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C. Fugazza et al.
Coup-TFII binds in vivo within the human β-globin locus and contributes to its three-dimensional conformation
The ChIP-sequencing data show that Coup-TFII binds in vivo within the β-locus, with the strongest peaks (pre- sent in both NT and OE cells but absent in KO cells) map- ping within the locus control region (LCR), at hypersensi- tive sites HS2, HS3 and HS4 (Figure 5A, B). Scanning of the core sequence of these elements using JASPAR software34 identified several potential Coup-TFII consensus sequences within these elements (Online Supplementary Figure S8), which were confirmed by ChIP assays (Figure 5B, C). To determine whether the level of Coup-TFII could influence the chromatin structure of the β-locus, we per- formed a 3C assay on the OE, NT and KO β-K562 cell lines described above. In this assay, the physical interac- tion within two nearby loci is measured on the basis of the relative crosslinking efficiency between a predefined anchor region and given downstream positions.
In our experiment, in the absence of Coup-TFII the rel- ative frequency of interactions of the γ-gene with the LCR is significantly lower (≈4%) than that of β (≈30%), point- ing to an effect on the β-locus favoring β-globin expression (in agreement with data in Figure 3B). By contrast, in the presence of Coup-TFII (OE and NT cells), the interaction of β with the LCR is around 15%. Of note, in KO cells, the absolute frequencies of interactions within the β-locus with the LCR increases, possibly reflecting concomitant overlapping differentiation effects.
Of interest, in OE cells, a significant interaction was observed in the Aγ-d region which has been implicated in hereditary persistence of fetal hemoglobin35 (Figure 5D). These results, together with ChIP-sequencing data identi-
fying Coup-TFII peaks in different regions within the β- locus, suggest a potential broad role of Coup-TFII in the differential regulation of globin genes and of the β-locus structure.
Discussion
In recent years, several repressors of embryonic/fetal globin genes have been identified in murine and human adult erythroid cells.6,36 These studies have not directly addressed the regulation of these genes in earlier develop- mental stages. In particular, there is no evidence so far of stage-specific factors activating human and mouse embry- onic/fetal globins prior to the switch to adult globin syn- thesis. Here we show that Coup-TFII is expressed in cells of yolk sac origin (Figure 1) and that its overexpression selectively activates embyonic/fetal globins in a variety of adult erythroid cells, including β039-thal cells (Figure 2). Coup-TFII overexpression increases γ-globin relative to β- globin expression and shows that Coup-TFII is not a repressor of γ-globin, as previously suggested by in vitro DNA binding studies.37
This evidence helps to reconcile the previously unex- plained observation that a mutation specifically abolishing Coup-TFII in vitro binding to the γ promoter (-107-108)38 failed to generate the phenotype of hereditary persistence of fetal hemoglobin in a mouse line transgenic for the human β-locus. In the absence of Coup-TFII, the γ- to β- globin ratio is decreased, pointing to a possible alternative interaction between γ- and β-globin genes with the LCR.39
To answer the question of whether Coup-TFII binds directly to the β-locus in vivo, we carried out ChIP-
Figure 6. Hypothetical model for the differential γ-globin activation/silencing in embryonic vs. adult erythroid cells. In embryo/fetal cells, Coup-TFII is expressed and recognizes the GGTCA consensus sequences within the locus control region (LCR) and possibly at other positions within the β-locus. The resulting β-locus architecture favors the expression of γ-genes. In adult cells, the same positions are occupied by Bcl-11A. The switch between the two factors results in γ-silencing. The overex- pression of Coup-TFII is capable of reverting γ-silencing in adult cells, suggesting that the erythroid cellular environment is similar in embryo/fetal and adult cell types, possibly because of the presence of a large set of common factors/cofactors. Whether Coup-TFII and Bcl-11A co-exist in some transient cellular stage during devel- opment or are distinctly expressed in different cells, remains to be elucidated. Continuous-line arrows point to the strong binding sites within the LCR; dashed-line arrows point to the other possible binding sites within the locus.
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haematologica | 2021; 106(2)