwise. Summits were also called in MACS2 and the highest summit for each peak was identified.

Genome browser coverage track (bigwig) files were generated using Deeptools (version 3.3.0) bamCoverage with parameters:–binsize 10–normalizeUsing RPGC (reads per genomic content)–ignoreDuplicates–blackListFileName hg38.blacklist.bed (obtained from http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/hg38-human/)–scaleFactor x. Spike-in-ratios (number of reads aligned to human genome)/(number of reads aligned to yeast genome) were calculated for each experiment. Scale factors were calculated by using spike-in-ratio (control or MTBP-ΔC)/spike-in-ratio (MTBP-WT) to normalize the reads to MTBP-WT. Scale factors ranged between 0.82 and 1.09. Three replicated genome browser track files were combined using bigWigMerge and the resultant bedgraph file was converted to a bigwig file with bedGraphToBigWig. Quantification of peaks was performed with multiBigwigSummary in Deeptools using the BED-file mode with MTBP peaks identified in MACS2.

Locations of TSSs were downloaded and extracted from GENCODE (gencode.v32.basic.annotation.gtf). Regions from 2 kb upstream to 2 kb downstream of each TSS were designated as promoter-TSS. Locations of enhancer and super-enhancers from HCT-116 cells were obtained from Hnisz et al. (2013).

Annotation of peaks and determination of the location of peaks relative to TSSs were performed with HOMER (annotatePeaks.pl) using the GENCODE (v32) basic gene annotation file. HOMER (findMotifsGenome.pl) was used to search for motifs enriched in the MTBP peaks. Locations of AP-1, RUNX, and TEAD motifs in the genome were identified using HOMER (scanMotifGenomeWide.pl). G4 motifs in human genome were mapped using G4Hunter with a strict threshold of 2 (Bedrat et al., 2016).

5 × 10⁶ DLD-1 cells (MTBP-mAID/MTBP-WT-FLAG and MTBP-mAID/MTBP-ΔC-FLAG) were arrested in G1 in the presence of 1 μM Cdk4/6i for 20 hr. Cells were washed and released from the arrest into fresh medium containing 500 μM auxin, 10 μM EdU, 2 mM HU, and 0.5 μg/ml aphidicolin for 3 hr (WT_EdU and ΔC_EdU). HU and aphidicolin cause arrest in early S-phase and aphidicolin further limits elongation at existing nascent strands. One batch of MTBP-WT cells was also incubated without release from G1 in the presence of 1 μM Cdk4/6i as well as 500 μM auxin, 10 μM EdU, 2 mM HU, and 0.5 μg/ml aphidicolin for 3 hr (Control_EdU). Following incubation, cells were washed and incubated in fresh medium lacking EdU, HU, and aphidicolin for another 30 min to allow for extension of replication forks. Cells were trypsinized, washed in PBS twice, and fixed with 70% ice-cold ethanol. After an overnight incubation, cells were washed in PBS and incubated in click-reaction buffer containing 100 μM biotin-dPEG₁₁-azide (Quanta Biodesign), 2 mM CuSO₄, and 10 mM sodium ascorbate in TBS for 15 min. After washing with TBS, cells were suspended in 0.3 mL 10 mM Tris-HCl (pH 8), 0.1 M NaCl, and 10 mM EDTA and incubated overnight at 65°C in the presence of 1% SDS and 0.3 mg/ml proteinase K. Genomic DNA was isolated by phenol/chloroform/isoamyl alcohol extraction, chloroform extraction, and finally ethanol precipitation. The DNA was sonicated to 300–500 bp with a Branson 450 sonifier. Biotinylated DNA was collected for 2 hr on 20 μl of Dynabeads MyOne Streptavidin T1 magnetic beads in 5 mM Tris-HCl (pH 7.5) containing 1 M NaCl, 0.5 mM EDTA, 0.1% Tween 20, and 1% bovine serum albumin. After extensive washing, the collected DNA was end-repaired and the appropriate adaptor was ligated to the DNA on the beads. Libraries were amplified and sequenced as described above. These experiments were conducted three times.

EdU-seq data was processed and aligned to the human hg38 genome as described for the CUT&RUN analyses. Genome browser coverage track (bigwig) files for EdU-seq were generated using Deeptools (version 3.3.0) bamCoverage with parameters:–binsize 50–normalizeUsing RPGC (reads per genomic content)–ignoreDuplicates–blackListFileName hg38.blacklist.bed (obtained from mitra.stanford.edu/kundaje/release/blacklists/hg38-human). Thus, reads for each experiment were normalized to the whole genome (RPGC, reads per genomic content). Using Deeptools bigwigCompare, log2 ratios of WT_EdU signal over control_EdU signal and ΔC_EdU signal over control_EdU signal were calculated for each replicate. The three replicates were combined to produce EdU_seq_WT.bw and EdU_seq_ΔC.bw files. Peaks of EdU-seq for MTBP-WT were called using HOMER findPeaks (region -size 6000 -minDist 12000 -F 2). Peaks from the three replicates were merged using HOMER mergePeaks to generate the Initiation_zones.txt file. For quantification of average read scores over control, average read scores were computed with Deeptools multiBigwigSummary with three bigwig files each for WT_EdU, ΔC_EdU, and control_EdU in the BED-file mode using the initiation zones file. Control zones were generated from random genomic loci using regioneR.

YY1 HiChIP data was downloaded from {"type":"entrez-geo","attrs":{"text":"GSE99521","term_id":"99521"}}GSE99521 ({"type":"entrez-geo","attrs":{"text":"GSM2774000","term_id":"2774000"}}GSM2774000) (Weintraub et al., 2017). Paired-end tags (PETs) with confidence scores > 0.9 were selected. PETs connecting adjacent bins were removed. PETs within initiation zones and randomly generated control zones of matching sizes were counted. Control zones from random genomic loci were made using regioneR.