Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo

Abstract

The locus control region of the ␤-globin cluster contains five DNase I hypersensitive sites (5 HS1-5) required for locus activation. 5 HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5 HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5 HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5 HS3.

Vascular Implications of the Krüppel-Like Family of Transcription Factors

“…For example, an experiment using transgenic mice showed that KLF1, but not Sp1, preferentially binds to the ␤-globin locus site in vivo, despite the fact that KLF1 and Sp1 bind to the locus in biochemical studies in vitro. 46 We have also shown that the KLF5-binding element in the SMemb gene also binds Sp1 in vitro.…”

Joining the loops: β‐Globin gene regulation

“…The exact mechanism of EKLF action in globin gene regulation is unclear because of several contradictory reports, most likely originating from different experimental designs. In mice, EKLF activates a human b-globin reporter construct containing HS3 of the LCR and is required for formation of this HS in the construct (31). Similarly, HS3 is not formed at the full transgenic human locus in EKLF knockout mice (30) (32).…”

Krüppel-like Factors: Three Fingers in Many Pies

“…As a result, the second level of specificity is via their respective activation/repression domains, which are unique to each member, and thus likely determine their resultant protein/protein interactions. This has been directly tested for KLF1, where in vivo tests of ␤-globin promoter activation by transient transfection assays (64) and in transgenic mice (65) demonstrate that the Sp1 transactivation domain cannot substitute for the KLF1 transactivation domain. Third, the zinc finger region can also play a role in KLF/ protein interactions, whether bound to DNA (e.g.…”