Journal: Nature

Article Title: Reduced cyclin D3 expression in erythroid cells protects against malaria

doi: 10.1038/s41586-026-10110-9

Figure Lengend Snippet: a , Top, vector content and expression of luciferase reporter genes driven by the extreme allele combinations: on the one hand the WT rs9349205-G|rs112233623-C (G|C) alleles on the other hand the derived rs9349205-A| rs112233623-T (A|T) alleles of the CCND3 enhancer in the erythroid cell line HUDEP-2, with empty vector as a control construct. Below, histograms show averages of the relative luminescence activity of the combinations of the two extreme alleles at rs9349205 and rs112233623 described above in absence (-) or presence of GATA1 and FOG-expressing plasmids; SMAD3 and p300 expressing plasmids; SMAD3 expressing plasmid alone; or empty expression vector. All values are plotted relative to the WT (G|C) construct. Note that the activity of the empty pGL4 vector is barely detectable. The mean ± s.e.m is shown (n = 7 (vectors); n = 3 (vectors + SMAD3), n = 3 (vectors+SMAD3 + p300), n = 4 (vectors + GATA1 + FOG) biologically independent experiments). A two-sided Student’s t -test was used, with level of significance indicated by asterisks (*P < .05;**P < .01; ****P < .0001, ns, not significant). The statistical results for all comparisons are provided in Supplementary Table . b , In silico prediction of binding to rs112233623 allele variants, showing derived allele T hindering binding of SMAD3 while favouring GATA1 binding. c , d , Representative electrophoretic mobility shift assays (EMSA) showing binding of SMAD3 and GATA1 proteins with labelled oligonucleotide probes (*) containing the WT (C) or derived (T) allele of rs112233623 (n = 3 biologically independent experiments). Competitor unlabelled oligonucleotides were used at the indicated fold excess to demonstrate specificity of binding. c , SMAD3 binds to the WT rs112233623-C allele (lanes 2 and 8); is weakly supershifted by anti-SMAD3 antibody (lanes 3 and 9); and is competed away by an excess of unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 10-12, at 25X, 50X and 100X respectively), but not by an excess of oligonucleotide containing the derived rs112233623-T allele (lanes 13-15, at 25X, 50X and 100X respectively). d , GATA1 binds to the rs112233623-T derived allele (lanes 2, 8 and 9), is supershifted by anti-GATA1 antibody (lane 3); and is competed away by an excess of unlabelled DNA oligonucleotides containing the derived rs112233623-T allele (lanes 10-12, at 25X, 50X and 100X respectively) compared to unlabelled DNA oligonucleotides containing the WT rs112233623-C allele (lanes 13-15, at 25X, 50X and 100X respectively). GATA1 does not appear to bind to the WT rs112233623-C allele (lane 5). The samples derive from the same experiment and, the gels were processed in parallel. For gel source data, see Supplementary Fig. . e , f , ChIP–qPCR for SMAD3 or GATA1 binding to the CCND3 enhancer region surrounding rs112233623 ( CCND3 ) and to a SimpleChIP Human α Satellite as negative control in erythroblasts derived from individuals homozygous for the rs112233623-T decrease of expression (DoE) allele versus the WT rs112233623-C allele (WT). e , ChIP was performed using an antibody against SMAD3, results are represented as percentage of input and nonspecific IgG used as negative control. The mean ± s.e.m is shown (n = 3 biologically independent experiments). A two-sided two-Sample t-test was used; significant differences are indicated (*P < .05; ns, not significant). The statistical results for the comparisons are provided in Supplementary Table . f , ChIP assays were conducted with an antibody against GATA1, results are represented as percentage of input and nonspecific IgG used as negative control (n = 2 biologically independent experiments).

Article Snippet: ChIP DNA was purified and subsequently quantified by qPCR using primers designed to amplify a region surrounding rs112233623 in the CCND3 enhancer and SimpleChIP Human α Satellite Repeat Primers (4486, Cell Signaling Technology).

Techniques: Plasmid Preparation, Expressing, Luciferase, Derivative Assay, Control, Construct, Activity Assay, In Silico, Binding Assay, Electrophoretic Mobility Shift Assay, ChIP-qPCR, Negative Control