Identification of KAP-1-associated complexes negatively regulating the Ey and β-major globin genes in the β-globin locus

Deregulations of erythroid differentiation may lead to erythroleukemia and other hemoglobinopathies, yet the molecular mechanisms underlying these events are not fully understood. Here, we found that KAP-1-associated complexes contribute to the regulation of the β-globin locus, the key events of erythroid differentiation. We show that RNAi-mediated knockdown of KAP-1 in mouse erythroleukemia (MEL) cells increases expression of the Ey and β-major globin genes during hexamethylenebisacetamide (HMBA) induced differentiation process. This indicates that at least part of KAP-1-associated complexes negatively regulates β-globin gene expression during definitive erythroid differentiation. ChIP-PCR analysis revealed that one or more KAP-1-associated complexes are targeted to the promoter region of the Ey and beta-major globin genes. Since KAP-1 is only a scaffold molecule, there must be some transcriptional regulators allowing its targeted recruitment to the β-globin locus. To further discover these novel regulators, proteins interacting with KAP-1 were isolated by endogenous immunoprecipitation and identified by LC-ESI–MS/MS. Among the proteins identified, MafK and Zfp445 were studied further. We found that KAP-1 may contribute to the repression of Ey and β-major globin gene transcription through recruitment to the promoters of these two genes, mediated by the interaction of KAP-1 with either Zfp445 or MafK, respectively.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (67 K)Download as PowerPoint slideHighlights► It is very important to uncover the mechanisms of the regulations of β-globin locus. ► KAP-1 complexes negatively regulate β-globin gene during erythroid differentiation. ► KAP-1 complexes are targeted to the promoter region of βmaj and Ey genes. ► KAP-1 interacting proteins were isolated and identified by endogenous IP-LC–MS/MS. ► Among them, MafK and Zfp445 bind to βmaj and Ey promoters, repressing them respectively.