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. 2023 Jun 19;51(14):7269–7287. doi: 10.1093/nar/gkad523

Figure 5.

Figure 5.

Depending on the genomic context, SMCHD1 contributes to gene silencing or protects against position effects. For the different pGL3 constructs, fragments corresponding to the regions that are differentially methylated (A–C) or corresponding to SMCHD1 binding sites (D) were cloned downstream of the luciferase reporter gene in vectors lacking an enhancer (pGL3 promoter). Firefly luciferase expression was determined 48 h post-transfection of the different constructs in HEK293 and HEK SMCHD1 KO cells. The pGL3 control vector was used as a transfection control. Firely luciferase levels were normalized to expression of the Renilla luciferase used as transfection control. Values corresponding to the normalized luciferase activity (expressed in relative luminescence units, RLUs) are the average of three independent assays, each realized as technical triplicates (n = 9). Error bars represent standard error. Statistical significance was determined using a Mann–Whitney test (****P-value <0.00001, ***P-value <0.0001, **P-value <0.001, *P-value = 0.01). (A) For the D4Z4 macrosatellite, regions that are differentially methylated in patients carrying a mutation in SMCHD1 were tested (DR1, 5P; a scheme of the D4Z4 repeat is presented in Supplementary Figure S13). (B) The DR1 sequence contains 31 CG sites. Fragments corresponding to CG1–10, CG10–20 and CG21–31 or deleted of 10 of these CGs (ΔCG1–10, ΔCG10–20 and ΔCG21–31) were tested. (C) HOX gene DMRs. (D) Putative SMCHD1 binding sites overlapping or not with CTCF binding sites. (E, F) For evaluation of protection against position effect, we used a vector carrying a hygromycin resistance gene and an eGFP reporter gene. Sequences to be tested are cloned downstream of the eGFP gene and transfected into HEK or HEK KO cells. Stable eGFP expression was measured by flow cytometry (FACS) for an extended period of time in cells grown in the presence of hygromycin B. Representative spectra of the % of eGFP-positive cells are presented. For each condition, eGFP expression was compared to values obtained in cells transfected with the empty vector (pCMV). In HEK SMCHD1 KO cells, eGFP expression was also measured 72 h after transfection of an SMCHD1 expression vector (gray curves). (E) D4Z4 (left upper panel), DR1–5P (right upper panel), DR1 (left lower panel) and 5P (right lower panel). (F) Results obtained for the HOXA13 (left) and HOXC4/5/6 (right) DMRs.