Supplementary MaterialsFigure S1: Quantification of sign intensities from mitotic chromatin certain versus unbound RBPJ and RBPJ derivatives shown in Shape 1. of experimental structure. (C) Reactions had been resolved inside a 5% indigenous polyacrylamide gel. The 147 bp DNA fragment that will not support the RBPJ-binding theme was utilized as unlabeled DNA rival.(TIFF) pgen.1004204.s002.tif (1.7M) GUID:?B51CEA81-015B-4E4B-9094-6D180919360C Shape S3: Titration of RBPJ for nucleosome binding assays. Two primary mononucleosomes had been found in the binding assays: RBPJ-end consists of an RBPJ-binding theme at positions 127C134, which is situated close to the entry/exit sites of the nucleosomal DNA, and RBPJ-minus nucleosomes, which do not contain an RBPJ-binding motif. Varying amounts of RBPJ were used in the binding assays as indicated.(TIFF) pgen.1004204.s003.tif (1.0M) GUID:?5EC3746C-DB6B-48E3-B8DD-347CAD7A8799 Figure S4: Characterization of the rabbit anti-RBPJ antibody. (A) Anti-RBPJ antibody specificity as revealed by western blot analysis. F9 cells treated with shRNA targeting RBPJ (+) or a non-specific shRNA (?) for 60 hours. Lysates were resolved in a NuPAGE 4C12% Bis-Tris gel, and western blots were probed with the anti-RBPJ and an anti-GAPDH antibody. (B) Western blot analysis showing RBPJ immunoprecipitation from crosslinked cells. 293T cells expressing Flag-RBPJ were cross-linked with 1% formaldehyde, and after sonication lysates were subjected to immunoprecipitation with the rabbit anti-RBPJ antibody or control rabbit IgG. The input to IP ratio loaded on the gel was 14. The western blot was probed with a mouse anti-Flag antibody (M2).(TIFF) pgen.1004204.s004.tif (932K) GUID:?9710B7C3-CE02-4E17-81BC-8EE8949D34AC Figure S5: Purity of mitotic cell preparations as revealed by immunofluorescence microscopy. Nocodazole arrested F9 cells were immunostained with antibodies against serine 10 phosphorylated histone H3 (green) and elongating RNA polymerase II (red). DNA was counterstained with DAPI. The field shown contains about 85 mitotic cells and no interphase cells, indicating that this mitotic cell preparation was greater than 98% pure.(TIFF) pgen.1004204.s005.tif (5.8M) GUID:?ADE1635A-8E5C-4C7F-96D0-9ACF95ACE109 Figure S6: Pie charts illustrating the genomic distribution of RBPJ occupancy, as determined by gene annotation. (A) Distribution of total RBPJ occupancy on chromatin of asynchronous cells. (B) Distribution of total RBPJ occupancy on mitotic chromatin. (C) Distribution of RBPJ occupancy common to asynchronous and mitotic cells. (D) Distribution of RBPJ occupancy Fisetin enzyme inhibitor unique to asynchronous cells. (E) Distribution of RBPJ occupancy unique to mitotic cells.(TIFF) pgen.1004204.s006.tif (2.4M) GUID:?8935F30A-6FEB-4D7C-9D4F-DAFB58D4D664 Figure S7: Association of the CTCF protein with the Naprt1 and Tcerg1 promoters, as revealed by ChIP-qPCR. (A) Naprt1 contains both CTCF- and RBPJ-binding motifs (shown in red and blue, respectively), with the CTCF-binding motif positioned at the center of the RBPJ ChIP sequencing peak. (B) Anti-CTCF ChIP-qPCR demonstrating that CTCF binds to the Naprt1 and Tcerg1 promoters, but not to Hes1 or actin, in both asynchronous and mitotic F9 cells.(TIFF) pgen.1004204.s007.tif (919K) GUID:?04442243-D0F6-4D32-8546-CDA9FB6C139C Figure S8: RBPJ binds to Notch responsive genes in asynchronous and mitotic F9 cells. Screen shots from the UCSC Genome Browser revealing RBPJ occupancy at Notch responsive genes. The position of the RBPJ-binding motif within each peak is indicated with an asterisk. The transcription factors Hes7 (A) and HeyL (B) are representative Notch-target genes of the Hes and Hey families. Timm13 (C), Nmnat2 (D) and Fbxl19 (E) are from Castel and Mourikis et al. . Coordinates of the regions shown are (A) chr11: 68,930,148-68,935,117, (B) chr4:122,908,000-122,912,999, (C) chr10:80,359,119-80,366,062, (D) chr1:154,936,858-154,940,532, and (E) chr7:134,889,218-134,893,011.(TIFF) pgen.1004204.s008.tif (973K) GUID:?14E800B4-9E07-4B33-AB92-0F80648A6BA3 Figure S9: Comparisons of RBPJ ChIP-seq results obtained from F9 cells to those of the TLL cell lines, T6E and G4A2. (A) Venn diagram illustrating the overlap of RBPJ occupancy sites in F9 cells Argireline Acetate (this study) and in T6E and G4A2 cells . (B) Screen shots taken from the UCSF Genome Browser showing side-by-side comparison of RBPJ occupancy at five regions. Also included are duplicates from asynchronous and mitotic F9 cells as well as input controls. The RBPJ- and CTCF-binding motifs are designated with red and dark squares, respectively. The coordinates of the five loci from remaining to correct are (1) chr16:30,055,655-30,076,174, (2) chr14:76,549,884-76,555,073, (3) chr8:86,183,791-86,188,980, (4) chr8:4,273,375-4,278,564 and (5) chr18:42,668,310-42,675,184. Area 1, 4 and 5 support the promoters of Hes1, Tcerg1 and Timm44, respectively. Fisetin enzyme inhibitor No transcript is available associated with area 2, as well as the peaks demonstrated in area 3 lay within an intron of Gipc1.(TIFF) pgen.1004204.s009.tif (1.1M) GUID:?5E3AB9BD-8E0C-4B6E-89FF-A4296A3EA731 Desk S1: Move analysis of RBPJ binding peaks common to asynchronous and mitotic F9 cells.(XLSX) pgen.1004204.s010.xlsx (9.6K) GUID:?C028C1F8-4FF5-4C5F-A76C-0D576DB10D94 Desk S2: Annotation of RBPJ binding peaks in asynchronous F9 cells.(XLSX) pgen.1004204.s011.xlsx (973K) GUID:?ECDCF513-38FF-4E3A-B740-D31195983243 Desk S3: Annotation of RBPJ binding peaks in mitotic F9 cells.(XLSX) pgen.1004204.s012.xlsx (560K) GUID:?D6E7C792-C485-4F4D-A55A-C196EA1D6D70 Desk S4: Move analysis of RBPJ binding peaks in asynchronous F9 cells. (A) All RBPJ peaks. (B) RBPJ peaks including the RBPJ binding Fisetin enzyme inhibitor motif. (C) RBPJ peaks including the CTCF binding motif. (D) RBPJ peaks including RBPJ and CTCF binding motifs.(XLSX) pgen.1004204.s013.xlsx (12K) GUID:?5A50FC50-2793-4815-A873-46AE14261AF7.