Báo cáo khoa học: Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA

Site-directed Mutagenesis and Footprinting Analysis of the Interaction of the Sunflower KNOX Protein HAKN1 with DNA

Tác giả: Mariana F. Tioni, Ivana L. Viola, Raquel L. Chan, and Daniel H. Gonzalez

Lĩnh vực: Sinh học Tế bào và Phân tử

Nội dung tài liệu: Nghiên cứu này khám phá sự tương tác giữa protein HAKN1, cụ thể là vùng homeodomain của nó, với DNA. Sử dụng các phương pháp như đột biến định hướng, phân tích footprinting bằng gốc hydroxyl và thí nghiệm missing nucleoside, các nhà nghiên cứu đã làm sáng tỏ trình tự DNA mà HAKN1 ưu tiên liên kết, cũng như các vị trí cụ thể trên protein chịu trách nhiệm cho sự tương tác này. Kết quả chỉ ra rằng HAKN1 liên kết với vùng 8 cặp base DNA, với lõi TGACA là quan trọng nhất. Các đột biến trên protein HAKN1 đã được tạo ra để xác định vai trò của các acid amin cụ thể, đặc biệt là Ile50, Asn51, Lys54 và Arg55, trong việc nhận diện trình tự DNA và ảnh hưởng đến ái lực liên kết. Dựa trên những phát hiện này, một mô hình tương tác HAKN1-DNA đã được đề xuất, trong đó helix III của homeodomain đóng vai trò chính trong việc tiếp xúc với các base DNA.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 13 July 2004, revised 31 August 2004, accepted 21 September 2004)
• doi:10.1111/j.1432-1033.2004.04402.x
• Homeobox genes encode a group of eukaryotic tran- scription factors generally involved in the regulation of developmental processes [1]. These genes contain a region coding for the homeodomain, a 60 amino acid protein motif that interacts specifically with DNA [2]. The homeodomain folds into a characteristic three- helix structure. Helices I and II are connected by a loop, while helices II and III are separated by a turn, resembling prokaryotic helix-turn-helix transcription factors. However, unlike helix-turn-helix-containing proteins, most homeodomains are able to bind DNA as monomers with high affinity, through interactions made by helix III (the so-called recognition helix) and a disordered N-terminal arm located beyond helix I [3-6].
• In plants, the first homeobox was identified in the maize gene Knotted1 (kn1; [7]). Dominant mutations in kn1, which is normally active only in meristematic cells, affect leaf development due to its aberrant expression in these organs [8]. Additional kn1-like genes (also termed knox genes) have been isolated from maize and other monocot and dicot species (reviewed in [9]), indicating that this class of genes constitutes a family present throughout the plant kingdom. The knox family of genes can be subdivided into two classes, I and II, by sequence relatedness and expression patterns [10]. Based on the expression pat- terns [11-13], analysis of mutants [14-17] and over- expression studies [18-21] it was proposed that class I knox genes are involved in the maintenance of meristematic cells in an undifferentiated state. Indeed, over- expression of some class I genes in Arabidopsis and tobacco produces the proliferation of meristems on the surface of leaves.
• The proteins encoded by knox genes belong to the three-amino-acid loop extension (TALE) superclass. Members of this superclass contain three extra amino acids within the loop connecting helices I and II [22] and are present in several eukaryotic kingdoms, sug- gesting that they represent an early evolutionary acqui- sition.
• Concerning their interaction with DNA, studies with proteins from barley [23], tobacco [24], rice [25] and maize [26] indicate that they bind related sequences containing a TGAC core (GTCA in the complement- ary strand), considerably different from the sequence TAAT recognized by most homeodomains [27]. Eluci- dating the structural basis for this difference would help to understand at the molecular level how KNOX transcription factors recognize their DNA target site.
• In this study, we analysed the interaction of the homeodomain of HAKN1, a sunflower class I KNOX protein [28], with DNA. Based on studies of wild-type and mutant forms of the homeodomain, we propose a model for the complex between HAKN1 and its target site. This model must be applicable to all KNOX homeodomains, as important amino acids are con- served within this family.
• Results
• Expression and DNA binding analysis of the HAKN1 homeodomain
• The homeodomain of the KNOX transcription factor HAKN1 was expressed in Escherichia coli as a fusion with the maltose binding protein using vector pMALc2. The fusion protein was purified by affinity chromatography in amylose resin and used for DNA- protein interaction studies. A 24-bp oligonucleotide (HAKN1 binding site; BS1) containing the sequence TGT(G/C)ACA was used as DNA target. This seq- uence was designed against a compilation of sequences bound by KNOX transcription factors from different species, and contains the TGAC (GTCA) core that is present in all of them.
• Figure 1A shows an electrophoretic mobility shift assay performed with HAKN1 and oligonucleotide BS1 or variants containing changes at single positions (sequences shown in the right panel). We have arbi- trarily numbered from 1 to 7 those positions present in the strand that contains the central G. Two shifted
• Fig. 1. Binding of HAKN1 to different oligo- nucleotides. (A) Electrophoretic mobility shift assay performed with 30 ng of HAKN1 and oligonucleotides containing different variants of the sequence TGT(G/C)ACA (numbers indicated above each lane). (B) Competition assay of HAKN1 binding to BS1 using a 15-fold molar excess of different oligonucleotides (numbers indicated above each lane) as competitors. The sequence of the 7-bp core present in each oligonucleo- tide is shown in (C) for reference. Modifica- tions with respect to BS1 are shown within black boxes.
• bands of similar intensity were observed in this experi- ment. The relative intensity of the low mobility com- plex varied when different protein preparations were used. We speculate that this behavior may arise from aggregation of the protein. Nevertheless, different pro- tein preparations showed the same specificity and affin- ity when considering the amount of bound protein as the sum of both shifted bands. These bands displayed similar footprinting patterns (see below), suggesting that a single HAKN1 homeodomain is bound to DNA in both complexes. This is strengthened by the fact that only monomer-DNA complexes were observed in crosslinking experiments (data not shown).
• Analysis of the interaction of HAKN1 with different oligonucleotides indicates that modifications in the outermost positions (1 and/or 7) do not significantly affect binding (Fig. 1A, lanes BS1, 1,7, 7T, 1 and 7C), while certain inner nucleotides, notably those located at positions 4–6, are critical for binding (Fig. 1A, lanes 4, 6A and 5). Regarding position 7, the change of A for T does not seem to affect binding, while the intro- duction of C partially reduces the amount of complex formed. Mutations at positions 2 (not shown) and 3 (lane 3) have only a moderate effect. Similar obser- vations could be made in experiments in which the binding to oligonucleotide BS1 was competed with a 15-fold molar excess of different oligonucleotides (Fig. 1B). These results indicate that HAKN1 mainly recognizes the GAC (GTC) trinucleotide and displays lower specificity at outer positions. The GAC triplet is contained within the TGAC sequence, found to be part of the binding sites of the barley KNOX protein Hooded [23] and of maize Knotted1 [26]. This element is also present in the sequence GTNAC, postulated to be important for the binding of the tobacco protein NTH15 to DNA [24], provided that N is G or C.
• Analysis of DNA binding by hydroxyl radical footprinting and interference assays
• A more detailed picture of the binding of HAKN1 to its target site was obtained by the analysis of footprint- ing patterns after cleavage of free and protein-bound DNA with hydroxyl radicals generated by Fe-EDTA complexes. For this purpose, a dimer of the corres- ponding oligonucleotide ligated through its EcoRI cohesive site was cloned into the BamHI site of pBlue- script SK. Cleavage with HindIII and Xbal produces a 94-bp fragment that contains two HAKN1 binding sites in opposite orientations. After HAKN1 binding to the 94-bp oligonucleotide, labeled specifically at one of its 3′ ends by filling-in the HindIII site, the complex was subjected to hydroxyl radical attack, and free and
• bound DNA were separated, recovered from the gel and analysed by denaturing polyacrylamide gel electro- phoresis (Fig. 2A). Because the oligonucleotide con- tains two sites in opposite orientation, both strands of the binding site can be observed in a single footprint- ing assay. Analysis of the cleavage patterns indicates that HAKN1 protects six nucleotides from the strand carrying the sequence TGTGACA (hereafter named the top strand). The protected area includes GACA and two adjacent nucleotides (GA) towards the 3′ end (Fig. 2A). On the bottom strand, the protected region covers two additional nucleotides, AC complementary to GT in TGTGACA (Fig. 2A). For both strands, the highest protection is observed within the GAC core, suggesting that the protein makes closer contacts in this region. This agrees with the important role of these nucleotide positions in determining the binding strength of HAKN1 to DNA shown by electrophoretic mobility shift assays. When the oligonucleotide labeled at its Xbal site (at the opposite 3′ end) was used, foot- printing patterns were identical to those described above, indicating that HAKN1 makes equivalent contacts with both binding sites present in the 94-bp fragment.
• Footprinting analysis was also performed with a similar oligonucleotide containing two mutated sites [BS(mut1,7); AGTGACT instead of TGTGACA, mutations underlined). The results obtained were essentially the same (not shown), suggesting that HAKN1 contacts the nucleotide adjacent to the GAC core and its complement on the other strand whether they are A or T.
• Information about the nucleotide positions that influence binding of HAKN1 to DNA was obtained from missing nucleoside (interference) experiments. Here, DNA is treated with hydroxyl radical-generating agents before protein binding, thus producing a popu- lation of molecules with single cleavages along the phosphodiester backbone. This population is incubated with the protein of interest and subjected to an elec- trophoretic mobility shift assay from which the free and bound fractions are recovered. Molecules with cleavages at positions important for binding are then under-represented in the bound fraction and, depend- ing on the binding conditions, over-represented in the free fraction. Figure 2B shows a missing nucleoside experiment using HAKN1 and the 94-bp DNA frag- ment containing two binding sites previously labeled in one of its 3′ ends (HindIII or Xbal sites) and treated with Fe-EDTA. It is noteworthy that there is a good correlation between the region protected by HAKN1 and the nucleotide positions important for binding. This means that all nucleotides in the protected area
• Fig. 2. Hydroxyl radical footprinting and interference analysis of HAKN1 binding to DNA. An oligonucleotide containing two HAKN1 binding sites (BS1) in opposite orientations was labeled in the 3′ end of either strand (HindIII or Xbal sites) and subjected to hydroxyl radical attack either after (A) or before (B) HAKN1 binding. Free (F) and bound (B) DNA were separated and analysed. A portion of the same fragment digested with defined restriction enzymes was used as a standard (S) to calculate the position of the footprint. Letters to the right of each panel indicate the DNA sequence (5′ end in the upper part) of the corresponding strand in this region. In the lower part, the sequence of the binding site is shown and the protected positions are indicated in bold and underlined. The GAC (GTC) core that shows the highest protec- tion is shaded.
• establish contacts that contribute to binding efficiency. Again, the GAC core seems to be particularly import- ant, but outside positions are also required (Fig. 2B). Within the core, modifications to G and A or their complements influence binding more markedly. These results agree with the fact that mutations of these two nucleotides abolish binding of HAKN1 to DNA. On
• the other hand, because nucleotides at outside posi- tions can be mutated without significant loss in bind- ing efficiency, it can be assumed that they mainly participate in nonspecific contacts, such as those estab- lished with the sugar-phosphate backbone.
• The results of footprinting and missing nucleoside experiments also indicate that HAKN1 does not make
• symmetrical contacts with its target site. The protein establishes contacts with both strands at the right side of the GAC core, while only one strand seems to be contacted at the left side. This lack of symmetry and the extension of the contacts most probably indicate that only one molecule of HAKN1 is bound at each target site.
• Binding of HAKN1 single-site mutants to DNA
• The picture that emerges from our results is that HAKN1 binds an 8-bp region of DNA with a tGACa (tGTCa) specificity core. An interesting question is how the HAKN1 homeodomain interacts with this sequence and which amino acids are involved in sequence-specific contacts. To answer this, we have analysed the effect of single-site mutations on HAKN1 binding to TGACA and variants of this sequence. It is logical to assume that changes in amino acids involved in the interaction must influence binding efficiency. In addition, some substitutions may alter binding specific- ity, indicating the existence of contacts between a given residue and defined positions within the DNA.
• Residue 50 (53 in TALE homeodomains) is usually involved in determining the different specificities among related homeodomains [27,29-31]. In homeo- domains that bind the canonical TAAT sequence, residue 50 interacts with nucleotides located 3′ to this site [27,31]. We reasoned, then, that changing Ile50, present in HAKN1 and all KNOX proteins, may influ- ence sequence preferences at external positions of the core. As a first approach, we mutated Ile50 to Ser, pre- sent in the yeast TALE protein MATa2 [32]. The ana- lysis of binding of I50S-HAKN1 to variants of the HAKN1 binding site indicates a preference for an oligonucleotide containing the sequence TGACT, while the wild-type HAKN1 homeodomain binds TGACA and TGACT with similar efficiency (Fig. 3A). This suggests that residue 50 interacts with the 3′ region of the top strand (and/or the 5′ region of the bottom strand), outside the GAC core. This is also evident in competition experiments (Fig. 3B), where oligonucleo- tides BS(mut1,7) and BS(mut7T) compete more effi- ciently than variants with A [BS1 and BS(mut1)] or C [BS(mut7C)] at this position. Changes at other posi- tions within the target DNA sequence produced sim- ilar effects on binding than with the wild-type protein (Fig. 3).
• To further explore the hypothesis that residue 50 is oriented towards the 3′ end of the top strand, we also mutated Ile50 to Lys, present in Drosophila bicoid [33]. I50K-HAKN1 shows a net preference for an oligonucleotide containing the sequence TGACCC [BS(mut7C)] over the original TGACAG, present in
• Fig. 3. DNA binding preferences of HAKN1 mutants at position 50. (A) Electrophoretic mobility shift assay of 150S-HAKN1 (30 ng) binding to BS1 and BS(mut1,7). (B) Binding of 150S-HAKN1 to BS(mut1,7) was com- peted with a 100-fold molar excess of oligo- nucleotides with different sequences (depicted in Fig. 1). (C) Binding of 150K- HAKN1 (30 ng) to different oligonucleotides was analysed by an electrophoretic mobility shift assay. In (D), the binding of different amounts (50, 100 and 250 ng) of either HAKN1 or 150K-HAKN1 to oligonucleotides BS1 and BS(mut7C) is shown. Oligonucleo- tide sequences are shown in Fig. 1.
• BS1 and BS(mut1) (Fig. 3C). This result confirms that residue 50 interacts with nucleotides adjacent to the TGAC core. Binding analysis with different oligonuc- leotides indicated that 150K-HAKN1 is also able to interact with oligonucleotide BS(mut6G), that contains a TGAG core (Fig. 3C). In fact, when higher protein concentrations were used in the assays, binding to TGAGAG was considerably better than to TGACAG (not shown), suggesting that Lys50 may also be able to contact the fourth position of the core, thus changing the preference for G. The inclusion of Lys at position 50, in addition to promoting a change in specificity, resulted in a protein with increased affinity towards its preferred binding site (Fig. 3D). An additional, fast- migrating band observed in this experiment is present in free DNA and may represent noncovalent ol