Figure 7. GATA-3 and Bcl11b bind to and mediate repression of the Cdkn2b locus in developing thymocytes.

(A) Location of primers spanning all predicted GATA-3 bindings sites upstream of Cdkn2b and Cdkn2a TSS as identified by rVista, genomatix MatlInspector and manual scan in VectorNTI. (B) Putative GATA-3 RES were identified by rVista, genomatix MatlInspector and manual scan in VectorNTI. ChIP assay was performed on wild type DN1, DN2 and DN3 thymocytes with anti-GATA-3 and control IgG antibodies. Enrichment was quantified by qPCR, normalized to β-actin and calculated as percentage of input. Enrichment was further compared to a known GATA-3 DNA target located in a Tcrb enhancer region. Data is presented as fold increase of GATA-3 enrichment over the control IgG. (C) Chromatin was immunoprecipitated with anti-GATA-3, anti- H3K27^3me or control IgG antibodies from a combination of wild type DN1, DN2 and DN3 thymocytes. Enrichment for GATA-3 (mean ± SEM, n = 3) and (D) repressive H3K27^3me epigenetic signature (mean ± SEM, n = 2) at promoter regions of Cdkn2a/b and percentage of input samples before ChIP (1% of total starting cell population) were quantified by qPCR and data normalized to β-globin gene expression (*p < 0.05, Student t-test). (E) Location of primers spanning Bcl11b RES upstream of Cdkn2b as identified by ChIP-seq (See Supplemental Figure 2B) and USC genome browser. (F) Chromatin immunoprecipitation from wild type DN1, DN2 and DN3 cells with anti-CTIP2 (Bcl11b) or control IgG antibodies. Enrichment for Bcl11b (mean ± SEM, n = 3) at promoter region of Cdkn2b, along with 1% of percentage input, were quantified by qPCR and data normalized to β-globin gene expression (*p < 0.05, Student t-test).