Explicit equilibrium modeling of transcription-factor binding and gene regulation

We have developed a computational model that predicts the probability of transcription factor binding to any site in the genome. GOMER (generalizable occupancy model of expression regulation) calculates binding probabilities on the basis of position weight matrices, and incorporates the effects of cooperativity and competition by explicit calculation of coupled binding equilibria. GOMER can be used to test hypotheses regarding gene regulation that build upon this physically principled prediction of protein-DNA binding

Contribution of Transcription Factor Binding Site Motif Variants to Condition-Specific Gene Expression Patterns in Budding Yeast

It is now experimentally well known that variant sequences of a cis transcription factor binding site motif can contribute to differential regulation of genes. We characterize the relationship between motif variants and gene expression by analyzing expression microarray data and binding site predictions. To accomplish this, we statistically detect motif variants with effects that differ among environments. Such environmental specificity may be due to either affinity differences between variants or, more likely, differential interactions of TFs bound to these variants with cofactors, and with differential presence of cofactors across environments. We examine conservation of functional variants across four Saccharomyces species, and find that about a third of transcription factors have target genes that are differentially expressed in a condition-specific manner that is correlated with the nucleotide at variant motif positions. We find good correspondence between our results and some cases in the experimental literature (Reb1, Sum1, Mcm1, and Rap1). These results and growing consensus in the literature indicates that motif variants may often be functionally distinct, that this may be observed in genomic data, and that variants play an important role in condition-specific gene regulation

The CArG Boxes in the Promoter of the Arabidopsis Floral Organ Identity Gene APETALA3 Mediate Diverse Regulatory Effects

APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box. The APETALA3 promoter contains three close matches to the consensus CArG box sequence. To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions. 496 base pairs of APETALA3 promoter sequence 5′ to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers. A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to INTRODUCTION The developmental fate of the organs in the Arabidopsis flower is controlled by the homeotic floral organ identity genes. When the activity of a particular floral organ identity gene is lost due to mutation, there is a homeotic conversion of one organ type to another. For example, the APETALA3 (AP3) and PISTILLATA (PI) genes are necessary for the proper development of petals that develop in the second whorl and stamens that develop in the third whorl of the flower. In ap3 and pi mutants, sepals and carpels develop in positions normally occupied by petals and stamens respectively (Bowman et al., 1989; Jack et al., 1992). Accumulating genetic and molecular evidence suggests that the AP3 and PI proteins together make up the B class organ identity function and these two proteins are sufficient to direct the identity of petals and stamens in the flower. In support of this, ectopic expression of AP3 and/or PI throughout the flower leads to homeotic transformations. Specifically misexpression of AP3 (i.e. 35S::AP3) results in the development of stamens in place of carpels in the fourth whorl and misexpression of PI (i.e. 35S::PI) results in the development of petaloid sepals in place of sepals in the first whorl of the flower (Jack et al., 1994; Krizek and Meyerowitz, 1996). 35S::AP3 leads to fourth whorl organ identity changes because PI is transiently expressed in whorl four during early stages of flower petals and stamens in the flower. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects. Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors. Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged. By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor

The Mads World of Floral Regulators in Gerbera Hybrida

The flowering process of plants is of great importance - both for the sexual reproduction of plants and human nutrition. Floral diversity has fascinated scientists for centuries, but the first important steps towards explaining the molecular puzzle of flowering were taken only thirty years ago when plant MADS box genes were discovered, and the classical ABC model was proposed. The focus of research has shifted from studying single genes to working on whole networks of genes and proteins - genomics and proteomics. The genomics of Arabidopsis (Arabidopsis thaliana), the well-known model plant, is advanced, but phenomena present in Arabidopsis might be lacking in other species, and vice versa. The model plant, Gerbera (Gerbera hybrida), belongs to the large Asteraceae (sunflower) family. While Gerbera flowering process shares several features with Arabidopsis and other model plants, it also shows great specialization. Gerbera bears a complex inflorescence containing hundreds of flowers of three different types, which differ in morphology and sex. The major aim of this thesis was to characterize Gerbera floral developmental genes, with special interest in genes related to A and E functions. The ABC model and the extended ABCDE model are applicable to Gerbera. It has been previously shown that the Gerbera B and C function genes behave as the model predicts. The E function genes affect the development of the whole flower in Arabidopsis, being redundantly active in all four floral whorls. However, the Gerbera E function genes, GRCD1 and GRCD2, are non-redundant and specialized in their tasks. Based on GRCD4 and GRCD5 expression patterns and PPI data presented in this thesis we proposed them to provide general E function in Gerbera. However, later this hypothesis was shown not to be completely accurate by RNAi transgenic lines that showed GRC4 and GCRD5 to be involved in Gerbera petal development. The ABCDE model proposes that A function genes determine the developmental fate of sepals and petals. This function, however, is the most problematic of the model since a true A function seems only to be present in Arabidopsis. Majority of homologous genes from the other model plants execute only partial functions of AP1. Gerbera contains several genes related to AP1 and its homologues CAL and FUL, but none of them supply the A function in the sense of the ABC model. All Gerbera AP1- and FUL-like genes GSQUAs display wide expression patterns, some of them present in all floral organs. Different GSQUA genes were transformed into Gerbera, but only GSQUA2 overexpression lines produced a recurrent phenotype that was an early flowering, dwarf Gerbera. The function of GSQUA2 was shown to be linked to floral transition. Phylogenetic analysis showed Gh-SOC1 to be distantly paralogous to Arabidopsis SOC1. In contrast to Arabidopsis SOC1, Gh-SOC1 was expressed only in the floral parts of Gerbera and it did not promote flowering but altered inflorescence identity towards vegetative characteristics. Temporal expression pattern late in floral development and floral abundancy suggested Gh-SOC1 to have a role in the late stages of Gerbera floral organ development. The results presented in this thesis add to our understanding of inflorescence and floral development of Gerbera. The ABCDE model is applicable to Gerbera for B and C function, A function does not seem exist in Gerbera and E function is differentiated from Arabidopsis general E function. Despite close sequence similarity to Arabidopsis SOC1, Gh-SOC1 function in Gerbera is related to floral development.Kasvien kukkiminen vaikuttaa sekä kasvien lisääntymiseen että ihmiskunnan ravinnonsaantiin. Kukkien monimuotoisuus on kiinnostanut tutkijoita satojen vuosien ajan, mutta ensimmäiset askeleet kukankehitykseen vaikuttavien geenien toiminnan selvittämiseksi otettiin n. 30 vuotta sitten kun MADS box-geenit löydettiin ja kukankehityksen ABC-malli esiteltiin. Kukankehityksen säätely tunnetaan hyvin mallikasvi Arabidopsiksella, mutta tietoa ei voi suoraan soveltaa muihin kasveihin. Gerbera kuuluu laajaan mykerökukkaisten kasvien heimoon. Sen kukat muistuttavat Arabidopsiksen ja muiden mallikasvien kukkia, mutta niillä on myös omia erikoispiirteitään. Gerberan monimutkainen, satoja kukkia sisältävä kukinto koostuu kolmesta eri kukkatyypistä, jotka eroavat rakenteeltaan ja toiminnaltaan. Tämän väitöskirjan tavoite on kuvata Gerberan kukinnon kehittymiseen vaikuttavia, laajennetun ABCDE-mallin A- ja E-funktion geenejä. ABCDE-malli selittää myös Gerberan kukinnon kehitystä. Aiemmin on osoitettu Gerberan B- ja C-funktioiden toimivan mallin mukaisesti. E-funktioon liittyvät geenit vaikuttavat koko kukan kehitykseen Arabidopsiksessa ja pystyvät osittain korvaamaan toisiaan. Gerberan E-geenit, GRCD1 ja GRCD2 ovat kuitenkin erikoistuneet toimimaan vain yhdessä kukkakiehkurassa. Tässä työssä Gerberan E-geenien (GRCD4 ja GRCD5) pääteltiin vastaavan Arabidopsiksen yleistä E-funktiota. Myöhempi tutkimus osoitti GRCD4- ja GRCD5 geenien liittyvän Gerberan teriön kehitykseen. ABCDE-mallin mukaan A-funktion geenit määräävät kukan verhiön ja teriön kehityksen. A-funktio on kuitenkin mallin kiistellyin osa ja se näyttää pätevän vain Arabidopsikseen. Muiden mallikasvien A-funktiota koodaavat geenit toteuttavat vain osan Arabidopsiksen AP1-geenin toiminnoista. Gerberassa on useita AP1-geenin sukuisia geenejä (GSQUA), mutta mikään niistä ei toimi kuten AP1. Kaikki GSQUA-geenit ilmentyvät laajasti kukassa, osa kaikissa kukan osissa. GSQUA2-geenin ilmentäminen Gerberassa sai aikaan varhain kukkivan pienikokoisen kasvin. GSQUA2-geenin toiminnan osoitettiin vaikuttavan gerberan siirtymiseen kasvullisesta kehitysvaiheesta kukintaan. Gh-SOC1 ja Arabidopsiksen SOC1 ovat paralogisia geenejä. Toisin kuin SOC1, joka aikaistaa kukintaa Arabidopsiksessa, Gh-SOC1 ilmentyy vain Gerberan kukkaosissa eikä aikaista kukkimisaikaa vaan muuttaa kukintoa kasvulliseen suuntaan. Gh-SOC1:n ilmentyminen viittaa sen rooliin kukan osien kehityksen myöhäisessa vaiheessa. Tämän väitöskirjan tulokset antavat lisätietoa Gerberan kukinnon ja yksittäisten kukkien kehityksestä. ABC-mallia voidaan soveltaa Gerberaan B- ja C-funktion osalta, A-funktiota Gerberassa ei ole ja E-funktio poikkeaa Arabidopsiksen E-funktiosta. GhSOC1 liittyy kukan kehitykseen, toisin kuin Arabidopsiksen SOC1

SRF and MCM1 have related but distinct DNA binding specificities.

The mammalian transcription factor SRF and the yeast regulatory protein MCM1 contain DNA binding domains that are 70% identical; moreover, both proteins can bind the serum response element in the human c-fos promoter. Here we present an analysis of MCM1 sequence specificity by selection of sites from random sequence oligonucleotides. In this assay the MCM1 DNA binding domain selects binding sites containing the consensus (NotC)CCY(A/T)(A/T)(T/A)NN(A/G)G, distinct from the SRF binding consensus CC(A/T)6GG. Carboxylethylation interference analysis of a set of selected sites suggests that MCM1 contacts DNA in its major groove throughout one helical turn. These differences in specificity are largely due to sequence differences between the N terminal basic parts of the SRF and MCM1 DNA binding domains. Comparison of the relative binding affinities of MCM1 and SRF for a panel of representative binding sites showed that many high affinity MCM1 sites have negligible affinity for SRF and vice versa. Thus MCM1 and SRF have significantly different sequence specificities

The evolutionary origin of "floral quartets": clues from molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon

The identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of floral homeotic MADS-domain proteins that bind to specific cis-regulatory DNA-elements (‘CArG-boxes’) of their target genes, thus constituting floral quartets. Gymnosperms possess orthologues of floral homeotic genes enconding MIKC-type MADS-domain proteins, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. To better understand the ‘abominable mystery’ of flower origin, in this project a comprehensive study was carried out to detect the dimerization and DNA-binding of several classes of MADS-domain proteins from a gymnosperm, Gnetum gnemon of the Gnetales. Determination of protein-protein interactions by pull-down assays revealed complex patterns of heterodimerization among orthologues of class B, class C and class E floral homeotic proteins and Bsister proteins, while homodimerization was not observed. In contrast, electrophoretic mobility shift assays (EMSAs) revealed that all proteins tested except one bind to CArG-boxes also as homodimers, suggesting that homodimerization is relatively weak, but facilitated by DNA-binding. Proteins able of DNA-based homodimerization include orthologues of class B and C proteins; B and C proteins also form heterodimers in vitro and in yeast, which is in sharp contrast to their orthologues from angiosperms, which require class E floral proteins to ‘glue’ them together in multimeric complexes. Remarkably, the heterodimers of B and C proteins from G. gnemon are not capable of binding to CArG-boxes, suggesting that DNA-binding in vivo is based on homodimers, while heterodimerization of B and C proteins may constitute multimeric, DNA-bound complexes by mediating the interaction between two DNA-bound homodimers. EMSAs and DNase I footprint assays indicated that both B with C proteins and C proteins alone but not B proteins alone can induce DNA-looping to form tetrameric protein-DNA complexes similar to floral quartets. These data suggest that at least some of the gymnosperm orthologues of floral homeotic proteins may have the capability of forming higher-order complexes and that gymnosperm B and C proteins control male organ identity and C proteins controls female organ identity, respectively, by forming quartet-like complexes composed of two homodimers, each bound to a CArG-box

Molecular and Genetic Analysis of Flower Development in Arabidopsis thaliana and the Diploid Strawberry, Fragaria vesca

In a world with a warming climate and a rapidly growing population, plant biology is becoming a field of increasing importance. Deciphering the molecular and genetic mechanisms behind the development of the flower, the fruit and seed progenitor, will enhance the agricultural productivity needed to ensure a sustainable food supply. My PhD research ties in with this need by furthering the basic knowledge of the mechanisms underlying flower development in two ways. First, using Arabidopsis thaliana, the classic model plant, I investigated the regulation of a gene, SPATULA (SPT), necessary for the proper development of the gynoecium, the female flower organ that, upon fertilization, directly gives rise to fruit. For flower and fruit to properly develop, the expression of SPT, must be tightly regulated both spatially and temporally. My research examined the mechanism of transcriptional repression of SPT in the sepals and petals by several interacting transcription factors (LEUNIG, SEUSS, APETALA2) and the molecular and genetic interaction between ETTIN and SPT in patterning gynoecium. The second focus of my research was to develop Fragaria vesca (the diploid strawberry), as a model Rosaceae for the study of flower and fruit development. Arabidopsis has much value as a small, fast growing, flowering plant with a multitude of genetic and genomic resources, however the flower of this mustard family weed is not representative of all crop flowers. The Rosaceae family, including many agriculturally important fruit trees such as apple, peach, blackberry, and strawberry, warrants its own model plant to investigate the distinct mechanisms behind their unique reproductive biology. Toward developing F. vesca as the model plant for studying Rosaceae flowers, I characterized and described developmental progression of F. vesca flowers morphologically through scanning electron microscopy and histological analysis as well as molecularly through transcriptomes and in situ hybridization. In addition, I pioneered a small-scale mutagenesis screen of F. vesca that will lead to future genetic resources. My thesis work places the groundwork for future discoveries in F. vesca and Rosaceae and benefits research, education, and agricultural applications for the Rosaceae and the plant biology communities