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. Author manuscript; available in PMC: 2023 May 16.
Published in final edited form as: Nat Immunol. 2022 Oct 31;23(11):1628–1643. doi: 10.1038/s41590-022-01322-y

Fig. 3. Notch1 expression depends on a conserved enhancer.

Fig. 3

(a) The 600bp region targeting DNSe for deletion as well as histone modifications (ChIPseq), chromatin accessibility (ATAC-seq), and gene expression (RNAseq) are shown in DN3 thymocytes. Sequence conservation between mouse (mm10) and human (hg19) are depicted by dark blue (mouse) and magenta pink (human) with conserved regions highlighted in pink. (b-c) Expression of Notch1, DNS1, DNS2, and DNS3 are shown in WT and ΔDNSe DN3 thymocytes (mean +/− SEM). (d) Statistically significant (FDR <0.01) long distance interactions at Notch1 locus identified by HiC in DN3 thymocytes are shown together with histone modifications and chromatin accessibility. Red arrows highlight a significant interaction between Notch1 promoter and 20kb upstream region. (e) A model of regulation of Notch1 and DNS1-3 transcripts by the DNSe in DN thymocytes. (f) Absolute numbers of total thymocytes, B220+CD19+ B cell precursors and B220hiCD19hi mature B cells from WT and ΔDNSe thymi are provided at different ages with unpaired t-test (two-tailed) used for statistical analysis. N.S. not significant (g) Absolute numbers of total γδT cells and immature (CD24+) or mature (CD24−) γδT cell subsets (Vγ1.1, Vγ2, and others) in the thymus of 28–35 days old WT and ΔDNSe mice are shown with statistical analysis performed as in (f). N.S. not significant (h) Proportional distribution of each γδT cell subset is shown.

ATACseq data shown in (a and d) for DN3 thymocytes was obtained from ImmGen (GSE100738) with other datasets described in Fig. 1. Data shown in (b-c) for ΔDNSe were generated from two independent experiments with pooled samples from at least two biological replicates. Data for WT DN3 is also used in Fig. 1g. HiC analysis of published data (GSE79422) is shown in (d). Data in (f) were generated from 4 independent experiments with mice at different age groups (WT; N=3, N=5, N=5 at 28, 38–60, >70 days, respectively, ΔDNSe; N=3, N=5, N=6 or 4 at 28, 38–60, >70 days, respectively), in (g and h) were generated from 3 independent experiments with WT; N=8 and ΔDNSe; N=8.