Fig 2: The binding of NFIB and the Pre-RC on chromatin.a Genome tracks show the signals of nascent strands, chromatin accessibility, NFIB, and ORC1. PRKAR2A and ECSIT are the Refseq gene names. b qChIP verification of chromatin binding of NFIB and pre-RC at the origins shown in A using antibodies against the indicated proteins. The data are presented as the means ± SD for triplicate experiments. P values were determined by an unpaired two-tailed T test. c U2OS cells were synchronized at the G2/M phase and released for different hours. qChIP experiments were performed with antibodies against the indicated proteins to examine the chromatin binding dynamics of NFIB and ORC1. Error bars represent mean ± SD for triplicate experiments. P values were determined by an unpaired two-tailed T test. d U2OS cells were infected with lentiviruses carrying the indicated shRNAs or transfected with the indicated siRNAs. qChIP experiments were performed using antibodies against the indicated proteins. Error bars represent mean ± SD for triplicate experiments (*P < 0.0001, 2-way ANOVA). e Knockdown efficiency of the indicated factors was verified by western blotting.

Fig 3: Depletion of NFIB alters chromosome contacts/compartments and replication profile.a Left: Heatmap of NS-seq signals in NFIB-depleted versus control U2OS cells. The fold change cutoff of signal intensity was set to 1.2. S1, peak signals decreased; S2, peak signals increased; S3, peak signals unaltered. Right: Heatmap of NS-seq signal distribution surrounding N1 peaks in NFIB-depleted versus control U2OS cells. b Different time points of synchronized NFIB-depleted U2OS cells were analyzed by Repli-seq. Control or NFIB-depleted U2OS cells were synchronized via thymidine/aphidicolin block followed by release for 0.5 h, 4 h, or 6 h to enrich cells in the early, middle, or late S phase, respectively. Graph showing the replication timing of NFIB-associated active origins (S1 peaks overlapped with N1 peaks, S1.N1, n = 415), other S1 peaks (S1.non-N1, n = 14,203), S2 peaks (n = 24,410) and total NS-seq peaks (all, n = 48,273). c Comparison between replication initiation patterns after NFIB was depleted in U2OS cells. Genome tracks showing the signals of Repli-seq, NFIB ORC1, and H3K4me3 at selected regions of chromosome 5. d Scatter plot of PC1 eigenvector of G1/S boundary or S phase U2OS cells without (shCTR) or with NFIB depletion (shNFIB) for each 250 kb genome bin. Red points: changed regions. Black points: unchanged regions. e Histogram showing the number of regions (250 kb bin) where the PC1 eigenvector significantly changed. f Knight-Ruiz (KR) balanced Micro-C contact map of chromosome 1: 162 to 183 Mb at a 100-kb resolution. g Violin plot showing the distribution of N1 peaks in different chromatin regions in G1/S phase (bin size 250 kb).