Iron is required for in vitro differentiation of both myeloid cell lines and primary CD34 cells. This iron requirement has been linked to the transcriptional induction of the cyclin dependent kinase inhibitor gene, p21WAF1/CIP1 (

J. Cell. Physiol.
). We conducted experiments to clarify the role of iron in p21 gene transcription during differentiation of HL-60 cells. Transient transfections of p21/luciferase deletion mutants into HL-60 cells identified iron-responsive areas in the p21 gene promoter at -119 to +16 bp. This region contains 6 binding sites for Sp family transcription factors. Mithramycin, an inhibitor of Sp-DNA interactions, blocked both expression of p21 and differentiation in HL-60 cells induced with phorbol myristate acetate (PMA) thereby confirming a key role for Sp family proteins in p21 transcription and cell differentiation. Gel mobility/supershift assays demonstrated that both Sp1 and Sp3 bound to the p21 promoter under baseline conditions. Chromatin immunoprecipitation (ChIP) experiments with quantitative PCR revealed complex changes of both Sp1 and Sp3 binding to the p21 promoter after PMA treatment. Overall, both Sp1 and Sp3 binding decreased. However, after PMA treatment the ratio of Sp3 bound relative to Sp1 changed significantly at different promoter sites. This ratio increased in the promoter region containing binding sites Sp1-4, Sp1–5, and Sp1-6 at -70/-40 bp but not in the region with binding sites Sp1-1, Sp1-2, and Sp1-3 at -118/-69 bp. These changes were reversed by the iron chelator, desferrioxamine (DF), demonstrating their dependence on iron. In vivo footprinting experiments on the p21 promoter confirmed the occurrence of major changes in Sp factor binding by demonstrating appearance of unprotected guanines on the sense strand of the Sp1-1, Sp1-2, and Sp1-3 binding sites after induction by PMA, an effect also reversed by DF. These data indicate that interactions of Sp family proteins with the p21 promoter during HL-60 differentiation are strongly influenced by iron status. Sp family members regulate acetylation status of the p21 promoter by interactions with p300 and histone deacetylase 1 (HDAC1). Therefore, we examined role of iron status on recruitment of p300 or HDAC1 to the p21 promoter during HL-60 cell differentiation. Oligonucleotide pull-downs indicated that p300 but not HDAC1 associated with the p21 promoter during PMA induction. p300 association was not blocked by DF. To assess histone acetylation at the p21 promoter we performed ChIP with antibodies to acetylated H3 or H4 histone. Acetylation of both H3 and H4 histones occurred within 1 hr of PMA induction and was not blocked by DF. Summarizing:

  1. Induction of differentiation in HL-60 cells by PMA was accompanied by complex alterations of Sp1 and Sp3 binding to the p21 promoter.

  2. Iron deprivation disrupted the pattern of Sp1 and Sp3 binding induced by PMA.

  3. Iron deprivation did not inhibit p300 recruitment or histone acetylation at the p21 promoter.

Taken together with the observed inhibition by mithramycin of both p21 expression and cell differentiation these results indicate that Sp family interactions with the p21 promoter are required for HL-60 cell differentiation and that these interactions are iron-dependent. Disruption of these interactions by iron deprivation blocks p21 expression and HL-60 cell differentiation. The precise molecular mechanism influenced by iron deprivation remains unknown but does not involve effects on histone acetylation.

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