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X. Yu et al.
is the fact that there are no CpG rich regions in the proxi- mal g-globin promoter, and in fact the entire β-globin locus is CpG poor. Previous work has shown that MBD2 does not bind directly to sequences in the β-globin gene locus in β-YAC transgenic mice;32 this is expected since there are no CpG rich regions in the locus. This suggests that MBD2-NuRD may exert its effect through regulation of other silencers. Here we investigated whether MBD2 depletion changes the expression of known g-globin silencers, and found that MBD2KO does not affect the expression of LRF and actually increases expression of BCL11A and KLF1. It remains possible that BCL11A inter- acts functionally with MBD2-NuRD in human erythroid cells, as depletion of either results in high levels of HbF without impairing differentiation. Current studies to iden- tify targets and interaction partners of MBD2 through which g-globin silencing is mediated are ongoing.
The data presented here firmly establish that MBD2- NuRD is a potent repressor of HbF expression in adult human erythroid cells, while MBD3-NuRD is not. Specific mutations in the intrinsically disordered region and CC domains of MBD2 necessary for association of other NuRD components that recruit HDAC and chro- matin remodeling sub-complexes abrogate the silencing
effect of NuRD on the g-globin gene (Figure 7). Finally, we re-enforce the finding that MBD2 knockdown in pri- mary human erythroid cells results in an 8-10-fold increase in %g/g+β mRNA expression without affecting erythroid differentiation. The fact that MBD2 null mice show only minor phenotypic abnormalities (mild deficits in maternal nurturing, a lower than normal body weight, and altered B-cell differentiation) but are otherwise fully viable and fertile49,50 suggests that therapies targeting MBD2, especially in somatic tissues, may have acceptable side effects.
Acknowledgments
The authors wish to thank Amy Jones and Lindsey Paisley for expert assistance in preparation of this manuscript.
Funding
This work was supported by National Institutes of Health grants; R56 DK29902 (GDG) and R01 DK115563 (GDG and DCW) from the National Institute of Diabetes, Digestive and Kidney Diseases, R01 GM098264 (DCW) from the National Institute of General Medical Sciences, and P30 CA16059 (VCU Massey Cancer Center Core Grant) from the National Cancer Institute.
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