Kumagai A and Dunphy WG (2020), Binding of the Treslin-MTBP Complex to Specific...

XB-ART-58065

Cell Rep 2020 Sep 22;3212:108178. doi: 10.1016/j.celrep.2020.108178.

Show Gene links Show Anatomy links

Binding of the Treslin-MTBP Complex to Specific Regions of the Human Genome Promotes the Initiation of DNA Replication.

Kumagai A , Dunphy WG .

???displayArticle.abstract???
The processes that control where higher eukaryotic cells initiate DNA replication throughout the genome are not understood clearly. In metazoans, the Treslin-MTBP complex mediates critical final steps in formation of the activated replicative helicase prior to initiation of replication. Here, we map the genome-wide distribution of the MTBP subunit of this complex in human cells. Our results indicate that MTBP binds to at least 30,000 sites in the genome. A majority of these sites reside in regions of open chromatin that contain transcriptional-regulatory elements (e.g., promoters, enhancers, and super-enhancers), which are known to be preferred areas for initiation of replication. Furthermore, many binding sites encompass two genomic features: a nucleosome-free DNA sequence (e.g., G-quadruplex DNA or AP-1 motif) and a nucleosome bearing histone marks characteristic of open chromatin, such as H3K4me2. Taken together, these findings indicate that Treslin-MTBP associates coordinately with multiple genomic signals to promote initiation of replication.

???displayArticle.pubmedLink??? 32966791
???displayArticle.link??? Cell Rep
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: mtbp ticrr
GO keywords: DNA replication initiation [+]

???attribute.lit??? ???displayArticles.show???

	Graphical Abstract
	Figure 1. Characterization of Cell Lines Harboring Endogenously Tagged Versions of MTBP (A) The indicated cell lines were incubated in the absence (lane 1) or presence of auxin (lanes 2 to 4) for 16 h. Cell lysates were immunoblotted with the indicated antibodies. Immunoblots were analyzed with the Odyssey system. (B) The indicated cell lines were arrested with Cdk4/6i. After 20 h, cells were incubated with EdU for 20 min (t = 0) or released from arrest in the presence or absence of auxin for 6 h prior to incubation with EdU for 20 min (t = 6 h). Incorporated EdU was labeled with Alexa 488 azide and quantified (indicated in au [arbitrary units]). Boxplots depict second quartiles, medians, and third quartiles. Representative of two experiments. (C) The indicated cell lines from (A) were arrested with Cdk4/6i for 20 h. Cells were released from arrest in the presence of auxin and incubated at the indicated times for 20 min with EdU. Alexa 488-labeled cells were observed by fluorescence microscopy and classified as being in early, mid, or late S-phase (see patterns on right). At least 200 cells were examined per time point. Representative of two experiments. Scale bar, 5 μm.
	Figure 2. MTBP Associates with Tens of Thousands of Sites in the Human Genome (A) Browser profiles of regions containing LMNB2 (top) and the HoxA cluster (bottom). Normalized read counts were plotted for CUT&RUN analyses from MTBP-WT, MTBP-ΔC, and control samples. MTBP-WT peaks are indicated with black bars. Replication origins identified by Ini-seq are marked with red bars. (B) Venn diagram of peaks for MTBP-WT and MTBP-ΔC. (C) Heatmaps of read distributions for MTBP-WT and MTBP-ΔC centered at the MTBP peak summits. Peaks identified by MACS2 in only MTBP-WT and MTBP-ΔC datasets were denoted as WT only and ΔC only, respectively. (D) Average normalized read counts for MTBP-WT and MTBP-ΔC were centered at the summits for MTBP peaks. (E) Violin plots of normalized read counts per peak for MTBP-WT and MTBP-ΔC. Medians are shown as lines and mean values are indicated. Paired Wilcoxon test was performed.
	Figure 3. Nature of Binding Sites for MTBP in the Genome (A) Classification of MTBP binding sites: promoter-TSS (10,365 peaks); enhancer/super-enhancer (5,257 peaks); and others (14,236 peaks). (B) Heatmaps of MTBP-WT, MTBP-ΔC, Orc2, and BG4 (G4-ChIP) centered around MTBP peak summits (sorted from high to low on the basis of the BG4 data). (C) TSSs were separated into classes that either contain or lack a G4 structure within 1 kb upstream and downstream. Potential G4 structures were identified with G4Hunter. Heatmaps were centered around TSSs, oriented in the direction of transcription (left to right), and sorted from high to low on the basis of the MTBP-WT data. (D) TSSs from DLD-1 cells were ordered according to RNA-seq read scores (RPKM, reads per kilobase per million mapped reads). Heatmaps for MTBP-WT, MTBP-ΔC, Orc2, BG4, and H3K27ac were centered around the TSSs. Direction of transcription is left to right.
	Figure 4. Different Sequence Elements Correlate with the Localization of MTBP at TSSs versus Enhancers (A) Density plots of MTBP peaks around TSSs and in enhancers. Top two panels: densities of MTBP peaks that contain (red) or lack potential G4 structures (blue) were plotted around TSSs (left) and the centers of enhancers (right). Mean size of enhancers is 4,150 bp. Bottom two panels: densities of MTBP peaks that contain (blue) or lack an AP-1 motif (yellow) were plotted around TSSs (left) and the centers of enhancers (right). (B) Bar graphs for numbers of MTBP peaks with predicted G4 structures and AP-1 motifs according to location. (C) Locations of AP-1 motifs mapped around the summits of MTBP peaks in the indicated categories. (D) Heatmaps of read distributions for MTBP-WT, MTBP-ΔC, MNase-seq, DNase-seq, and ChIP-seq for CTCF and YY1 around MTBP peak summits (scale is indicated in kilobases).
	Figure 5. MTBP Associates Coordinately with Adjacent Nucleosome-Containing and Nucleosome-Free Regions (A) Browser profiles of normalized read counts for MTBP-WT, MTBP-ΔC, and H3K4me2 from CUT&RUN analyses in DLD-1 cells. (B) Venn diagram of peaks for MTBP-WT and H3K4me2 in DLD-1 cells. (C) Bar graphs for the distribution of MTBP peaks among H3K4me2-containing regions that contain or lack AP-1 or G4 motifs. (D) Heatmaps of read distributions for MTBP-WT, MTBP-ΔC, and H3K4me2 from CUT&RUN in DLD-1 cells. (E) For MTBP peaks containing an AP-1 site, regions 1 kb upstream to 1 kb downstream of the MTBP peak summits were oriented so that the AP-1 site would be on the right side. Values from the indicated datasets were scaled from 0 to 1 to show the distribution of each signal around the MTBP peak summits. For original data, see Figure S5C. (F) For MTBP peaks containing a TSS, regions 1 kb upstream to 1 kb downstream of the peak summits were oriented so that the TSS would be on the right side. Values from the indicated datasets were scaled from 0 to 1 to show the distribution of each signal. For original data, see Figure S5D. (G) Cartoons summarizing features of MTBP binding sites at TSSs and enhancers.
	Figure 6. MTBP Is Concentrated in Early-Replicating Zones in DLD-1 Cells (A) Replication timing tracks from HCT-116 cells, plotted as log2(Early/Late), were downloaded from a public database (see Method Details) and segmented as follows: early (blue); mid (yellow); and late (gray). EdU-seq tracks for MTBP-WT and MTBP-ΔC, plotted as log2(WT or ΔC/control), and CUT&RUN track read coverages for MTBP-WT and MTBP-ΔC are shown. Bottom, blowup of one chromosomal region. (B) Densities of MTBP-WT peaks in early, mid, and late replication timing zones. Boxplots depict second quartiles, medians, and third quartiles. Mean values are also shown. (C) Average reads over control of EdU-seq (WT and ΔC) for each initiation zone. Boxplots depict second quartiles, medians, and third quartiles. Mean values are also listed. Paired Wilcoxon test was performed. (D) Average EdU-seq reads around MTBP-WT peaks in the following regions: promoter-TSS (green); enhancer/super-enhancer (red); and others (blue). Reads in 10,000 randomly chosen positions were also plotted (orange). (E) MTBP-WT peaks associated with the 5′ (light blue) or 3′ side of TSSs (dark blue), respectively, were separated into two groups. Normalized counts for MTBP signals in each group were plotted. (F) Average EdU-seq reads (log2 EdU/control) around TSSs with MTBP-WT located at the 5′ (light blue) or 3′ side (dark blue) and around control TSSs (orange). (G) Cartoon illustrating that MTBP can associate with either side of a TSS.
	Figure 7. MTBP-Containing, Early-Replicating Zones Contain More DNA Loops (A) Densities of MTBP-WT peaks (left) and YY1 peaks (right) in initiation and control zones. Boxplots depict second quartiles, medians, and third quartiles. Mean values are also shown. p values were determined by Wilcoxon test. (B) Heatmap of EdU-seq centered around midpoints of initiation zones. CUT&RUN reads for MTBP-WT and H3K4me2 from DLD-1 cells as well as ChIP-seq data for YY1, H3K4me1, H3K4me2, H3K4me3, H3K9ac, and H3K27ac were plotted in parallel. Initiation zones were sorted according to the size. (C) Violin plots of PET counts per 10 kb from YY1 HiChIP experiments in initiation and control zones. Medians are shown with lines and mean values are also indicated. p values were determined by Wilcoxon test. (D) Model for structure of initiation zones. Treslin-MTBP and YY1 are enriched in initiation zones. YY1 could form dimers connecting distal DNA elements. Treslin-MTBP could potentially activate MCMs located at both nearby and distal locations.
	Figure S1. Construction and Characterization of Auxin-Regulated Cell Lines, Related to Figure 1 (A) For preparation of MTBP-mAID/MTBP-mAID and MTBP-mAID/MTBP-WT-FLAG cell lines, DLD-1 cells were transfected with pX330-Cas9-MTBP-Exon22 and combinations of either pBluescript-MTBP-mAID-Neo and pBluescript-MTBP-mAID-Hygro or pBluescript-MTBP-mAID-Neo and pBluescript-MTBP-WT-FLAG-Hygro, respectively. Clones resistant to G418 and Hygromycin were selected for genotype testing. (B) For preparation of MTBP-mAID/MTBP-ΔC-FLAG cells, DLD-1 cells were transfected with pX330-Cas9- MTBP-Exon22 and pBluescript-MTBP-mAID-Neo. G418-resistant clones were selected for insertion of mAID at both alleles of MTBP. Cells with mAID at both alleles were transfected with pX330-Cas9-MTBP-Exon19 and pBluescript-MTBP-ΔC-FLAG-Hygro. Clones resistant to both G418 and Hygromycin were screened for the presence of one copy each of MTBP-mAID and MTBP-ΔC-FLAG. (C) MTBP-mAID/MTBP-mAID, MTBP-mAID/MTBP-WT-FLAG, and MTBP-mAID/MTBP-ΔC-FLAG cells were arrested with Cdk4/6i for 20 hr. At this point, cells were maintained in Cdk4/6i (t=0) or washed to remove Cdk4/6i and thereafter incubated in the absence (column 1) or the presence of auxin (columns 2-4). Cells were incubated for 20 min with EdU at the indicated times. Incorporated EdU was labeled with Alexa 488 azide and DNA was stained with DAPI. Total nuclear EdU fluorescence (au) and nuclear DNA content were determined with CellProfiler. Red, G1; Blue, S-phase; and Green, G2/M. At least 1,500 nuclei were examined for each time point. Representative of two experiments.
	Figure S2. Characterization of MTBP Binding Sites, Related to Figure 2 (A) Venn diagram of three replicates of MTBP-WT CUT&RUN experiments. Peaks were called on each of three replicates using MACS2. Peaks that were called at least two out of three times were selected. (B) All of the peaks identified in pooled files by MACS2 for MTBP-WT (left) and MTBP-ΔC (right) are shown in blue and selected reproducible peaks are shown in yellow. For MTBP-WT and MTBP-ΔC, more than 99.7% and 99.0% of the selected peaks, respectively, have a q-value (FDR) of < 10-6 . (C) Read scores at each peak for MTBP-WT and MTBP-ΔC were generated with Deeptools multiBigWigSummary and plotted. Each dot corresponds to a peak. (D) Binding of Xenopus Treslin, TopBP1, and Orc2 to biotinylated fragments of double-stranded DNA of the indicated lengths bound to streptavidin beads in Xenopus NPE fractions. DNA fragments of overlapping sequence were generated from pBluescript by PCR. (E) Genome ontology analysis using HOMER AnnotatePeaks is plotted. Genome segments were determined according to the GENCODE basic gene annotation file (v32). (F) Venn diagram of MTBP peaks and Ini-seq peaks showing that 38.7% of the MTBP binding sites overlap with initiation sites that were detected by Ini-seq
	Figure S3. MTBP Is Enriched at Transcriptional-Regulatory Elements, Related to Figure 3 (A) ChromHMM analysis of MTBP peaks. With the chromatin-state discovery tool ChromHMM and publicly available DNase-seq and ChIP-seq datasets (H3K4me3, H3K27ac, H3K4me1, H3K36me3, H3K9me3, H3K27me3, and CTCF) from ENCODE, 200 bp genomic segments were classified into twelve states and annotated as shown on the right. (B) MTBP, MTBP-ΔC, Orc2 ChIP-seq, and BG4 ChIP-seq average reads around TSSs were plotted. (C) Peak scores of MTBP peaks at TSSs and corresponding reads from RNA-seq (RPKM) from DLD-1 cells were plotted.
	Figure S4. Identification and Characterization of Motifs in MTBP Binding Sites, Related to Figure 4 (A) Results of motif-searching analysis. In HOMER, findMotifsGenome.pl was used to search for motifs enriched in the MTBP peaks. (B) Locations of AP-1 (TGAG/CTCA), a variation of AP-1 (version 2; TGAG/CTAA), RUNX (AACCACA), and TEAD (A/GCATTCC) motifs were plotted around MTBP peak summits. (C) AP-1 motif peaks are 145 bp (± 10 bp) away from the MTBP peak summits. Each peak file was examined and separated into three classes according to whether the AP-1 motif was on the left, right, or both sides. The bottom figure shows the two profiles of MTBP peaks that have an AP-1 site on the left or right, respectively. (D) TSSs are located around 145 bp (± 10 bp) away from MTBP peak summits. Analysis was performed in the same manner as in panel C.
	Figure S5. Analysis of Histone Markers at the MTBP Binding Sites, Related to Figure 5 (A) Heatmap analysis of read distributions for various histone markers from HCT-116 cells around the summits of MTBP peaks. (B) Venn diagram of DNase-seq and various ChIP-seq peaks that overlap with MTBP peaks. (C) Various signal profiles around MTBP summits that have AP-1 sites. The peaks are oriented so that AP-1 sites are on the right of MTBP summits. (D) Various signal profiles and G4 motifs around MTBP summits near TSSs. The peaks are oriented so that TSSs are on the right of MTBP summits.
	Figure S6. Analysis of EdU-seq Patterns in DLD-1 Cells, Related to Figure 6 (A) Distributions of different classes of MTBP binding sites (Promoter-TSS, Enhancer/Super-enhancer, and Others) in replication timing domains (Early, Mid, and Late). (B) Comparison of the read counts for the three replicates of EdU-seq for MTBP-WT (left three panels) and MTBPΔC (right three panels). Analysis was performed using Deeptools multiBigWigSummary. (C) Size distribution of the initiation zones for MTBP-WT determined by EdU-seq. (D) Heatmap analysis of MTBP-WT, MTBP-ΔC, MNase-seq, H3K4me2 CUT&RUN from DLD-1 cells, DNaseseq, and various ChIP-seq peaks around TSSs containing MTBP at either the 5′ or 3′ side, respectively. MTBP peaks were segregated according to whether their summits were on the 5′ or 3′ side. Heatmaps were oriented so that transcription was from left to right.
	Figure S7. Characterization of Early-Replicating Zones in DLD-1 Cells, Related to Figure 7 (A) Heatmaps of reads of EdU-seq, MTBP, and YY1 as well as locations of TSSs were centered around the midpoint of initiation zones. TSSs were plotted according to the direction of transcription as indicated with arrows. Locations of TSSs were extended 2 kb upstream and downstream for visibility. (B) Genome browser profiles of MTBP, YY1, YY1 loops, and EdU-seq. Profiles were mapped to hg19. Loops connecting adjacent bins were removed. YY1 loops with PET scores of 10 and above are shown.

References [+] :

Abdurashidova, Start sites of bidirectional DNA synthesis at the human lamin B2 origin. 2000, Pubmed