Supplemental data for "Differential Histone Distribution Patterns in Induced Asymmetrically Dividing Mouse Embryonic Stem Cells"
Those additional supplemental data includes figures, raw image galleries, quantification tables and movie, which are complementary to the main-text figures 1-4 and supplemental figure S1-S4. Validation of the E14TG2a mESC line used in this study. (A) Representative images of the mESC morphology and alkaline phosphatase (AP) staining when cultured in complete medium (CM) with LIF+2i. (B) Cells cultured in N2B27 medium with bFGF/ActivinA for five days show EpiSCs cell morphology and decreased AP staining. (C) Growth curve of mESCs and mEpiSCs, with cell doubling time indicated in the plot. Scale bar in (A) and (B): 100 um. Expression of histone-Dendra2 fusion proteins have no apparent effect on the cell cycle progression of mESCs. (A) Western blot analysis of non-transgenic wild-type (WT) mESCs and transgenic mESCs expressing H3-Dendra2, H4-Dendra2, H2A-Dendra2, H2B-Dendra2 and H3.3-Dendra2, respectively. The sizes of histone proteins are ~15 KD, the sizes for Histone-Dendra2 fusion proteins are ~40 KD, as indicated in the panel. (B) The cell cycle profiles obtained using DNA content analysis (PI staining) with FACS (Fluorescence Activated Cell Sorting). (C) Growth curves of non-transgenic WT-mESCs and each histone-Dendra2 transgenic mESC lines, with cell doubling times indicated in the panel. (D) The percentage of EdU-positive cells with 30-minute EdU pulse labeling in H3-Dendra2, H4-Dendra2, H2A-Dendra2, H2B-Dendra2 and H3.3-Dendra2 transgenic mESCs. (E) The “stemness” identity of the mESC line carrying the H3-Dendra2 transgene, confirmed by immunofluorescent signals for ESC markers, including Nanog, Rex1, Oct4, Sox2 and SSEA-1. Scale bar in (E): 20 um. Schematic depiction of a model for different histone distribution patterns with Wnt3a-induced ACD of mESCs. (A) With Wnt3a bead, non-overlapping histone H3 and H4 regions could be explained by regional conservative mode of nucleosome reassembly during DNA replication, shown as [Model (1) in (E)]. (B) The separable signals are lost in symmetrically dividing mESCs, which could be due to dispersive mode of nucleosome reassembly during DNA replication, shown as [Model (2) in (E)]. (C) The symmetric histone distribution patterns of H2A and H2B during ACD of mESCs. (D) DNA replication-independent model, which applies to H3.3. (E) Distinct nucleosome reassembly modes [(1) conservative versus (2) dispersive] during DNA replication could have regional specificities: the conservative model could occur at gene regions that need to be differentially expressed in the two daughter cells derived from Wnt3a-induced asymmetrically dividing mESCs, such as stemness genes, differentiation genes, or Wnt signaling pathway target genes. On the other hand, for those genes whose expression is similar between the two daughter cells, such as housekeeping genes and genes silenced in mESCs, the dispersive model could be applied to make sure the sister chromatids inherit the identical epigenetic information.
Steps to reproduce
For reproducing quantification on .czi files: 1/Indicate the range of z-planes where the cell locates. (e.g. change the value in z_start/z_ends; Default is to calculate all z-planes;Preferably preprocess the stack to contain only the ROI.) 2/Run main_czi_singlecell.czi and select the image stack on the pop-up file browser . 3/ Select the ROI with the pop-up crop tool.