Protocols for protein-DNA binding analysis of a zinc finger transcription factor bound to its cognate promoter

Abstract

Here, we describe protocols to interrogate the binding of the zinc fingers of the transcription factor ZBTB7A to the fetal γ-globin (HBG) promoter. We detail the steps for performing electrophoretic mobility shift assays (EMSAs), X-ray crystallography, and isothermal titration calorimetry (ITC) to explore this interaction. These techniques could readily be applied to the structural studies of other zinc finger transcription factors and cognate DNA sequences. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).

Keywords: Biophysics; Molecular Biology; Protein Biochemistry; Protein expression and purification; X-ray Crystallography.

Graphical abstract

Figure 1

Procedure for performing electrophoretic mobility shift assays using a radiolabeled DNA probe to analyze ZBTB7A ZF-DNA binding The experimental steps include DNA probe labeling with ATP [γ-³²P], probe purification, the preparation of a 6% non-denaturing polyacrylamide gel, EMSA sample preparation and loading, gel drying and gel imaging.

Figure 2

Example EMSA ZBTB7A ZF1-4 binds to the -200γ WT probe in lane 2 (∗∗). ZBTB7A ZF1-4 binding is reduced in lanes 3 and 5 with the use of mutant probes. A pcDNA3 ‘empty’ control was used in lane 1 to observe background binding of endogenous COS-7 proteins. Free probe that is not bound to any protein (∗) migrates to the bottom of the gel. If working with different ZBTB7A ZF truncations, DNA-probe bands may appear at different migration positions in the gel due to the smaller size of the truncated protein. Published in Yang et al. (2021).

Figure 3

Procedures for the expression and purification of ZBTB7A and ZBTB7A-DNA complex The purification steps include lysis of cells by high-pressure cell disrupter, incubation of the supernatant with the GST column, cleavage by TEV enzyme through dialysis, and size exclusion chromatography for ZBTB7A and ZBTB7A-DNA complex, respectively. Use SDS-PAGE to evaluate the purity of protein or protein-DNA complex.

Figure 4

Structure determination of ZBTB7A in complex with -200 element with a C-194A mutation The crystallization process consists of crystal screening and optimization by hanging drop method, picking the crystal that is suitable for X-ray diffraction, data collection at SSRF and structure determination.

Figure 5

ITC evaluation of HPFH-associated mutations in 150 mM NaCl