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Fig. 3 Differences in chromatin accessibility of Gata2b result in up-regulation of Gata2b expression in gata2b+/− HSPCs. (A) Uniform manifold approximation and projection (UMAP) analysis of single-nucleus ATAC sequencing (snATAC-seq) showing all 15 cell types. UMAP atlas was separated between wild-type (WT) and gata2b+/− cells. (B) Coverage plots showing significant differences in gata2b chromatin accessibility between WT and gata2b+/− HSPCs with the location of the gata2b gene block shown in blue. The normalized peak signal range is 0–40. Highlighted genomic regions within grey blocks indicate the location of two putative enhancers, enhancer 1 (E1) and enhancer 2 (E2). The links between the blocks illustrate predicted cis-regulatory interactions between the gata2b gene body and its upstream region, based on the examination of genome co-accessibility. Link colours correspond to predicted co-accessibility scores, with yellow indicating stronger predicted interactions. Black arrows indicated the co-accessibility between E2 and E1. Red arrows indicated the co-accessibility between E1 and transcription start site. which is stronger in gata2b+/− kidney marrow (KM). (C) Accessibility tracks of the same region shown in (B) in bulk ATAC-sequencing data. The peak signal range is 0–750. Blue blocks show the gata2b gene and red blocks show the putative enhancers as highlighted in (B). (D) Violin plot displaying the quantification and statistics of gata2b mRNA level in HSPCs between WT and gata2b+/− in 12 months postfertilization (mpf) scRNA-seq. The average log2 fold change of WT versus gata2b+/− is −0.34; t test was used to calculate p value. (E) The dot plot displays the log2 fold-change values of differentially activated transcription factor (TF) motifs in ‘HSPCs’ cluster between WT and gata2b+/− KM. The TF motifs are ranked in increasing order based on log2 fold-change values. Dotted lines indicate the threshold for significance, with absolute log2 fold-change values ≥0.1, the default parameter in function FindMarkers. The blue and red colours represent TF motifs that are statistically significant (p value <0.05) with absolute log2 fold-change values ≥0.1. (F) Representative figure of identification of CD41:GFPint population (in green) and its distribution in the forward (FSC-A) and side-scatter (SSC-A) KM population. (G) Quantification of the percentage of CD41:GFPint cells in WT (n = 12) and gata2b+/− (n = 12) KM single live cells. (H) Representative flow cytometry plots of cell cycle analysis by anti-Ki67 and DAPI staining in WT and gata2b+/− CD41:GFPint KM. (I) Quantification of the percentages of WT (n = 3) and gata2b+/− (n = 3) CD41:GFPint cells in different cell cycle stages. Data represent mean ± standard error of the mean. t Test was used to calculate p value. *p < 0.05; **p < 0.01.