A downstream element in the human β-globin promoter: Evidence of extended sequence-specific transcription factor IID contacts

A downstream element in the human β-globin promoter: Evidence of extended sequence-specific transcription factor IID contacts

^*Division of Hematology/Oncology, The Children's Hospital Medical Center, and Dana Farber Cancer Institute, Department of Pediatrics, Harvard Medical School, Howard Hughes Medical Institute, Boston, MA 02115; and ^†Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854

Contributed by Stuart H. Orkin

Abstract

We describe here the identification and characterization of a functional downstream element in the human adult β-globin promoter. The existence of this element was indicated by two mutations at +22 and +33 downstream of the β-globin transcriptional start site in humans with β-thalassemia. In vitro transcriptional analysis of these mutants, plus a third at +13, indicates that all three decrease transcription from the β-globin promoter. Scanning mutagenesis from +10 to +45 indicates that this region contains a functional cis element(s) in vitro, and we designated this element the DCE (downstream core element). The DCE functions in concert with the β-globin CATA box and initiator element, as well as in a heterologous, TATA-less context. A second set of mutants indicates that a particular geometry of the DCE and core promoter is necessary for promoter function. Lastly, DCE mutants show reduced affinity for transcription factor IID (TFIID). These data indicate that TFIID makes sequence-specific contacts to the DCE and that TFIID binding is necessary for DCE function.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Howard Hughes Medical Institute, Division of Hematology/Oncology, Enders, Room 761, 300 Longwood Avenue, Boston, MA 02115. E-mail: orkin{at}rascal.med.harvard.edu.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.120181197.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.120181197
Abbreviations:

TFIID,

transcription factor IID;

DCE,

downstream core element;

DPE,

downstream promoter element;

Inr,

initiator element;

βInr,

β-globin initiator element