Alumni Dissertations and Theses

 
 

Alumni Dissertations and Theses

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  • Systems biology-based study of provitamin A carotenoid biosynthesis in Arabidopsis thaliana

    Author:
    Oren Tzfadia
    Year of Dissertation:
    2011
    Program:
    Biology
    Advisor:
    Eleanore Wurtzel
    Abstract:

    Due to their great nutritional and health value, understanding the regulatory mechanisms and recognizing new points of control in the carotenoid pathway can be the goal of breeding plans for increasing carotenoids accumulation in crop plants. Systems biology is an inter-disciplinary field, which integrates computational models and tools with molecular biology and different types of data including in silico transcriptomics, co-expression correlation, metabolomics, proteomics and phylogenetic information in order to develop hypotheses with statistically sound robustness. In the first steps of my work I describes the sequential use of freely available databases to explore the regulation of carotenoid biosynthesis in Arabidopsis during chloroplast development. The findings suggested that coordinated transcriptional regulation of genes along the isoprenoid-related biosynthesis pathways, play a major role in coordinating the synthesis of functionally related, chloroplast-localized isoprenoid-derived compounds. Next I aspired to find candidate genes that are participating in or regulating the carotenoid pathway. A model was developed to integrate several types of high-throughput data, in order to optimize candidate gene ranking in an effort to best define associated genes for a specific studied pathway. The candidate ranking was achieved by using an iterative algorithm (called MORPH), which is built on implementation of machine learning techniques. Application of the method on several biological pathways in Arabidopsis proved the ability of the algorithm to capture experimentally proven gene candidates related to known biological pathways. The robustness of the predictions provided by MORPH creates an exciting research methodology to explore regulation of biological pathways in plants. Although the development of the computational algorithm was initially triggered by the specific needs of our laboratory, namely, for close analysis of the carotenoid pathway, the algorithm is suitable for almost any biological pathway in plants. Moreover the method could be applied to any other model system that has enough available high-throughput data.

  • Carotenogenesis in Maize Endosperm: Natural Genetic Variation as a Tool for Predictive Metabolic Engineering

    Author:
    Ratnakar Vallabhaneni
    Year of Dissertation:
    2009
    Program:
    Biology
    Advisor:
    Wurtzel Eleanore
    Abstract:

    Vitamin A deficiency is a global health burden. This deficiency can be alleviated through provitamin A carotenoid biofortification of maize and other Poaceae crops. However, the predictive metabolic engineering or breeding is limited by the incomplete understanding of endogenous pathway regulation. If the pathway regulation was better understood, enhancement of carotenoid biosynthesis could be controlled by limiting rate-controlling steps and timing of expression in carotenogeneic tissues. Maize carotenogenesis was investigated using a novel approach to discover genes encoding limiting biosynthetic steps in the nutritionally targeted seed endosperm. A combination of bioinformatics and transcript profiling were first used to identify, map and study expression analysis of gene families encoding enzymes in maize and other grasses. These enzymes include the methylerythritol 4-phosphate MEP pathway for synthesis of IPP and GGPP, the downstream carotenoid biosynthetic pathway and as well as those involved in degradation of carotenoids. A novel approach was used to capture the genetic and biochemical diversity of a large germplasm collection, representing 80% of maize genetic diversity, without having to sample the entire collection. This core collection was used for statistical testing of correlation between carotenoid content and candidate gene transcript levels. Multiple pathway bottlenecks for isoprenoid biosynthesis and carotenoid biosynthesis controlling both total carotenoid content and individual carotenoids were discovered in specific temporal windows of endosperm development. Transcript levels of paralogs encoding isoprenoid IPP and GGPP-producing enzymes, DXS3, DXR, HDR, and GGPPS1, were found to positively correlate with endosperm carotenoid content. For carotenoid pathway enzymes, transcript levels for CrtISO inversely correlated with seed carotenoid content, as compared to positive correlation of PSY1 transcripts. Among genes encoding enzymes controlling individual carotenoids, LCYE was shown to control the ratio of two branches of the pathway, and HYD3 was shown to be associated in converting provitamin A compounds to non-provitmain A compounds. Three natural alleles of HYD3 in 51 maize lines explained 78% of variation and ~11-fold difference in beta-carotene relative to beta-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of beta-carotene. Further downstream, since ZEP depletes the carotenoid pool in subsequent conversion to ABA, ZEP transcripts were examined. Carotenoid accumulation was found to be inversely associated with ZEP1 and ZEP2 transcript levels. Additionally, degradation of carotenoids is another mechanism to reduce the level of carotenoids in any tissue. Degradation of carotenoids is controlled by a small gene family of eleven carotenoid cleavage genes. Although there is no direct evidence between mRNA levels of these genes and total carotenoid content, preliminary analysis suggested that copy number may be associated with reduced endosperm carotenoids. Degradation of ABA by the action of ABA hydroxylases was also studied. The enzymatic activity of ABA 8'-hydroxylase is considered as one of the key steps in maintaining ABA levels necessary for physiological processes. ABA8Ox genes were identified in maize and respective gene expression was found in all tissues, with a high degree of specificity to each tissue and some degree of overlap. ZmABA8Ox1a and ZmABA8Ox1b were shown to be the major transcript components for regulating ABA catabolism in drought-stressed roots. Extension of the maize results using phylogenetic analysis identified orthologs in other Grass species that may serve as potential metabolic engineering targets.

  • VARIATION IN HABITAT THRESHOLDS: AN ANALYSIS OF MINIMUM HABITAT REQUIREMENTS OF NORTH AMERICAN BREEDING BIRDS.

    Author:
    Yntze van der Hoek
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Lisa Manne
    Abstract:

    Many species show dramatic changes in population extinction or persistence probability at particular habitat amounts. These `extinction thresholds' could be translated to conservation targets, under the condition that we can derive generalities. I investigated the level of variation in landscape-level habitat thresholds for a suite of North American, forest-associated, breeding birds. Records from Breeding Bird Atlases and the availability of remotely-sensed land cover data allowed me to compare habitat thresholds for 25 species across the states of Massachusetts, Michigan, New York, Ohio, Pennsylvania, and Vermont. I show that variation in thresholds is considerable (Chapter II, III), as thresholds range from 7 to 90% forest cover between species, within regions, and even from 12 and 90% forest cover within species across regions (results for White-throated Sparrow (Zonotrichia albicollis)). I found no universal trend in this variation, although a few species showed a significant increase in threshold amounts with increasing forest cover in the landscape. In Chapter IV, I show that it is possible to assess vertical habitat structure with light detection and ranging (lidar) data. The availability of detailed habitat metrics, such as maximum canopy height and canopy heterogeneity, allowed me to detect detailed extinction thresholds for five species of cavity breeding birds and the Cerulean Warbler (Setophaga cerulea), a species of great conservation concern. Models also showed that some species persist at low forest cover, even though they demonstrate a peak in extinction probability at intermediate levels of forest cover (Chapter V). These peaks in extinction probability correspond with a peak in change in amount of forest cover over time, indicating that change in habitat might be predictive of extinction probability. Estimation of species-specific thresholds, I propose, provides information that can potentially be used to set management targets (Chapter VI). I conclude that we should be wary of extrapolation of thresholds and emphasize that estimation of thresholds should be considered a tool for understanding the process of habitat loss, not a goal in itself.

  • SYSTEMATICS AND PHYLOGENY OF ELAPHOGLOSSUM SECTION LEPIDOGLOSSA (DRYOPTERIDACEAE)

    Author:
    Alejandra Vasco-Gutierrez
    Year of Dissertation:
    2010
    Program:
    Biology
    Advisor:
    Robbin Moran
    Abstract:

    Elaphoglossum (Dryopteridaceae) is one of the most diverse genera of ferns, yet it is noteworthy for lacking monographic studies for any of its subgroups. This study was an attempt to find clades of convenient size for monographic study within Elaphoglossum section Lepidoglossa, one of the largest sections of the genus. To do that the phylogeny of the section was investigated using three chloroplast markers. These molecular phylogenetic analyses identified 13 clades, two of which were selected for monographic revision: the Elaphoglossum ciliatum group and Elaphoglossum subsection Muscosa. The Elaphoglossum ciliatum group turned out to have nine species characterized by laminar scales reduced to minute dots, spiny perispores, and resinous rhizomes. Elaphoglossum subsection Muscosa turned out to have 15 species characterized by flat scales with acicular marginal cells, scaly laminae, and spores about twice as long as other species of Elaphoglossum. Besides constituting a framework from which to choose morphologically recognizable clades of convenient size for monographic study, the phylogeny provided important new insights into the systematics and taxonomy of Elaphoglossum section Lepidoglossa.

  • Mechanisms underlying the adverse impact of mitochondrial dysfunction and its prevention on the ubiquitin/proteasome pathway: relevance to Parkinson disease

    Author:
    Hu Wang
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    MARIA FIGUEIREDO-PEREIRA
    Abstract:

    Neuroinflammation has long been accepted as a probable factor in Parkinson disease(PD). Besides neuroinflammation, mitochondrial impairment and ubiquitin/proteasome pathway (UPP) dysfunction have also been recognized as important contributors to the pathogenesis of PD. Parkin, which plays a central role in the link between mitochondria and the UPP, is mutated in both familial and sporadic forms of PD. The function of Parkin was first described as an E3 ubiquitin ligase that delivers its substrates to the proteasome to be degraded. However, recent findings discovered another important role for Parkin, which is its involvement in mitochondrial quality control and mitochondrial dynamics. Since neuroinflammation, mitochondrial impairment, UPP dysfunction and Parkin are all involved in PD pathogenesis, it is of great interest and importance to investigate the interaction among these pathogenic elements. Therefore, the MAJOR AIMS of these studies were to: (1) Investigate whether Parkin is affected by neuroinflammation, mitochondrial and UPP dysfunction, and assess approaches to protect from these insults. (2) Determine the mechanisms by which mitochondrial impairment disturbs the UPP. Rat E18 midbrain and cerebral cortical neuronal cultures were used to carry-out our specific aims. We used a pharmacological approach to mimic neuroinflammation, as well as mitochondrial and proteasomal dysfunction. The neuronal cultures were treated with: (1) Prostaglandin J2 (PGJ2), an endogenous product of inflammation, which inhibits mitochondrial complex I and impairs proteasomal function. (2) Mitochondrial toxins that target the electron transport chain (ETC): a) Oligomycin, which inhibits ATP synthase (complex V); b) Antimycin, which inhibits complex III; c) Rotenone, which inhibits complex I; (3) Epoxomicin, a specific and irreversible proteasomal inhibitor. The results from the first aim reveal that: (1) Upon mitochondrial dysfunction a new form of Parkin (newParkin) is detected that is generated by calpain cleavage of full length Parkin. To our knowledge, we are the first to report this new form of Parkin. (2) NewParkin generated upon mitochondrial impairment translocates to mitochondria, and dissociates from the 26S proteasome. (3) Phosphorylation attenuates Parkin cleavage induced by mitochondrial impairment. (4) The endogenous product of inflammation PGJ2 and the proteasomal inhibitor epoxomicin, also lead to Parkin cleavage but under these conditions, cleavage is mediated by caspase and not calpain activation. (5) Increasing intracellular cAMP with the lipophilic peptide PACAP27 mitigates some of the adverse effects of the product of inflammation PGJ2, including caspase activation, caspase-mediated cleavage of Parkin, and loss of neuronal viability. (6) Partially replenishing intracellular ATP with the nearly planar creatine analog cyclocreatine, diminishes Parkin cleavage triggered by mitochondrial impairment. The results from the second aim reveal that: (1) Mitochondrial toxins which deplete intracellular ATP levels, lead to the downregulation of protein ubiquitination, by adversely affecting the first step of the ubiquitination cascade, i.e. E1-mediated ubiquitin activation, which is ATP-dependent. (2) Mitochondrial toxins downregulate 26S proteasome assembly via selective processing of the Rpn 10 subunit of the 26S proteasome by calpain cleavage. To our knowledge, we are the first to identify this unique calpain substrate. (3) The 26S proteasome deficit induced by the mitochondrial toxins is accompanied by a rise in 20S proteasome levels. (4) These events were induced by acute (16h) and long term (up to seven days) mitochondrial impairment. In summary, our results addressed the complex relationship among neuroinflammation, mitochondrial impairment, proteasomal dysfunction and Parkin, all of which are relevant to Parkinson Disease (PD). We identified the PARKIN-mitochondria-UPP link as highly vulnerable to stress conditions, revealing a relevant mechanistic pathway to exploit for protecting against the progressive nature of PD. We identified two pharmacological approaches to diminish the adverse effects of an impaired PARKIN-mitochondria-UPP link, by increasing intracellular cAMP as well as ATP levels with PACAP27 and cyclocreatine, respectively. Our findings provide a new basis for the development of novel and more effective therapeutic strategies that prevent/stop neurodegeneration in PD.

  • Fine-scale genetics, population dynamics, and management of suburban white-tailed deer (Odocoileus virginianus)

    Author:
    Mark Weckel
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Robert Rockwell
    Abstract:

    Overabundant populations of white-tailed deer Odocoileus virginianus can cause broad declines in native biodiversity, the lack of advanced woody regeneration, and shifts in successional trajectories. These problems are especially pronounced in the suburbs of the Northeastern U.S. In the northern suburbs of New York City, land managers have begun implementing small scale (< 20 km2), bow-only hunts to reduce overabundant deer herds. The success of these controlled hunts will depend on deer socio-population dynamics, the efficacy of bow hunting, and the sustained participation of bow hunters. I used a multi-disciplinary approach to address these issues and to evaluate the utility of bow hunting as a tool for managing suburban deer. I used surveys to evaluate why hunters from Westchester, NY and Fairfield, CT participated in controlled hunts. Members were primarily motivated by the chance to see wildlife, opportunities for recreation, and a passion for archery. Most (71%) reported that their enjoyment had increased since first joining their controlled hunt. Nevertheless, I documented several trends that threaten the long-term sustainability of these programs. First, 78.2% of survey respondents were over the age of 40, possibly suggesting few young recruits. Second, the opportunity to hunt previously unhunted land, a transitory incentive, was the most common reason for participating in controlled hunts. Third, respondents whose doe harvest was limited by choosing to spend time hunting outside of the controlled hunts were also more likely to have seen fewer deer when participating in those controlled hunts (G-test = 13.2, df = 4, P = 0.01). This suggests that if herd reduction is successful, and opportunities for seeing and harvesting deer become fewer, that hunter participation and effort may decline as well. To evaluate the impact of deer management activities, land managers need accurate measures of deer abundance. I proposed modifications to Jacobson et al.'s (1997) camera trap method to estimate the abundance of the Mianus River Gorge Preserve (MRGP; Westchester County, NY) deer herd. This method uses photographs to provide a minimum count of distinctive branch-antlered males, and then uses the photographic rate of males, females, and fawns to estimate demographic ratios. These ratios are used to extrapolate from the number of individual branched-antlered males to the number of females, fawns, and spike males. I modified this technique to 1) generate measures of uncertainty for parameter estimates via bootstrapping camera stations, and 2) address the concern that demographic ratios will be biased if groups of animals differed in their probability of being photographed. For each demographic group, I standardized photographic rates by a measure of detection probability using linear regression. I evaluated the performance of using standardized vs. raw photographic rates by estimating female abundance using both sets of ratios. I compared the results to an independent estimate of MRGP female abundance based on mark-resight methods. Using standardized rates generated a female estimate ( = 60.43 44, 95% PI = 44.78-77.38) closest to the mark capture estimate ( = 49.50, 95% PI = 38.70-63.40), although precision intervals were wide regardless of whether raw or standardized ratios were used. The concept that deer can be successfully managed at fine-scales (<20 sq km) is based on the rose-petal hypothesis (RPH). Under this theory, female deer are believed to exist in spatially exclusive, matrilineal social-units whose members are philopatric and exhibit low rates of dispersal. In theory, repopulation of the removal area should be slow as immigration is low and because deer immediately adjacent to the removal area will not shift their movements towards the void. Previous studies have demonstrated that the RPH model leaves a genetic fingerprint such that female deer separated by short distances (< 1.0 km) are genetically related. I used molecular techniques and Moran I's spatial autocorrelation analyses to evaluate whether an overabundant deer herd in suburban Westchester County was structured following the RPH. At the MRGP, tissue collected from deer that were sampled at the same spatial coordinate were genetically similar (r2=0.21). However, Moran's I index of genetic relatedness became non-significant (e.g. no different from 0) by 230 m. High deer densities may have resulted in overlapping ranges of non-related social units thus weakening a broader genetic signature. To evaluate the short and long-term impact of bow hunting, I used projection models to simulate harvests on deer herds exhibiting density-dependent growth and survival across a range of carrying capacities and immigration rates. For scenarios believed to be representative of the MRGP (low immigration, ~ 7%, and carrying capacity = 13.8 female km-2), maximum herd reduction was modest (~20%) when simulated harvest rates approximated those observed at the MRGP. Sustaining harvests over multiple decades is the biggest challenge facing bow-only hunts. Hours per female harvest increased rapidly as population declined (CF(t) =0.0031NF(t); R2=0.56). As controlled bow hunts are executed by volunteer sportsman, realized reductions will be contingent hunters' incapability or unwillingness to increase effort. Consequently, bow hunting will likely result in deer densities lower than historical peak values, yet higher than is currently assumed necessary for forest regeneration.

  • DEVELOPMENTAL ALTERATIONS OF RAPHÉ NUCLEI IN AUTISTIC SUBJECTS 5-15 YEARS OF AGE- METHODS AND TECHNICAL LIMITATIONS

    Author:
    Jarek Wegiel
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Probal Banerjee
    Abstract:

    The role of the serotonergic system in autism is supported by more than 500 reports. They reveal a link between serotonergic system alterations and social deficits, repetitive behavior, hyperactivity, anxiety and obsessive compulsive behavior observed in autism. However, in spite of evidence of altered development of brain serotonergic system and contribution of these alterations to the autism phenotype, the raphé nuclei, which are the source of brain serotonin, have not been examined. The aim of this stereological and quantitative immunofluorescence- based study of raphé nuclei in autistic subjects 5 to 15 years of age and age matched control subjects was to (a) establish methods of preparation, staining, and analysis of fixed human brainstem samples obtained from brain banks, and (b) characterize the pattern of developmental abnormalities which may contribute to the autistic phenotype. Routine neuropathological brainstem dissection results in partial or complete loss of raphé nuclei integrity. From 9 autistic and 6 control subjects only four pairs 5 to 15 years of age were qualified for the study of raphé nuclei. Formalin-fixed brainstem was dehydrated and embedded in polyethylene glycol and cut into serial 50-um-thick sections. They were stained to estimate cell volume, and immunostained and examined by fluorescence microscopy to estimate the amount of tryptophan hydroxylase (TPH) which is a measure of serotonin synthesis level. 3-D reconstruction demonstrated topography and size of raphé nuclei and explained why preservation of raphé nuclei located in the midline required modification of brainstem sampling. Nucleator applied to TPH (+) sections revealed 24% smaller neuronal soma volume in the dorsal raphé nuclei of autistic subjects than in control group. Application of immunofluorescence and ImageJ software (NIH) revealed significant increase in tryptophan hydroxylase (TPH) immunofluorescence in spite of smaller size of raphé neurons. These data indicate developmental impairment of neuron growth comparable to that observed in cortex and in subcortical structures. Enhanced TPH immunofluorescence in raphé neurons was consistent with enhanced immunoreactivity in serotonergic fibers in several brain regions of autistic subjects (Azmitia et al. 2011). Pathology detected in raphé neurons suggests that target brain areas were exposed to altered levels of serotonin, which may modify function of cerebral cortex and subcortical structures and contribute to the autistic phenotype.

  • Spatial Ecology of Long-tailed Ducks and White-winged Scoters Wintering on Nantucket Shoals, Massachusetts

    Author:
    Timothy White
    Year of Dissertation:
    2013
    Program:
    Biology
    Advisor:
    Timothy White
    Abstract:

    A substantial proportion, perhaps 30%, of the North American breeding population of Long-tailed Ducks (Clangula hyemalis) winter in the vicinity of Nantucket Island, Massachusetts. These birds spend the night on Nantucket Sound and commute during daylight hours to the Nantucket Shoals, which extend about 65 km offshore from the southeastern corner of Nantucket. Strip transects done from a single-engine plane in 1997 and 1998 indicated that Long-tailed Ducks foraged over the shallower (~ 20 m depth) portions of the Nantucket Shoals, up to 70 km offshore. Diet analyses of ten birds collected in February 1999 and five in December 2006 showed that they fed principally (106.6 +/- 42.0 individuals per crop) on Gammarus annulatus, a pelagic amphipod that often forms large aggregations, and is consumed by several species of fish and marine mammals. I conducted high-intensity aerial surveys to map the distribution and abundance of foraging sea ducks on Nantucket Shoals, MA in February 2008 - Spring 2011. For these surveys, west/east transect orientation ran perpendicular to bathymetric gradients, and covered a greater extent of Nantucket Shoals than did our preliminary surveys in 1997 and 1998. Core foraging areas of Long-tailed Ducks and White-winged Scoters (Melanitta fusca) overlapped along southwest Nantucket Shoals (mean foraging depth (m) ± SD: Long-tailed Ducks = 27.20 ± 8.25; White-winged Scoters = 29.8 ± 7.23), a zone where clams and pelagic amphipods are historically abundant. I mapped the spatial dispersion of Long-tailed Ducks and White-winged Scoters to determine if and where foraging aggregations persisted through space and time in relation to prey hotspots. Spatial dispersion of both species was strikingly concordant. Since previous studies have found very little dietary overlap between Long-tailed Ducks and White-winged Scoters, I sought to explain this high degree of spatial concordance. I also examined the potential physical and biological mechanisms (e.g., structural fronts, primary productivity) that may influence the spatial organization of Long-tailed Ducks, White-winged Scoters and their prey on Nantucket Shoals.

  • Computational analyses of the components of Sinorhizobium meliloti ExoR-ExoS/ChvI pathway: the ExoR and ExoS proteins

    Author:
    Eliza Wiech
    Year of Dissertation:
    2014
    Program:
    Biology
    Advisor:
    Shaneen Singh
    Abstract:

    Abstract COMPUTATIONAL ANALYSES OF THE COMPONENTS OF Sinorhizobium meliloti ExoR-ExoS/ChvI PATHWAY: THE ExoR AND ExoS PROTEINS by Eliza M. Wiech Adviser: Professor Shaneen M. Singh The Sinorhizobium meliloti periplasmic ExoR protein and the ExoS/ChvI two-component system form a regulatory mechanism that directly controls the transformation of free-living to host-invading cells. In the absence of crystal structures, understanding the molecular mechanism of interaction between ExoR and the ExoS sensor, which is thought to drive the key regulatory step in the invasion process, remains a major challenge. In this study, we present theoretical structural models of the active form of ExoR protein, ExoRm, as well as of the sensing domain of ExoS, ExoSp, generated using computational methods. Our model suggests that ExoRm possesses a super-helical fold comprising twelve α-helices forming six Sel1-like repeats, including two that were unidentified in previous studies. The structural model of ExoSp suggests that ExoSp is a single Per-ARNT-Sim (PAS) domain. Docking analysis was used to suggest models for ExoSp-ExoSp and ExoSp-ExoRm protein interactions and interfaces. Our studies reveal three novel insights: (a) a possible extended conformation of the ExoR third Sel1-like repeat that might be important for ExoR regulatory function (b) a buried proteolytic site that implies a unique mechanism of proteolysis, central to controlling ExoR function and (c) an elongated structure of helix H4 that is unique to ExoSp and might be crucial for the association with ExoRm. This study provides new and interesting insights into the structure of the S. meliloti ExoRm and ExoSp proteins, lays the groundwork for elaborating the molecular mechanism of ExoRm cleavage, ExoRm-ExoS interactions, and studies of ExoR homologs in other bacterial host interactions.

  • Transcriptional Regulation of Yeast CUP1 gene

    Author:
    Roshini Wimalarathna
    Year of Dissertation:
    2012
    Program:
    Biology
    Advisor:
    Chang-Hui Shen
    Abstract:

    The yeast CUP1 gene encodes a metallothionein required for cell growth at high copper concentrations. The induction of CUP1 with copper results in activator-dependent nucleosome repositioning. To further understand the mechanism of CUP1 activation, it is necessary to identify which chromatin remodeler(s) is/are involved in CUP1 induction. Here, we demonstrated that both ino80∆ and snf2∆ cells grew well in the absence of copper, but were inviable in the presence of copper, indicating that they are required for CUP1 expression. Furthermore, CUP1 mRNA did not significantly increase in mutants lacking either remodeler in the presence of copper, suggesting that they regulate CUP1 induction at the transcriptional level. Both isw1Δ and rsc3Δ cells displayed similar growth patterns as WT cells in the presence of copper. Further CUP1 mRNA was significantly increasing in both isw1Δ and rsc3Δ cells showing similar pattern as WT cells. This observation suggests that ISW1 and RSC3 remodeling complexes have no positive effect in CUP1 expression. We also demonstrated, using chromatin immunoprecipitation, that both INO80 and SWI/SNF are present at the promoter in the wild type cells and they were dependent on each other to be recruited to the CUP1 promoter. Both chromatin remodeling activity and targeted histone acetylation were not observed in ino80∆ or snf2∆ strains at the CUP1 promoter. These results suggest that both INO80 and SWI/SNF directly participate in CUP1 chromatin remodeling, and that histone acetylation is recruited after the arrival of chromatin remodelers. We also observed that more polII was recruited to the CUP1 promoter under inducing conditions and that such a recruitment was not observed in ino80∆ or snf2∆ strains. Furthermore, we observed that both Snf2p and Ino80p were activator-dependent. Our observations provide direct evidence for the involvement of both INO80 and SWI/SNF remodelers in CUP1 activation. In light of these findings, we propose a working model for CUP1 activation.