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Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activationPasini, Luigi <1978> 03 April 2008 (has links)
Transcription is controlled by promoter-selective transcriptional factors (TFs),
which bind to cis-regulatory enhancers elements, termed hormone response
elements (HREs), in a specific subset of genes. Regulation by these factors
involves either the recruitment of coactivators or corepressors and direct
interaction with the basal transcriptional machinery (1). Hormone-activated
nuclear receptors (NRs) are well characterized transcriptional factors (2) that
bind to the promoters of their target genes and recruit primary and secondary
coactivator proteins which possess many enzymatic activities required for
gene expression (1,3,4).
In the present study, using single-cell high-resolution fluorescent microscopy
and high throughput microscopy (HTM) coupled to computational imaging
analysis, we investigated transcriptional regulation controlled by the estrogen
receptor alpha (ERalpha), in terms of large scale chromatin remodeling and
interaction with the associated coactivator SRC-3 (Steroid Receptor
Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a
steroid-dependent transcriptional factor (16) that belongs to the NRs
superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2),
regulates transcription of distinct target genes involved in development,
puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the
nucleus and undergoes a rapid (within minutes) intranuclear redistribution
following the addition of either agonist or antagonist (17,18,19).
We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated
reporter gene array (PRL-array) containing multicopy hormone
response-binding elements for ERalpha that are derived from the physiological
enhancer/promoter region of the prolactin gene. Following GFP-ER
transfection of PRL-HeLa cells, we were able to observe in situ ligand
dependent (i) recruitment to the array of the receptor and associated
coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout
of the reporter gene. Addition of E2 causes a visible opening
(decondensation) of the PRL-array, colocalization of RNA Polymerase II, and
transcriptional readout of the reporter gene, detected by mRNA FISH. On the
contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI),
a dramatic condensation of the PRL-array was observed, displacement of
RNA Polymerase II, and complete decreasing in the transcriptional FISH
signal.
All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid
Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160
family member and a known oncogenic protein (4,34). SRC-3 is regulated by a
variety of posttranslational modifications, including methylation,
phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These
events have been shown to be important for its interaction with other
coactivator proteins and NRs and for its oncogenic potential (37,39). A number
of extracellular signaling molecules, like steroid hormones, growth factors and
cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by
a wide range of kinases, including extracellular-regulated kinase 1 and 2
(ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs)
(41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein,
whose cellular localization is regulated by phosphorylation and interaction with
ERalpha. Using a combination of high throughput and fluorescence microscopy,
we show that both chemical inhibition (with U0126) and siRNA downregulation
of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in
SRC-3 localization, whereas stimulation by EGF signaling enhances its
nuclear localization by inducing phosphorylation at T24, S857, and S860, known
partecipants in the regulation of SRC-3 activity (39). Accordingly, the
cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further
supports these results. In the presence of ERalpha, U0126 also dramatically
reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the
nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell
lysates; localization of SRC-3 at the ER-targeted prolactin promoter array
(PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also
function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of
estrogen receptor.
While a wealth of studies have revealed the molecular functions of NRs and
coregulators, there is a paucity of data on how these functions are
spatiotemporally organized in the cellular context. Technically and
conceptually, our findings have a new impact upon evaluating gene
transcriptional control and mechanisms of action of gene regulators.
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Insulin and TOR pathways regulate cellular and organismal growth through the myc oncogene in DrosophilaParisi, Federica <1981> 21 April 2009 (has links)
A large body of literature documents in both mice and Drosophila the
involvement of Insulin pathway in growth regulation, probably due to its role
in glucose and lipid import, nutrient storage, and translation of RNAs
implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover
several lines of evidence implicate this pathway as a causal factor in cancer
(Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008).
With regards to Myc, studies in cell culture have implied this family of
transcription factors as regulators of the cell cycle that are rapidly induced in
response to growth factors. Myc is a potent oncogene, rearranged and
overexpressed in a wide range of human tumors and necessary during
development. Its conditional knock-out in mice results in reduction of body
weight due to defect in cell proliferation (Trumpp et al. 2001).
Evidence from in vivo studies in Drosophila and mammals suggests a
critical function for myc in cell growth regulation (Iritani and Eisenman 1999;
Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al.
2001). This role is supported by our analysis of Myc target genes in
Drosophila, which include genes involved in RNA binding, processing,
ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al.,
2005, Hulf et al, 2005).
The fact that Insulin signaling and Myc have both been associated
with growth control suggests that they may interact with each other.
However, genetic evidence suggesting that Insulin signaling regulates Myc
in vivo is lacking.
In this work we were able to show, for the first time, a direct
modulation of dMyc in response to Insulin stimulation/silencing both in vitro
and in vivo. Our results suggest that dMyc up-regulation in response to
DILPs signaling occurs both at the mRNA and potein level. We believe
dMyc protein accumulation after Insulin signaling activation is conditioned to
AKT-dependent GSK3β/sgg inactivation. In fact, we were able to
demonstate that dMyc protein stabilization through phosphorylation is a
conserved feature between Drosophila and vertebrates and requires
multiple events. The final phosphorylation step, that results in a non-stable
form of dMyc protein, ready to be degraded by the proteasome, is
performed by GSK3β/sgg kinase (Sears, 2004). At the same time we
demonstrated that CKI family of protein kinase are required to prime dMyc
phosphorylation.
DILPs and TOR/Nutrient signalings are known to communicate at
several levels (Neufeld, 2003). For this reason we further investigated TOR
contribution to dMyc-dependent growth regulation. dMyc protein
accumulates in S2 cells after aminoacid stimulation, while its mRNA does
not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient
pathway regulates dMyc mostly post-transcriptionally.
In support to this hypothesis, we observed a TORC1-dependent
GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and
Nutrients on dMyc protein stability.
On the other hand, our data show that Rheb but not S6K, both
downstream of the TOR kinase, contributes to the dMyc-induced growth of
the eye tissue, suggesting that Rheb controls growth independently of S6K..
Moreover, Rheb seems to be able to regulate organ size during
development inducing cell death, a mechanism no longer occurring in
absence of dmyc. These observations suggest that Rheb might control
growth through a new pathway independent of TOR/S6K but still dependent
on dMyc.
In order to dissect the mechanism of dMyc regulation in response to
these events, we analyzed the relative contribution of Rheb, TOR and S6K
to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic
clones and we further confirmed an interplay between Rheb and Myc that
seems to be indipendent from TOR.
In this work we clarified the mechanisms that stabilize dMyc protein in
vitro and in vivo and we observed for the first time dMyc responsiveness to
DILPs and TOR. At the same time, we discovered a new branch of the
Nutrient pathway that appears to drive growth through dMyc but
indipendently from TOR.
We believe our work shed light on the mechanisms cells use to grow
or restrain growth in presence/absence of growth promoting cues and for
this reason it contributes to understand the physiology of growth control.
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Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor geneDuchi, Serena <1979> 21 April 2009 (has links)
During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity.
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Genetica molecolare dell'autismo: studi di associazione ed analisi di geni candidatiToma, Claudio <1978> 21 April 2009 (has links)
Autism is a neurodevelpmental disorder characterized by impaired verbal communication, limited reciprocal social interaction, restricted interests and repetitive behaviours. Twin and family studies indicate a large genetic contribution to ASDs (Autism Spectrum Disorders). During my Ph.D. I have been involved in several projects in which I used different genetic approaches in order to identify susceptibility genes in autism on chromosomes 2, 7 and X:
1)High-density SNP association and CNV analysis of two Autism Susceptibility Loci.
The International Molecular Genetic Study of Autism Consortium (IMGSAC) previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we evaluated the patterns of linkage disequilibrium (LD) and the distribution of haplotype blocks, utilising data from the HapMap project, across the two strongest peaks of linkage on chromosome 2 and 7. More than 3000 SNPs have been selected in each locus in all known genes, as well as SNPs in non-genic highly conserved sequences. All markers have been genotyped to perform a high-density association analysis and to explore copy number variation within these regions. The study sample consisted of 127 and 126 multiplex families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Association and CNV analysis implicated several new genes, including IMMP2L and DOCK4 on chromosome 7 and ZNF533 and NOSTRIN on the chromosome 2. Particularly, my contribution to this project focused on the characterization of the best candidate gene in each locus:
On the AUTS5 locus I carried out a transcript study of ZNF533 in different human tissues to verify which isoforms and start exons were expressed. High transcript variability and a new exon, never described before, has been identified in this analysis. Furthermore, I selected 31 probands for the risk haplotype and performed a mutation screen of all known exons in order to identify novel coding variants associated to autism.
On the AUTS1 locus a duplication was detected in one multiplex family that was transmitted from father to an affected son. This duplication interrupts two genes: IMMP2L and DOCK4 and warranted further analysis. Thus, I performed a screening of the cohort of IMGSAC collection (285 multiplex families), using a QMPSF assay (Quantitative Multiplex PCR of Short fluorescent Fragments) to analyse if CNVs in this genic region segregate with autism phenotype and compare their frequency with a sample of 475 UK controls.
Evidence for a role of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.
2)Analysis of X chromosome inactivation.
Skewed X chromosome inactivation (XCI) is observed in females carrying gene mutations involved in several X-linked syndromes. We aimed to estimate the role of X-linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 164 affected girls. The study sample included families from different european consortia. I analysed the XCI inactivation pattern in a sample of italian mothers from singletons families with ASD and also a control groups (144 adult females and 40 young females). We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X-linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X-linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27-Xq28 region, with a peak Z score of 1.75 close to rs719489. In this region FMR1 and MECP2 have been associated in some cases with austim and therefore represent candidates for the disorder. I performed a mutation screen of MECP2 in 33 unrelated probands from IMGSAC and italian families, showing XCI skewness. Recently, Xq28 duplications including MECP2, have been identified in families with MR, with asymptomatic carrier females showing extreme (>85%) skewing of XCI. For these reason I used the sample of probands from X-skewed families to perform CNV analysis by Real-time quantitative PCR. No duplications have been found in our sample. I have also confirmed all data using as alternative method the MLPA assay (Multiplex Ligation dependent Probe Amplification).
3)ASMT as functional candidate gene for autism.
Recently, a possible involvement of the acetylserotonin O-methyltransferase (ASMT) gene in susceptibility to ASDs has been reported: mutation screening of the ASMT gene in 250 individuals from the PARIS collection revealed several rare variants with a likely functional role; Moreover, significant association was reported for two SNPs (rs4446909 and rs5989681) located in one of the two alternative promoters of the gene. To further investigate these findings, I carried out a replication study using a sample of 263 affected individuals from the IMGSAC collection and 390 control individuals. Several rare mutations were identified, including the splice site mutation IVS5+2T>C and the L326F substitution previously reported by Melke et al (2007), but the same rare variants have been found also in control individuals in our study. Interestingly, a new R319X stop mutation was found in a single autism proband of Italian origin and is absent from the entire control sample. Furthermore, no replication has been found in our case-control study typing the SNPs on the ASMT promoter B.
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Identificazione di un QTL principale per resistenza a ruggine bruna sul cromosoma 7B di frumento duroMantovani, Paola <1978> 05 May 2009 (has links)
Leaf rust caused by Puccinia triticina is a serious disease of durum wheat (Triticum durum) worldwide. However, genetic and molecular mapping studies aimed at characterizing leaf rust resistance genes in durum wheat have been only recently undertaken. The Italian durum wheat cv. Creso shows a high level of resistance to P. triticina that has been considered durable and that appears to be due to a combination of a single dominant gene and one or more additional factors conferring partial resistance. In this study, the genetic basis of leaf rust resistance carried by Creso was investigated using 176 recombinant inbred lines (RILs) from the cross between the cv. Colosseo (C, leaf rust resistance donor) and Lloyd (L, susceptible parent). Colosseo is a cv. directly related to Creso with the leaf rust resistance phenotype inherited from Creso, and was considered as resistance donor because of its better adaptation to local (Emilia Romagna, Italy) cultivation environment.
RILs have been artificially inoculated with a mixture of 16 Italian P. triticina isolates that were characterized for virulence to seedlings of 22 common wheat cv. Thatcher isolines each carrying a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci, in order to determine their specialization with regard to the host species. The characterization of the leaf rust isolates was conducted at the Cereal Disease Laboratory of the University of Minnesota (St. Paul, USA) (Chapter 2).
A genetic linkage map was constructed using segregation data from the population of 176 RILs from the cross CL. A total of 662 loci, including 162 simple sequence repeats (SSRs) and 500 Diversity Arrays Technology markers (DArTs), were analyzed by means of the package EasyMap 0.1. The integrated SSR-DArT linkage map consisted of 554 loci (162 SSR and 392 DArT markers) grouped into 19 linkage blocks with an average marker density of 5.7 cM/marker. The final map spanned a total of 2022 cM, which correspond to a tetraploid genome (AABB) coverage of ca. 77% (Chapter 3).
The RIL population was phenotyped for their resistance to leaf rust under artificial inoculation in 2006; the percentage of infected leaf area (LRS, leaf rust susceptibility) was evaluated at three stages through the disease developmental cycle and the area under disease progress curve (AUDPC) was then calculated. The response at the seedling stage (infection type, IT) was also investigated. QTL analysis was carried out by means of the Composite Interval Mapping method based on a selection of markers from the CL map. A major QTL (QLr.ubo-7B.2) for leaf rust resistance controlling both the seedling and the adult plant response, was mapped on the distal region of chromosome arm 7BL (deletion bin 7BL10-0.78-1.00), in a gene-dense region known to carry several genes/QTLs for resistance to rusts and other major cereal fungal diseases in wheat and barley. QLr.ubo-7B.2 was identified within a supporting interval of ca. 5 cM tightly associated with three SSR markers (Xbarc340.2, Xgwm146 e Xgwm344.2), and showed an R2 and an LOD peak value for the AUDPC equal to 72.9% an 44.5, respectively. Three additional minor QTLs were also detected (QLr.ubo-7B.1 on chr. 7BS; QLr.ubo-2A on chr. 2AL and QLr.ubo-3A on chr. 3AS) (Chapter 4).
The presence of the major QTL (QLr.ubo-7B.2) was validated by a linkage disequilibrium (LD)-based test using field data from two different plant materials: i) a set of 62 advanced lines from multiple crosses involving Creso and his directly related resistance derivates Colosseo and Plinio, and ii) a panel of 164 elite durum wheat accessions representative of the major durum breeding program of the Mediterranean basin. Lines and accessions were phenotyped for leaf rust resistance under artificial inoculation in two different field trials carried out at Argelato (BO, Italy) in 2006 and 2007; the durum elite accessions were also evaluated in two additional field experiments in Obregon (Messico; 2007 and 2008) and in a green-house experiment (seedling resistance) at the Cereal Disease Laboratory (St. Paul, USA, 2008). The molecular characterization involved 14 SSR markers mapping on the 7BL chromosome region found to harbour the major QTL. Association analysis was then performed with a mixed-linear-model approach. Results confirmed the presence of a major QTL for leaf rust resistance, both at adult plant and at seedling stage, located between markers Xbarc340.2, Xgwm146 and Xgwm344.2, in an interval that coincides with the supporting interval (LOD-2) of QLr.ubo-7B.2 as resulted from the RIL QTL analysis. (Chapter 5).
The identification and mapping of the major QTL associated to the durable leaf rust resistance carried by Creso, together with the identification of the associated SSR markers, will enhance the selection efficiency in durum wheat breeding programs (MAS, Marker Assisted Selection) and will accelerate the release of cvs. with durable resistance through marker-assisted pyramiding of the tagged resistance genes/QTLs most effective against wheat fungal pathogens.
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Identificazione di mutanti di interesse agronomico in orzo mediante approcci di genetica diretta e inversaBovina, Riccardo <1980> 26 May 2009 (has links)
No description available.
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Complexity of N-Myc transcriptional function in childhood neuroblastomaIraci, Nunzio <1979> 21 April 2009 (has links)
Myc is a transcription factor that can activate transcription of several hundreds genes by direct binding to their promoters at specific DNA sequences (E-box). However, recent studies have also shown that it can exert its biological role by repressing transcription. Such studies collectively support a model in which c-Myc-mediated repression occurs through interactions with transcription factors bound to promoter DNA regions but not through direct recognition of typical E-box sequences. Here, we investigated whether N-Myc can also repress gene transcription, and how this is mechanistically achieved. We used human neuroblastoma cells as a model system in that N-MYC amplification/over-expression represents a key prognostic marker of this tumour. By means of transcription profile analyses we could identify at least 5 genes (TRKA, p75NTR, ABCC3, TG2, p21) that are specifically repressed by N-Myc. Through a dual-step-ChIP assay and genetic dissection of gene promoters, we found that N-Myc is physically associated with gene promoters in vivo, in proximity of the transcription start site. N-Myc association with promoters requires interaction with other proteins, such as Sp1 and Miz1 transcription factors. Furthermore, we found that N-Myc may repress gene expression by interfering directly with Sp1 and/or with Miz1 activity (i.e. TRKA, p75NTR, ABCC3, p21) or by recruiting Histone Deacetylase 1 (Hdac1) (i.e. TG2). In vitro analyses show that distinct N-Myc domains can interact with Sp1, Miz1 and Hdac1, supporting the idea that Myc may participate in distinct repression complexes by interacting specifically with diverse proteins. Finally, results show that N-Myc, through repressed genes, affects important cellular functions, such as apoptosis, growth, differentiation and motility. Overall, our results support a model in which N-Myc, like c-Myc, can repress gene transcription by direct interaction with Sp1 and/or Miz1, and provide further lines of evidence on the importance of transcriptional repression by Myc factors in tumour biology.
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L'analisi di Linkage come metodo per la mappatura di malattie genetiche: studio di famiglie con paraparesi spastica ereditaria e anomalie del corpo callosoPippucci, Tommaso <1977> 12 June 2007 (has links)
No description available.
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Trapianto di cellule staminali autologhe geneticamente modificate per il trattamento di patologie metaboliche del fegato: approccio di terapia genica ex vivo per la sindrome di Crigler Najjar tipo IVenturi, Beatrice <1982> 07 June 2010 (has links)
No description available.
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Lo studio di famiglie con patologia genetica per l'identificazione del gene malattia: sindrome di mungan e trombocitopenia autosomica dominante (thc2)Castegnaro, Giovanni <1981> 07 June 2010 (has links)
No description available.
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