Combination of a zinc finger and homeodomain required for protein-interaction

Abstract

The Zinc Finger Homeodomain Enhancer-binding Protein (Zfhep) is involved in skeletal patterning, immune cell, muscle, and brain development, and is necessary for life. Zfhep contains a single central homeodomain (HD) adjacent to an isolated zinc finger, the function of which is unknown. The placement of a zinc finger so close to a homeodomain is novel in nature. The aim of this work was to characterize the Zfhep homeodomain (HD) or the zinc finger homeodomain (ZHD), with respect to DNA-binding and protein-protein interactions. Glutathione-S-transferase (GST) fusion proteins containing either just the HD or both the zinc finger and HD (ZHD) were expressed in E. coli. The GST fusion protein affinity-binding assay demonstrated that Zfhep ZHD interacts specifically with the POU domain of the Oct-1 transcription factor. The adjacent zinc finger is required since Zfhep HD alone does not interact with Oct-1 POU domain. Furthermore, ZHD does not bind to the POU homeodomain lacking the POU specific region. These results demonstrate that the Zfhep zinc finger homeodomain motif functions as a protein-binding domain in vitro, and suggests that Zfhep may modulate the activity of POU domain transcription factors. However, neither the Zfhep ZHD nor the HD bound DNA in EMSA or selected a DNA-binding site from a pool of random oligonucleotides. This is the first demonstration of a function for the HD region of Zfhep, which is the first case of a bi-partite domain requiring both a zinc finger and a HD for binding to protein.

Fig 1

Domain structure of Zfhep. The Zfhep structure contains tandem repeats of zinc fingers near the N- and C-terminal ends (ZFN and ZFC), and a central ZHD. The amino acid sequence of the central zinc finger region in the Zfh-1 subfamily typically is similar to the zinc finger consensus (C-X_2,4-C-X₃-F/L-X₈-H-X_2-5-C/H) [13]. Sequences are from; 1, rat Zfhep (Q62947); 2, mouse Zfhx1a (NP 035676); 3, hamster BZP (Q60542); 4, Drosophila Zfh-1 (P28166); 5, mosquito (EAA06926); 6, mouse SIP-1 (NP 056568).

Fig 2

SDS-PAGE of GST-fusion proteins. pGEX-2TK vector, or subclones of Zfhep were expressed in bacteria and purified by affinity chromatography. GST; GST-HD; or GST-ZHD proteins were analyzed by SDS-PAGE. High Range protein molecular size standards (Invitrogen, Carlsbad, USA) are shown (M).

Fig 3

Zfhep ZHD binds the Oct-1 POU domain. ³²P-ZHD (5 × 10⁵ cpm) was incubated with 20 μl GST, GST-POU_HD or GST-POU bound to glutathione-sepharose in the presence of 4 mg E. coli extract as competitor. Pellets were washed as described. ³²P-ZHD bound by the fusion protein was eluted in SDS sample buffer, separated by 12.5 % SDS-PAGE and analyzed by autoradiography. Lane 1 received 5 % ³²P-ZHD input in the reaction (Input). Full-length ³²P-ZHD is indicated by the arrow.

Fig 4

ZHD interaction with Oct-1 POU domain is specific. ³²P-ZHD (2.5 μl, approximately 2.3 × 10⁵ cpm) was incubated with 7 μl glutathione-sepharose beads with GST-POU (or GST alone) with or without a 100-fold excess non-radioactive ZHD (250 μl). Beads were washed and eluted as described. The SDS-PAGE gel lanes received either 5 % ³²P-ZHD input (Input), or ³²P-ZHD eluted from the GST beads (GST), GST-POU beads (POU), or GST-POU beads with 100-fold excess non-radioactive ZHD (100x).

Fig 5

The Zfhep HD alone does not bind GST-POU or GST-POU. ³²HD P-HD (5 × 10⁵ cpm) was incubated with 20 μl of either GST, GST-HD, GST-POU_HD or GST-POU bound to glutathione sepharose, and washed as described. ³²P-HD bound by the fusion protein was eluted in SDS sample buffer, separated by 15 % SDS-PAGE and analyzed by autoradiography. Five percent of the ³²P-HD input was loaded in lane 1 for comparison. Zfhep HD did not form homodimers under these conditions, as indicated by a lack of binding to GST-HD, nor did ³²P-HD bind to Oct-1 proteins.

Fig 6

Domains in Zfhep ZHD required for interaction with Oct-1 POU domain. A schematic of the domains that bind GST-POU are shown. Neither the HD alone, nor the zinc finger alone, are able to bind the Oct-1 POU domain.

Fig 7

Zfhep HD and ZHD do not bind DNA in EMSA. (A) The Zfhep HD and ZHD do not bind the ³²P-H2B probe. Each GST-fusion protein (5 μl in the first lane, and 10 μl in the second lane) was incubated with the ³²P-H2B oligonucleotide. GST-POU_HD and GST-POU were used as positive controls. The reaction mixture was separated on a 5 % non-denaturing gel, and exposed to film. (B) The Zfhep HD and ZHD do not bind the ³²P-AFP1 probe. 5 or 10 μl of the indicated GST-fusion protein was incubated with the ³²P-AFP1 oligonucleotide. The reaction mixture was separated on a 5 % non-denaturing gel, and exposed to film.

Fig 7

Zfhep HD and ZHD do not bind DNA in EMSA. (A) The Zfhep HD and ZHD do not bind the ³²P-H2B probe. Each GST-fusion protein (5 μl in the first lane, and 10 μl in the second lane) was incubated with the ³²P-H2B oligonucleotide. GST-POU_HD and GST-POU were used as positive controls. The reaction mixture was separated on a 5 % non-denaturing gel, and exposed to film. (B) The Zfhep HD and ZHD do not bind the ³²P-AFP1 probe. 5 or 10 μl of the indicated GST-fusion protein was incubated with the ³²P-AFP1 oligonucleotide. The reaction mixture was separated on a 5 % non-denaturing gel, and exposed to film.

Fig 8

Zfhep HD and ZHD cannot select a DNA binding site. (A) Zfhep HD did not select a DNA binding site from a pool of random oligonucleotides. Oligonucleotides obtained after eight rounds of binding were radiolabeled with ³²P and used in EMSA with either 5 μl (first lane) or 10 μl (second lane) of GST-HD or MBP-ZFC (positive control) fusion proteins. (B) The Zfhep ZHD did not select a DNA-binding site after five rounds of binding. GST-ZHD or GST-POU (the positive control) were used for five rounds of oligonucleotide selection. The resulting oligonucleotides were labeled and incubated with either 5 μl (first lane) or 10 μl (second lane) of the indicated GST-fusion protein. The binding reactions were separated on a 5% non-denaturing gel, and exposed to film. Labeled oligonucleotides without protein are run on adjacent lanes (DNA). Unbound oligonucleotides migrate to the bottom of the gel (Free DNA).