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Coup-TFII activates γ-globin
sequencing and 3C experiments in β-K562 cells, which offer the advantage of co-expression of Coup-TFII togeth- er with e-, γ- and β-globins, making it possible to evaluate the effects of the modulation of Coup-TFII levels. We demonstrated that Coup-TFII does indeed bind the GGTCA binding sites present within the LCR and that its KO favors LCR-β-globin interactions (Figure 5). The pres- ence of Coup-TFII binding at different positions suggests a possible widespread action of Coup-TFII in the tran- scriptional regulation of the β-locus. The functional rele- vance of these single binding sites will require their pre- cise editing, with the caveat that the presence of overlap- ping/adjacent binding sites, bound by different transcrip- tion factors/cofactors (as in the case of the CCAAT box region of γ genes), could complicate the interpretation of the results.
Of interest, the Coup-TFII consensus sequence retrieved by MEME (GGTCA) in ChIP-sequencing almost perfectly matches the one described for Bcl11a-XL40 and for TR2/TR4, two known repressors of γ-globin in adult cells.40,41 As for Bcl11a-XL (for which direct binding to the γ promoter could only be seen using the very sensitive CUT&RUN technique40), we could only detect very weak peaks on the CCAAT box region of the γ promoter, although previous in vitro binding and dimethyl sulfate interference experiments clearly mapped the Coup-TFII binding sites to the double CCAAT box γ-promoter region.14 The binding of Coup-TFII to the LCR (and pos- sibly its interaction with the weaker sites on the double CCAAT box region), suggests that it might concur to establish/maintain the architecture of the β-locus in early developmental stages, when preferential γ activation is required, as confirmed by the 3C experiment (Figure 5). The same regions (γ-promoter and LCR) bound by Coup- TFII within the LCR, are occupied in adult cells by the γ repressors Bcl11a-XL and/or TR2/TR4 that keep γ-globin expression switched off. Coup-TFII is expressed in an early developmental stage, whereas expression of Bcl11a- XL and TR2/TR4 is confined to adult cells. This would suggest that repression of γ-globin in the adult stage may involve a switch between factors binding to the CCAAT box within the γ promoters and potentially within the LCR, i.e., from Coup-TFII in erythroid cells of yolk sac origin to Bcl11a-XL and TR2/TR4 in adult erythroid cells. The existence of specific developmental activators and repressors could be made necessary by the presence in both embryonic/fetal and adult cells of a common set of activators/coactivators (such as Gata1 and its coactiva- tors) that create a “permissive environment” to which the γ promoter is responsive. This hypothesis would also help to explain why Coup-TFII, when overexpressed in
adult cells, is indeed capable of activating γ-globin, over- coming γ-globin repression (Figure 2).
Whether Coup-TFII and Bcl11a-XL and/or TR2/TR4 coexist in the same cell in a given transitory status in which they directly compete/substitute each other to allow the switch (from γ “ON” to γ “OFF”), or whether their expression is restricted to non-overlapping spatial and temporal windows of development, remains to be elucidated.
The novel role of Coup-TFII as an activator of fetal glo- bin genes provides new insight into the early activation of embryonic/fetal genes and, possibly, on the mechanisms of hemoglobin switching. In the light of these observa- tions, Coup-TFII could be considered as a target for γ-glo- bin reactivation. Although COUP-TFII has been exten- sively studied genetically, present knowledge of upstream regulatory signals and of possible ligands, remains poor. Retinoids,42-44 sonic hedgehog,45 ETS and their coactiva- tors46 activate COUP-TFII expression in different cellular contexts. Moreover, whereas structural data suggest that the Coup-TFII protein is an “orphan” receptor capable of an auto-repressive conformation, there is evidence that Coup-TFII ligands may exist47 and, in addition, descrip- tions of synthetic Coup-TF agonists have been recently published.48 Collectively, the evidence that Coup-TFII can be transcriptionally induced and functionally activated suggests that it may be an attractive novel target for γ-glo- bin reactivation in β-hemoglobinopathies.
Disclosures
No conflicts of interest to disclose.
Contributions
SE, CF, GB and MGM performed experiments, and ana- lyzed and discussed data; MFM performed FC analysis; LM performed human cultures; CC performed ChIP-seq experi- ments; FG performed immunofluorescence analysis; IFM and SG performed experiments. YN provided HUDEP cells; JS con- tributed with support and discussion of the research; PM, SO and SMLB critically discussed the manuscript; AR led the proj- ect and wrote the paper.
Funding
The authors would like to thank Dr. Andrea Ditadi for help with experimental work, Fondazione Cariplo grant n. 2012.0517 for support to AR, PM and JS, the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement n. 289611 (HEM_ID Project) for support to AR and JS and the Knut and Alice Wallenberg foundation for support to CC.
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