This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第17979号 / 医科博第48号 / 新制||医科||4(附属図書館) / 80823 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 五十嵐 樹彦, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

To investigate whether higher order packing interactions confer protein-DNA specificity, a modified Knob-Socket (KS) model was used to analyze the interface of bZIP-DNA crystal structures. The KS analysis identified a nine-residue quadripartite recognition core consisting of four contiguous KS pockets P1, P2, N3, and N4 that each pack one of the four DNA half-site bases in the target sequence. Only one base per base pair packs, and these interactions are split across the DNA strands: the first two positive strand positions 1p and 2p pack into P1 and P2 while the last two negative strand positions 3n and 4n pack into N3 and N4. Amino acid sequence analysis of the four KS pocket regions indicates that the primary mechanism recognition is packing or non-packing of the 5-methyl group of dT as well as 5-methylcytosine. P1 shows little packing of dT; P2 packs dT but including two Asn residues in this pocket seems to block packing in this region; N3 also packs dT, but including a Phe also blocks packing; N4 consistently packs dT. This analysis demonstrates that there is an amino acid code to DNA recognition, allowing for multi-residue recognition and packing of the 5-methyl group.

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In Tabak interagieren das Ankyrin-repeat Protein NtANK1 und der basische Leucin Zipper (bZIP)-Transkriptionsfaktor NtBZI-1. Diese Proteine sind in Auxin-vermittelter Genaktivierung und in Pathogenabwehr involviert. Ziel dieser Arbeit war es, die Funktion der homologen Ankyrin-repeat Proteine AKR2A und AKR2B aus Arabidopsis thaliana zu untersuchen. Dazu wurde die Interaktion zwischen AKR2A/B, und den homologen bZIP-Transkriptionsfaktoren der Gruppe C getestet. Mit verschiedenen Methoden, wie Hefe- und Protoplasten-two-hybrid und BiFC ( bimolecular fluorescence complementation ) konnte eine Interaktion der Arabidopsis Proteine nicht bestätigt werden. Lokalisationsstudien von YFP-AKR2A/B-Fusionsproteinen bestätigten, dass die Proteine im Cytoplasma lokalisiert sind. Sie besitzen ein funktionsfähiges Kernexportsignal und akkumulieren nach Inhibierung des Kernexports im Kern. Zur Funktionsaufklärung wurden AKR2-RNAi Pflanzen erzeugt, die sich phänotypisch vom Wildtyp unterscheiden: sie zeigen ein verringertes Wachstum und einen reduzierten Chlorophyllgehalt, abhängig von der Ausprägung des RNAi-Effektes. In elektronenmikroskopischen Untersuchungen ist zu erkennen, dass sich die Blattchloroplasten der AKR2-RNAi Pflanzen von denen des Wildtyps morphologisch unterscheiden und in ihrer Entwicklung unspezifisch beeinträchtigt sind. Eine Transkriptomanalyse der AKR2-RNAi Pflanzen zeigte, dass Gene des Endomembransystems herunterreguliert sind und viele Stress-induzierte Gene hochreguliert sind. Deshalb wurden die Pflanzen verschiedenen Stressbedingungen unterzogen und übereinstimmend stellte sich heraus, dass sie anfälliger gegenüber oxidativem Stress, Infektion mit dem biotrophen Bakterium Pseudomonas syringae und Infektion mit dem nekrotrophen Pilz Botrytis cinerea waren. Diese erhöhte Anfälligkeit kann als sekundärer Effekt aufgrund der beeinträchtigten Chloroplasten-Biogenese oder als spezifische Reaktion auf die reduzierte AKR2A/B-Proteinmenge interpretiert werden. Da AKR2A/B bereits als Importver mittler für chloroplastidäre Membranproteine beschrieben wurden (Bae et al., 2008), werden zusammenfassend mit den hier erhaltenen Daten multiple Funktionen für AKR2A und AKR2B diskutiert: Transport von Proteinen zu verschiedenen Endomembransystemen, eine Funktion im Signalaustausch zwischen Chloroplast und Kern, und eine Regulation der Transkriptionskontrolle im Kern.

<p>The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The <i>napA</i> gene encodes for a <i>Brassica napus</i> 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The <i>napA</i> promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to <i>napA</i> through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster.</p><p>We also show that the RY/G cluster is responsible for repression of <i>napA</i> expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the <i>napA</i> RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the <i>napA</i> RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.</p>

The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The napA gene encodes for a Brassica napus 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The napA promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to napA through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster. We also show that the RY/G cluster is responsible for repression of napA expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the napA RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the napA RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.