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. 2019 Mar 1;13:284–304. doi: 10.1016/j.isci.2019.02.027

Figure 7.

Figure 7

CRY1 Inhibits Binding of HIF-1α to Its Target Gene Promoters

(A and B) Cells were cotransfected with an expression vector for CRY1 along with either (A) the wild-type human PAI-1 promoter Luc construct (pGL3-hPAI-806/+19) or a PAI-1 promoter with a mutated hypoxia-responsive element (HRE) (pGL3-hPAI-HREm) as well as with (B) Luc reporter gene constructs containing three HIF-binding HREs (pGL3-HRE) or three mutated HREs (pGL3-HREm) in front of the SV40 promoter and cultured under normoxia (16% O2) and hypoxia (5% O2) for 24 h. The Luc activity of pGL3-hPAI-806/+19-, pGL3-hPAI-HREm-, pGL3-HRE-, or pGL3-HREm-transfected cells at 16% O2 was set to 1. Values are means ± SEM of at least three independent culture experiments, each performed in duplicate. Statistics, Student's t test for paired values: *significant difference, p ≤ 0.05.

(C) Cells were cotransfected with pGL3-HRE or pGL3-HREm Luc reporter gene constructs and expression vectors encoding CRY1, HIF-1α, and ARNT alone or in combination. In each experiment the Luc activity of pGL3-HRE- or pGL3-HREm-transfected cells at 16% O2 was set to 1. Values are means ± SEM of at least three independent culture experiments, each performed in duplicate. Statistics, Student's t test for paired values: ∗significant difference, p ≤ 0.05.

(D) Chromatin immunoprecipitation (ChIP) was performed on cell lysates from formaldehyde cross-linked wild-type or CRY1-deficient MEFs, cultured under normoxia or hypoxia overnight. DNA fragments were co-precipitated with antibodies against mouse HIF-1α and amplified by quantitative PCR using primers specific for the mouse PAI-1, VEGF-A, PGK-1, and GLUT-1 promoters containing the corresponding HRE. Chromatin immunoprecipitation without antibody or using IgG instead of antibody served as additional specificity controls. Differences in the HIF-1α DNA-binding efficiency in wild-type and ΔCRY1 MEFs were calculated by the fold enrichment method relative to the IgG control. Values are means ± SEM of three independent experiments. Statistics, Student's t test for paired values: *significant difference, p ≤ 0.05.