Loading of DNA-binding factors to an erythroid enhancer

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• The HS-40 enhancer controls developmental stage- and erythroid lineage-specific expression of human alpha-like globin genes.
• A model suggests competitive factor binding at the 3'-NA motif of HS-40 modulates zeta-globin promoter activation through enhanceosome configuration changes.

Purpose of the Study:

• To investigate the molecular mechanisms underlying HS-40 enhancer function and its role in globin gene regulation.
• To elucidate the specific roles of transcription factors AP1 and NF-E2 in the developmental regulation of the alpha-globin locus.

Main Methods:

• Genomic footprinting and expression analysis of K562 cells with HS-40 mutants.
• Gel mobility shift assays and transient cotransfection assays to study factor binding.
• Chromatin immunoprecipitation experiments to assess in vivo factor binding.

Main Results:

• Factors bound to HS-40 motifs cooperate to form a functional enhanceosome; mutations alter its configuration and function.
• A 3'-NA(II) mutation abolishes small MafK homodimer binding but allows NF-E2 and AP1 binding.
• NF-E2 binding affinity to the 3'-NA motif is reduced twofold by the 3'-NA(II) mutation due to increased dissociation rate.
• In vivo, only NF-E2 binding, not AP1, is detected near HS-40 in K562 cells.

Conclusions:

• The 3'-NA(II) mutation's effect on zeta-globin promoter activity is mediated by altered NF-E2 binding, not AP1.
• AP1 is excluded from a role in the developmental regulation of the human alpha-globin locus via the HS-40 enhancer's 3'-NA motif.
• Small Maf homodimers, besides NF-E2, are likely involved in regulating HS-40 function in vivo.