Roman-Ulrich Müller - assoc. RG

Characterizing the molecular functions of the tumor suppressor protein Folliculin using the model organism Caenorhabditis elegans

Summary

Recent evidence suggests that the Birt-Hogg-Dubé syndrome is a ciliopathy with the tumor suppressor protein mutated in this disease – Folliculin (FLCN) - localizing to primary cilia and influencing their function. Previous work from our group could establish the nematode Caenorhabditis elegans as a model organism for studying the molecular biology underlying this disease. In this project, we investigate the putative ciliary role of FLCN exploiting the power of this model for analyzing ciliopathy genes.

Introduction

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant multitumor syndrome that is characterized by the triad of benign skin tumors, recurrent pneumothoraces and renal cyst and tumor formation. Germline mutations in Folliculin (FLCN) are the underlying cause of this syndrome. However, its exact molecular functions have remained elusive. In a recent study we have been able to introduce the nematode C. elegans as a novel model organism for studying the biology of the BHD syndrome by characterizing a knockout strain of flcn-1, the worm homologue of FLCN. We will now continue this project using both cell culture and particularly the nematode model to examine the signaling pathways FLCN is involved in more closely. Polycystic kidney diseases such as the one occurring in Birt-Hogg-Dubé syndrome are generally considered to be the consequences of dysfunction or dysgenesis of primary cilia – i.e. ciliopathies. Importantly, C. elegans is one of the key model organisms for studies addressing the ciliary function of cystopathy genes. The nematode paired with studies in cultured cells are exploited in this project to gain a better understanding of the molecular function of the tumor suppressor protein FLCN, in particular as to a putative role in ciliary biology.

1. Determination of the FLCN interactome

Only few interacting proteins of FLCN are known by now. To allow for IP-MS analysis of the FLCN interactome we generated a single-copy transgenic HEK293T cell line expressing an FLCN::GFP fusion protein using TALEN technology. Putative interaction partners that co-precipitated with FLCN were identified using nLC-MS/MS. Interestingly, GO term analysis revealed terms associated with vesicle formation and amino acid metabolism to be among the most enriched biological processes (Fig. 1).

2. Characterization of a novel splice-site mutation

In parallel to our work in cell culture and C. elegans we focus on characterizing novel mutations found in affected families. In this context, human genetics analysis of a family affected by BHD syndrome revealed not mutation in the coding region. However, we found a sequence alteration in a splice site and could confirm its impact on splicing of the folliculin transcript (Fig. 2). We then went on to analyse the impact of this splicing defect on protein biology and could show that the mutant protein is strongly destabilized and shows a localization defect (Bartram et al. 2017).

3. C. elegans as a model for a putative ciliary function of FLCN

Recently, two publications have linked FLCN to ciliary functions and shown its ciliary localization in cultured cells. We set out to analyse FLCN mutant worms using established C. elegans phenotypes.  So far no overt phenotype in ciliary morphology and function (osmotic avoidance, velocity of IFT, mechanosensation, male mating behaviour, dye filling) could be observed (Fig. 3). Nonetheless analysis of a strain expressing a FLCN::GFP fusion protein under the control of the FLCN promoter we generated clearly shows expression of the protein in sensory neurons – the only ciliated cell type of the nematode. Consequently, we are currently completing our analysis of ciliary readouts.

Perspectives 

The identification of the FLCN interactome will now pave the way to elucidating the molecular function of this protein as well as the consequences of a loss of FLCN for cellular biology. In this context, C. elegans will greatly facilitate to gain a better understanding on whether and how FLCN and its interactors impact on ciliary functions. Elucidation of the functional consequences of actual mutations from BHD patients on binding to the interactors and the cellular functions of FLCN continue to be at the heart of our project. 

Selected publications

M.P. Bartram, T. Mishra, N. Reintjes, F. Fabretti, H. Gharbi, A.C. Adam, H. Göbel, M. Franke, B. Schermer, S. Haneder, T. Benzing, B.B. Beck and R.-U. Müller (2017), Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer, BMC Med. Genet. 

Gharbi H, Fabretti F, Bharill P, Rinschen MM, Brinkkötter S, Frommolt P, Burst V, Schermer B, Benzing T, Müller RU (2013). Loss of the Birt-Hogg-Dubé gene product folliculin induces longevity in a hypoxia-inducible factor-dependent manner. Aging Cell

 

 


Dr. Roman-Ulrich Müller

Dept. II of Internal Medicine / RG location - CECAD Building

Dr. Roman-Ulrich Müller

Work +49 221 478 89033

Fax (Work) +49 221 478 89041

Dept. II of Internal Medicine Nephrology, Rheumatology, Diabetology and general Internal Medicine
Kerpener Str. 62
50937 Cologne

http://www.kidneyresearchcenter.org/51/Research/Renal-Damage-and-Cancer/RNA-biology-in-renal-damage-and-cancer-%E2%80%93-Roman-Ulrich-Mueller.htm

Publications - Roman-Ulrich Müller

Link to PubMed

Group Members

Michael Ignarski (Postdoc)
Reza Esmaillie (PhD student)
Tripti Mishra (PhD student)
Lisa Seufert (PhD student)
Katrin Bohl (bioinformatician)
Serena Greco-Torres (technician)

Figure 1

CMMC Mueller
GO-term enrichment (FLCN interactome) reveals vesicle organization as well as amino acid metabolism and DNA strand elongation to be strongly enriched.

Figure 2

CMMC Mueller
Sequencing of the FLCN gene reveals a 3 base-deletion in a splicing acceptor site of exon 11 (modified from Bartram et al. BMC Med. Genet. 2017)

Figure 3

CMMC Mueller
A Representative kymograph showing the movement osm-6 ::GFP IFT complexes along the ciliary axis. B IFT velocity is not altered in FLCN mutant worms.