Rossella Ginevra Tupler
Rossella Ginevra Tupler
affiliation: Università di Modena-Reggio Emilia
research area(s): Genetics And Genomics, Molecular Biology
Course: Molecular and Regenerative Medicine
University/Istitution: Università di Modena-Reggio Emilia
University of Pavia, Italy B.S. 1977 Human Physiology
University of Brescia, Italy M.D. 1985 Medicine
University of Pavia, Italy Ph.D. 1990 Human Mol. Genetics
University of Pavia, Italy Specialization 1993 Medical Genetics

My training was in human molecular genetics in the laboratories of Drs. Marco Fraccaro, Erika Buhler and Peter StGeorge-Hyslop. I have been a Principal Investigator since 1992, using medical genetics, molecular genetics and molecular biology to investigate molecular mechanisms at the basis of one of most common myopathies, facioscapuloumeral muscular dystrophy (FSHD).
My laboratory has made several significant contributions: 1. Discovery that haploinsufficiency of the FSHD locus does not cause FSHD (Tupler et al et al., J Med Genet 1996), 2. Discovery that FSHD can be expressed differenly in presence of identical genetic background, (Tupler et al et al., J Med Genet 1998), 3. The first demonstration that transcriptional profile is profoundly altered in FSHD affected muscle (Tupler et al Proc Natl Acad Sci U S A 1999), 4. First prediction of of novel transcriptional regulators and splicing factors based on the first human genome sequence (Tupler et al., Nature 2001), 5. Discovery that transcriptional deregulation is at the basis of FSHD via reduction of repetitive elements (Gabellini et al Cell, 2002), 6. Generation of the first animal model for FSHD (Gabellini et al, Nature 2006) 7. Establishment of a standardized clinical evaluation of patients affected by Facioscapulohumeral Muscular Dystrophy to generate the FSHD clinical score (Lamperti et al, Muscle and Nerve, 2010).
Our current data demonstrate the high variability of clinical expression in FSHD, and we plan to focus our efforts in this grant on biochemical and genomic approaches to understanding the molecular basis of this variability.

Positions and Honors
1992 - 2005 Assistant Professor, Institute of General Biology and Medical Genetics, University of Pavia, Italy
1996 - 1998 Visiting Scientist, Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA.
1998 - 2002 Instructor, Program in Molecular Medicine, University of Massachusetts, Worcester, MA.
2002 " present Research Assistant Professor, Program in Gene Function and Expression, University of Massachusetts, Worcester, MA.
2005 " present Associate Professor of Medical Genetics, Department of Biomedical Sciences, University of Modena e Reggio Emilia, Italy
Other Experience and Professional Memberships

The laboratory main research interest is the molecular mechanisms leading to muscular dystrophy. In particular the research focuses on facioscapulohumeral muscular dystrophy (FSHD) the third most common hereditary myopathy. The disease has been causally related to the deletion of tandemly arrayed 3.3 kb repeat units (D4Z4) located on chromosome 4q35. FSHD rather than being the result of a classical mutation within a protein-coding gene, appears to be caused by a transcriptional misregulation of one or more key genes initiated by reduction of the D4Z4 repeats. Consistently, we have observed that genes mapping at 4q35 (FRG1, FRG2, ANT1) are over-expressed in the FSHD affected muscle. More specifically, the FRG1 gene was established as a key gene in FSHD, as transgenic mice over-expressing FRG1 developed a muscular distrophy having features that recapitulate human FSHD. A transcriptional repressor complex that binds D4Z4 repetitive elements was moreover identified and shown to bind in vivo and in vitro the D4Z4 repeats, and to mediate transcriptional repression of of 4q35 genes.
The final goal of this research is dissecting the FSHD pathogenic process through the analysis of the molecular events occurring at D4Z4. In particular this research project aims at the characterization of the machinery controlling gene expression at the 4q35 region.
Our previous study demonstrated that a multi-protein complex binds D4Z4 repetitive elements at 4q35 and is involved in transcriptional repression of 4q35 genes. Interestingly, the 4qter D4Z4 region is hypermethylated at the DNA level in normal cells, but is hypomethylated in both 4q-linked and phenotypic FSHD suggesting that other factors may influence chromatin organization at 4q35 interfering with expression of proximal genes. To define the molecular basis of this phenomenon we will:
a) Analyze modification of chromatin structure at 4q35 in response to various agents;
b) Define the functional organization of the promoter regions of ANT1, FRG1 and FRG2 genes.
To define the molecular basis of FSHD pathophysiology studies on FRG1 mice are ongoing.
1. Cagliani R, Fruguglietti ME, Berardinelli A, D'Angelo MG, Prelle A, Riva S, Gorni K, Orcesi S, Lamperti C, Pichiecchio A, Signaroldi E, Tupler R, Magri F, Govoni A, Corti S, Bresolin N, Moggio M, Comi GP. (2011) New molecular findings in congenital myopathies due to selenoprotein N gene mutations. J Neurol Sci. 300:107-13.
2. Lamperti C, Fabbri G, Vercelli L, D"Amico R, Frusciante R Bonifazi E, Fiorillo C, Borsato C, Cao M, Servida M, Greco F, Di Leo R, Volpi L, Manzolij, Cudia P, Pastorello E, Ricciardi L, Siciliano G, Galluzzi G, Rodolico C, Santoro S, Tomelleri G, Angelini C, Ricci E, Palmucci L, Moggio M, Tupler R. (2010) A standardized clinical evaluation of patients affected by Facioscapulohumeral Muscular Dystrophy: the FSHD clinical score. Muscle and Nerve, 42:213-7.
3. Forlani G, Giarda E, Ala U, Di Cunto F, Salani M, Tupler R, Kilstrup-Nielsen C, Landsberger N. (2010) The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis Hum Mol Genet 19:3114-23.
4. Darabi R, Baik J, Clee M, Kyba M, Tupler R, Perlingeiro RC (2009) Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy. Exp Neurol 220:212-6
5. Filosto M, Tonin P, Scarpelli M, Savio C, Greco F, Mancuso M, Vattemi G, Govoni V, Rizzuto N, Tupler R, Tomelleri G. (2008) Novel mitochondrial tRNA(Leu(CUN)) transition and D4Z4 partial deletion in a patient with a facioscapulohumeral phenotype. Neuromuscul Disord. 18:204-9.
6. Trevisan CP, Pastorello E, Ermani M, Angelini C, Tomelleri G, Tonin P, Mongini T, Palmucci L, Galluzzi G, Tupler RG, Marioni G, Rimini A. (2008) Facioscapulohumeral muscular dystrophy: a multicenter study on hearing function. Audiol Neurootol 13:1-6.
7. D'Antona G, Brocca L, Pansarasa O, Rinaldi C, Tupler R, Bottinelli R (2007)Structural and functional alterations of muscle fibres in the novel mouse model of Facioscapulohumeral Muscular Dystrophy. J Physiol. 584: 997-1009.
8. Trevisan CP, Pastorello E, Armani M, Angelini C, Nante G, Tomelleri G, Tonin P, Mongini T, Palmucci L, Galluzzi G, Tupler RG, Barchitta A. (2006) Facioscapulohumeral muscular dystrophy and occurrence of heart arrhythmia. Eur Neurol 56:1-5.
9. Gabellini D, D'Antona G, Moggio M, Prelle A, Zecca C, Adami R, Angeletti B, Ciscato P, Pellegrino MA, Bottinelli R, Green MR, Tupler R. (2006) Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature, 449:973-977.
10. Perini G.,Tupler R. (2006) Altered gene silencing and human diseases. Clin Gen 69:1-7.
11. Soragna D., Vettori A., Carraro G., Marchioni E., Vazza G., Bellini S., Tupler R., Savoldi F., Mostacciuolo M.L. (2003) A locus for migraine without aura maps on chromosome 14q21.2- q22.3. Am J Hum Genet 72: 161-167.

Project Title:
Role of FRG1 gene in development
The molecular mechanism underlying FSHD development is still not clear. FRG1 is a good candidate gene for FSHD pathophysiology. It has been shown in fact that transgenic mice overexpressing FRG1 in skeletal muscles, develop a muscular dystrophy with features characteristic of the human disease. Furthermore FRG1 overexpression in Xenopus seriously compromises muscle development. Additionallly FRG1 ablation is embryonic lethal in mouse indicating its crucial role in development.
Therefore we consider essential studying the physiological role of FRG1 in muscles function in order to develop new treatments for FSHD.
FRG1 is a protein very highly conserved among invertebrates and vertebrates: human FRG1 shares 42% identity with C. elegans, 81% identity with Xenopus and 97% identity with mouse. This level of conservation reveals that FRG1 might have a very important function that is preserved during the evolution.
Several lines of evidence suggest FRG1 involvement in RNA processing:
- FRG1 is a nuclear protein that localizes in Cajal bodies, in nucleoli and in nuclear speckles, site where RNA processing is taking place.
- Proteomic studies have found that FRG1 is a component of purified spliceosomes.
- We found that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing.

These data suggest a pivotal role of FRG1 in FSHD pathology mechanism. Thus understanding FRG1 physiological function in skeletal muscle is critical to identify new target for the diagnosis and treatment of the FSHD.

One of our goals is to study FRG1 function during development. To this purpose we have developed a conditional FRG1knock-out mice. This animal model will give us the possibility of characterizing the effects of FRG1 ablation in precise time-windows throughout mouse embryonic development.

In these time-windows FRG1 function will be monitored through the identification of FRG1 RNA targets.

Project Title:
Identification of proteins controlling FRG1 expression
Facioscapulohumeral muscular dystrophy (FSHD) is a common autosomal dominant myopathy. Reduction of tandemly arrayed 3.3 kb repeat units (D4Z4) at chromosome 4q35 has been identified as the causative genetic defect of this disease. The current model for FSHD pathogenesis is that loss of D4Z4 repeats causes improper transcriptional regulation of 4q35 genes. Consistent with this model, ANT1, FRG1 and FRG2 gene expression was elevated in muscle biopsies and myogenic celles from FSHD patients. The over-expression of these genes was not confirmed by subsequent studies, however this seems to be a common trend for all FSHD candidate genes. Notably, transgenic over-expression of FSHD region gene 1 (FRG1), leads to selective muscle wasting and alteration of the muscle contractile apparatus in mice, thus providing a molecular explanation for muscle weakness. Additional molecular studies on muscles from FRG1 transgenic mice allowed the identificaton of mis-splicing of several muscle transcripts that were also aberrantly spliced in muscles from FSHD patients.
Importantly many studies demonstrate that FRG1 function is evolutionary conserved and show the critical role of FRG1 gene in muscular development. This functional evidences of the importance of FRG1 on muscle development support its relevance in FSHD onset. The aim of the study is to identify regulatory elements that are involved in the regulation of FRG1 expression in normal myoblasts and during the myogenic differentiation using a genome wide shRNA screening. The long-term goal of this research is to shed light on the strict regulation of FRG1 gene in muscle cells and understand FRG1 role in FSHD pathogenesis.