Flavonoids are important and ubiquitous secondary metabolites and are known to participate in various developmental and stress response processes in plants. Common flavonoids include anthocyanins, proanthocyanidins and flavonols. This thesis aims to determine, at the molecular level, how the biosynthesis of flavonoids, in particular the proanthocyanidins, is regulated in poplar. Poplars accumulate large amount of flavonoids and the major flavonoid biosynthetic genes in poplar have been identified. Flavonoid biosynthesis is known to be regulated by MYB transcription factors. Previous work had identified MYB134 as a key regulator of proanthocyanidin synthesis in poplar. Here I describe experiments on five additional genes encoding MYB activators (MYB115 and MYB117), MYB repressors (MYB165 and MYB194), and one bHLH cofactor (bHLH131) as possible flavonoid regulators in poplar. The objective of this work is to determine the in planta functions of these new flavonoid regulators using reverse genetic methods, phytochemical and transcriptome analysis, to identify their target genes and to determine how these transcriptional regulators interact using promoter transactivation and yeast two- hybrid assays. MYB115 was identified as a second proanthocyanidin regulator. Similar to the effects of MYB134, overexpression of MYB115 in poplar led to increased proanthocyanidin content and upregulated flavonoid biosynthesis genes, but reduced the accumulation of salicinoids. Overexpression of repressor type MYBs, MYB165 or MYB194 led to reduced anthocyanin, salicinoid and hydroxycinnamic ester accumulation in leaves, while reducing proanthocyanidin content in roots. Transcriptome analysis demonstrated the downregulation of most flavonoid genes in these transgenics, as well as some shikimate pathway genes, confirming the broad repression function on the phenylpropanoid pathway. By contrast, MYB117 encodes an anthocyanin activator, and was shown to be specific to this branch of the flavonoid pathway. Overexpression of MYB117 in transgenic poplar increased accumulation of anthocyanin in all tissues, resulting in red poplar plants. One bHLH cofactor, bHLH131 was shown to interact with both MYB activators and repressors and required by MYB activators to activate flavonoid gene promoters. This indicate an important role of bHLH131 in the flavonoid biosynthesis. Proanthocyanidin MYB activators, MYB134 and MYB115 could activate each other. This indicates a positive feedback loop of proanthocyanidin MYB activators. Interestingly, repressor MYB165 suppressed expression of other flavonoid MYB repressors including MYB194 and MYB182, which shows a negative feedback loop of MYB repressors. The expression of bHLH131 was also regulated by MYB activators and repressors. These results reveal the complex interaction between these regulators. iii iv Unexpectedly, overexpression MYB134, MYB115 or MYB117 poplars upregulated flavonoid 3’5’-hydroxylase and cytochrome b5 genes, and lead to enhanced flavonoid B- ring hydroxylation and an increased proportion of delphinidin, myricetin Overexpression of flavonoid 3’5’-hydroxylase in poplar confirmed its function in enhancing B-ring hydroxylation. However, overexpression of cytochrome b5 in flavonoid 3’5’-hydroxylase- overexpressing plants did not further increase flavonoid B-ring hydroxylation. Thus its role in flavonoid B-ring hydroxylation remained unclear. These results show that flavonoid MYBs could also alter flavonoid structure. Together, these studies outline the complex regulatory network formed by flavonoid MYB activators and repressors, and bHLH cofactors controlling both flavonoid accumulation and structure. / Graduate / 2020-12-04

Repressors, defined as individuals possessing an increased cortical sensitivity to anxiety-provoking stimuli combined with the tendency to avoid such stimuli, may be differentiated from low- and high-anxious individuals. This study investigated behavioral performance, electrophysiological reactivity (EEG), and cardiovascular reactivity (heart rate, systolic and diastolic blood pressure) in three major coping style groups (repressors, low-anxious, and high-anxious) during low and high stressful conditions. Conditions were rest, cognitive stress (computerized Stroop Interference Task, with and without negative emotional stimuli), and physiological stress (cool water vs. ice cold water left hand immersions). Participants were 49 healthy, right handed university undergraduate women, chosen with Weinberger et al.'s (1979) criteria according to their differentially extreme scores on the Marlowe-Crowne Social Desirability Scale and the Spielberger Trait Anxiety Scale. Although mixed, findings supported the proposal that these three groups utilize differential attentional strategies and interpretive biases during both neutral and negative stimuli. In the word interference Stroop condition, reaction times (RTs) were longer for negative than neutral stimuli. Unexpected RT differences occurred in low and high error groups across the three coping styles. High error scoring repressors had longer RTs than low error/low-anxious; high-anxious/low error and high-anxious/high error groups did not differ. Repressors perceived the cold water as being less distressful and painful than the low- and high-anxious groups. No heart rate differences occurred. Diastolic and systolic blood pressure increased following cool and cold water immersion. Unexpectedly, the high-anxious group exhibited significantly lower diastolic blood pressure than the other groups, both at rest and following the stressors. EEG activation pattern differences were observed. During anticipation for cool immersion, in comparison to the high-anxious group, repressor and low-anxious groups showed less right parietal involvement suggestive of less arousal (Heller, 1993). During cool water immersion, frontal and parietal differences were observed across groups. During the Stroop Interference Task, repressors showed greater right frontal involvement than other groups. The data are most supportive of Eysenck and Derakshan's (1997) four-factor theory that proposes differential and attentional biases for these three groups. Suggestions for future research and practical applications in health fields are discussed. / Ph. D.

Recently, through reporter gene studies, the novel BTB/POZ protein, Kaiso, has been identified as a transcriptional repressor. The purpose of this study was to determine if Kaiso recruited the Histone Deacetylase Complex to mediate repression and if the previously identified Kaiso Binding Site (KBS; TCCTGCNA) is a physiological target regulated by Kaiso. The two objectives are complementary because an HDAC interaction identifies the mechanism of transcriptional regulation used by Kaiso and regulation of the KBS element identifies a novel, non-methylation dependent, physiological target under transcriptional regulation by Kaiso. Through coimmunoprecipitation and Western blot analyses, Kaiso does not interact with HDAC1, HDAC2 or mSIN3A. These results were surprising since all three of these proteins are common to a variety of repression complexes. mSIN3A is a common component of SIN3 mediated repression and HDAC1/HDAC2 are part of various repression complexes including SIN3, NuRD and CtBP. Although the remaining HDAC proteins were not assayed for an interaction, Kaiso transcriptional activity was demonstrated to be insensitive to the HDAC inhibiting drug, Trichostatin A (TSA). These results indicate either a non-HDAC mechanism of action or alternatively, transcriptional activation. Complementary to the observations of no Kaiso-HDAC interaction and TSA insensitivity was the findings that Kaiso activates transcription of the KBS cis-element in HCT116, HCA-7 and 293 cells, but not MOCK cells in reporter gene assays. Taken together, these results indicate that Kaiso is a dual functioning protein capable of both transcriptional activation and repression and that the mechanism of repression is not through the direct recruitment of HDAC proteins. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 232-250).

No description available.

The Escherichia coli transcription factor NikR is responsible for nickel-mediated repression of the nik operon. The crystal structure of NikR in complex with its operator sequence provided insight into the mechanistic details of nickel-activated NikR-DNA complex formation. The crystal structure revealed that the α3 helix and its preceding loop (residues 63-79) in two of the metal-binding domains—which become structurally ordered after high-affinity nickel binding—make non-specific contacts with the DNA phosphodiester backbone. The proposed mechanism of NikR binding to DNA suggests that the non-specific interactions between the DNA phosphodiester backbone and the positively-charged residues Lys64 and Arg65 anchor NikR to the DNA, thereby allowing the protein to initiate a one-dimensional search for its recognition sequence. The DNA-binding studies presented here strongly support an important role for Lys64 and Arg65 in NikR-DNA complex formation which is in agreement with the proposed model of NikR binding to DNA.

The Escherichia coli transcription factor NikR is responsible for nickel-mediated repression of the nik operon. The crystal structure of NikR in complex with its operator sequence provided insight into the mechanistic details of nickel-activated NikR-DNA complex formation. The crystal structure revealed that the α3 helix and its preceding loop (residues 63-79) in two of the metal-binding domains—which become structurally ordered after high-affinity nickel binding—make non-specific contacts with the DNA phosphodiester backbone. The proposed mechanism of NikR binding to DNA suggests that the non-specific interactions between the DNA phosphodiester backbone and the positively-charged residues Lys64 and Arg65 anchor NikR to the DNA, thereby allowing the protein to initiate a one-dimensional search for its recognition sequence. The DNA-binding studies presented here strongly support an important role for Lys64 and Arg65 in NikR-DNA complex formation which is in agreement with the proposed model of NikR binding to DNA.

In Drosophila, XX embryos express Sxl from the early promoter, SxlPe, and become females. At the same time, XY embryos with only one X chromosome become males. I investigated the role of repression in the establishment of the strict regulation of SxlPe. I found that the co-repressor Groucho, is responsible for amplification of the two-fold difference in X-encoded activator genes into an all-or-nothing difference in Sxl expression. Three new basic helix-loop-helix repressors of Sxl were identified: Hey, Cwo and the prooncogene Myc, all of which are maternally supplied. I have shown that Myc specific repression is important as early as cycle 10, which is 2 cycles earlier than the onset of normal Sxl expression.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 91-109).