Additional file 1 of Cellular reprogramming is driven by widespread rewiring of promoter-enhancer interactions
Creators: name: Wang, Miao
name: He, Bing
name: Hao, Yueling
name: Srinivasan, Divyaa
name: Shrinet, Jatin
name: Fraser, Peter
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. Transcriptional profile changes during Pre-B transdifferentiation. a Principal component analysis (PCA) RNAseq of each replicate from different time points. b SNPsplit identifies the percentage of total RNA reads that align to Cebpa gene region that contain Mus musculus specific SNP (mouse specific), Rat specific SNP (Rat specific), regions that do not contain any SNP (Unassignable), conflicting SNP information (Conflicting). c Volcano plots show upregulated and downregulated differentially expressed genes (DEGs). d GO ontology 5 Fuzzy c-means clusters of DEGs e qPCR analysis of Pre-B cell and macrophage marker gene expression during Pre-B cell trans-differentiation. f Gene expression level (TPM) of Pre-B cell-specific genes (Blnk, Cd19, Cd79a, Cd79b, Vpreb1, Vpreb2, Vpreb3), and macrophage-specific genes (Ccl6, Ctsc, Mmp8, Msr1) during Pre-B trans-differentiation. Fig. S2. Hi-C data analysis. aHiCUP pipeline analysis HiC and PCHi-C data, including the following three categories: Cis &lt; 10Kbp, Cis &gt; 10Kbp, and trans-interactions across four-time points. b Heatmaps show Hi-C matrix at 100kb and 25 kb resolution. Fig. S3. Altered TAD and A/B compartment during Pre-B transdifferentiation. a Number of TADs and size distribution at each time point. b GO enrichment analysis of genes changed TAD boundaries from 0h to 48h. c Pearson correlation coefficient of PC1 values (50kb resolution) of the entire genome. d Gene expression level (TPM) in the A and B compartments at four different time points. A two-sided unpaired t-test was performed for the significance test. ePercentage of compartment change for DEGs (differentially expressed genes).  f Log2 fold change of gene expression in dynamic compartments. stable (n= 19157), A to B (n= 450), and B to A (n= 474). g Gene ontology analysis of 2105 genes at 48h that switch from A to B and 1552 genes at 48h that switch from B to A. A two-sided unpaired t-test was performed for the significance test. Fig. S4. PCHi-C data analysis. a HiCUP pipeline analysis of PCHi-C data: Cis &lt;10Kbp, Cis &gt;10Kbp, and trans-interactions across four-time points. b Proportion of promoter-promoter (P-P) and promoter-other interactions (P-O) interactions as deduced from PCHi-C. c Expression values (log2(TPM+1)) of genes/promoters classified into 4 groups (at least one PIR overlaps with histone markers). H3K27ac: interaction between gene/promoter and PIRs only with H3K27ac marks. 0h (n=4829); 1h (n=5710);12h (n=5257); 48h (5735). H3K27Ac+H3K27me3: interaction between gene/promoter and PIRs with both H3K27ac and H3K27me3 marks. 0h (n=8075); 1h (n=7044); 12h (n=7926); 48h (n= 7727). H3K27me3: interaction between gene/promoter and PIRs only with H3K27me3. 0h (n=2757);1h (n=2286); 12h (n= 2545); 48h (n=2165). No marks: interaction between gene/promoter and PIRs without any histone modification. 0h (n=3667), 1h (n=4805); 12h (n=4403); 48h (n= 4291). The box plot represents 25 and 75 percentiles with the median. An unpaired two-sided t test is performed for the significance test. d Boxplot shows gene expression values (log2(TPM +1)) of promoters that do not contact any PIRs with H3K27ac categorized into four groups according to the number of PIRs marked by H3K27me3 at four different time points.  0: Promoters without any PIRs marked by H3K27me3. 0h (n=2551), 1h (n=2991); 12h (n=2711); 48h (n=2682).  1: Promoters with only one PIR marked by H3K27me3 0h (n=1247), 1h (n=1100); 12h (n=1188); 48h (n= 955). 1-5: Promoters with one to five PIRs marked by H3K27me3. 0h (n=749), 1h (n=672); 12h (n=697); 48h (n=567).  &gt;5: Promoters with more than five PIRs marked by H3K27me3. 0h (n=80), 1h (n=63); 12h (n=103); 48h (n=95).  An unpaired two-sided t-test is performed for the significance test. The box plot represents 25 and 75 percentiles with the median. Fig. S5. Integrated analysis of promoter interactions and epigenetic features. a Maf1 and Ssbp1 region that shows promoter interaction changes accompanied by H3K27me3(green), H3K27ac (yellow), and H3K4me1 at interacting regions, along with gene expression TPM of Maf1 and Ssbp1 during Pre-B transdifferentiation. Multiple Maf1 promoters in two HindIII digested fragments b Log2 fold change of gene expression based on the number of H3K4me1 peaks on the increased PIRs (48h vs 0h). The number of histone mark peaks is classified into three groups.  &lt;=1: Genes that gain chromatin interactions with less than one histone mark peaks (n=360). 1-5:  Genes that gain chromatin interactions with one to five histone mark peaks (n=313). &gt;5: Genes that gain chromatin interactions with greater than five histone mark peaks (n=69). The unpaired two-sided t-test was performed to test significant differences between groups. The box plot represents 25 and 75 percentiles with the median. Fig. S6. Decreased promoter interactions at 48h. a GO term for genes that lost promoter interactions at 48h. b Examples of significantly decreased B cell-specific gene promoter interactions (Pax5,and Clcf1) across time points. Fig. S7. C/EBPα ChIPseq data analysis. a The number of identified C/EBPα ChIP peaks at 1h, 1h, 12 h and 48h. b Comparison of 1h, 12,h or 48h C/EBPα ChIP peaks signal to 0h. M value represents log2 fold change of read density between two samples, and A value represents the average signal intensity. The red dots represent induced C/EBPα binding sites at 1h, 12h, and 48h time points. The blue dots represent C/EBPα binding sites at 0h. The grey dots represent unchanged C/EBPα binding sites at four time points. Fig. S8. Uncropped blots.