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. 1995 Jan 3;14(1):97–105. doi: 10.1002/j.1460-2075.1995.tb06979.x

Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specific protein.

S M Jane 1, A W Nienhuis 1, J M Cunningham 1
PMCID: PMC398056  PMID: 7828600

Abstract

The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma-globin promoter is integral to the competitive silencing of a linked beta-promoter in embryonic/fetal stage erythroleukemia (K562) cells. We now report the biochemical purification of SSP from K562 cell nuclear extract and demonstrate that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti-CP2 antiserum disrupts the formation of the SSP-SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. We have defined the molecular weight of the partner protein as 40-45 kDa in UV and protein cross-linking experiments. An element analogous to the human SSE has previously been demonstrated in the chicken beta A-gene-promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF-E4. Comparative studies between human CP2 and chicken NF-E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE and formation of this complex is ablated by the addition of anti-CP2 antiserum or a monoclonal antibody to NF-E4. Western analysis of partially purified NF-E4 using anti-CP2 antiserum or the NF-E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF-E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP-SSE complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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