PI: Radislav Sedláček Phenogenomics: Systematic Phenotypic Analysis of Mice and Rat Mutant Models for the Annotation of Gene Function
Ubiquitylation-Mediated Processes in Health and Disease
Using mutant mouse models we are addressing the role of several ubiquitin ligases whose function and role have not been described – the field of Ub-ligases and deubiquitinases is of key importance for cellular processes and many of the genes in the Ub-ligase and deubiquitinase families (altogether around 900 members) have not been investigated so far, or investigated only poorly. A major focus of these studies is to understand the role of ubiquitination in regulating intestinal barrier function, immunity, and to characterize links with human inflammatory bowel disease. In our current work we focus on cullin-RING ubiquitin ligases involved in GIT homeostasis and pathological processes since the cullin family has been largely associated with different types of cancer in GIT and thus represents a promising pharmacological target. We use RNA in situ hybridization, qRT-PCR and LacZ reporter systems to localize expression of components of cullin-RING –ubiquitin ligase complexes together with conditional transgenic models to specifically assess the role of cullin dependent ubiquitination in GIT. We are studying also other U3 ligases using conditional, mouse models, among them Btbd3, Trim15, Rnf121, Wdsub1, Mex3b, and others.
Proteases in Physiology and Disease
Another part of our work is focused on proteases, particularly on matrix metalloproteinases (MMP), a disintegrin and metalloproteinase (ADAM), and currentyly alsao on kallikreins (Klk). While MMP and Klk proteases are largely responsible for controlling extracellular matrix -cell interactions affecting cell differentiation, survival, migration, and other processes, the ADAM proteinases such as ADAM 10 and ADAM17 (TACE) release ligands and their receptors from the cell surface thus guiding bioavailability of many important regulatory molecules. Balance among the proteases and their natural inhibitors determines if tissues and organ architecture are to be built up or disrupted, or whether biological processes are to be initiated or terminated. This balance is pivotal for tissue homeostasis and disturbance may lead to development of various pathologies.
Functional Redundancy of the Kallikrein Locus
The mammalian kallikrein gene cluster exists as a gene cluster (on chromosome 19 in humans) comprise a class of extracellular proteases that mediate tissue homeostasis. They are often dysregulated during cancer and can serve as excellent diagnostic markers.
We are addressing the functional redundancy of kallikreins by introducing two or more concurrent gene deletions. We expect these experiments not only to resolve the role of kallikreins in important physiological functions such as wound healing, but also to yield insight into their epigenetic co-regulation.
Metalloproteinases in Gastrointestinal Tract and Immunity
This part of our work is focused especially metalloproteinases that process extracellular matrix (ECM) proteins or release (shed) ligands and their receptors from the cell surface. Interactions between cells and matrix control cell differentiation, survival, migration, and activation via cell surface receptors and adhesion molecules. Especially adhesion molecules sense changes in the composition of extracellular matrix that is affected by proteases and their inhibitors. Balance between these two molecule classes determines if tissues and organ architecture are to be maintained, built up or disrupted. Thus, this balance is crucial for tissue homeostasis and its disturbance may lead to development of various pathologies such as cancer, chronic inflammation, or fibrosis.
Our research activities focus on how proteases process the ECM proteins and how these changed matrix affect the biology of various cell types. Our work is focused specifically on proteases function in epithelia/epidermis and currently also liver and intestine pathogenesis. Inflammatory reactions in these tissues are of our special interest as regulated proteolytic activity in the epidermis and many epithelia is crucial not only to maintenance of the body and organ barriers, but also to regulation of local inflammatory reactions.
Molecular Mechanisms of Craniofacial Development
Our main focus is to investigate the recently discovered collective epithelial migration as an important morphogenetic mechanism during craniofacial development. We put the first evidence that signalling molecules Fgf8 and Shh act, beside other functions, also as the key regulators of collective epithelial migration during initiation of tooth development (Figure 2). In our current research we are focused on unveiling another locations and processes in orofacial area where collective epithelial migration is involved (tongue, palate or salivary gland) and on the description of how this mechanism is related to pathological behaviour of invasive epithelial cells in certain types of cancer. The important part of our research is thus revealing general molecular mechanisms behind initiation of epithelial migration; in particular, we study the role of cullin-RING ubiquitin ligases in regulation of Shh, Fgf and Wnt signaling pathways involved in collective epithelial migration.
Stem Cell Pluripotency and Early Embryonic Development
Recently, the major effort has been dedicated to the Fam208a (D14Abb1e) gene and heterochromatic gene silencing. FAM208A (D14Abb1e) was identified in an ENU mutagenesis screen for genes that alter expression of a variegating reporter transgene in mice. Classified as a new suppressor of variegation, its mutation not only leads to deficient transgene silencing, but also to a failure of embryonic gastrulation. Using unique mouse models we are currently addressing the molecular mechanisms by which Fam208a influences embryonic development, stem cell pluripotency, and embryonic robustness to environmental stressors and teratogens.
Interactions between Tumour Cells and the Immune System
This part of our work studies interactions between tumour cells and the immune system as well as the impacts of anti-tumour chemotherapy on these interactions. Special attention is paid to the mechanisms of DNA demethylation and the IFNγ signalling pathway. Our objective is to elucidate DNA demethylation events upon IFNγ signalling pathway activation. We are proposing that changes in DNA methylation of selected genes, as we have recently reported for the APM genes, represent an early and active event upon IFNγ signalling induction.
Another topic studied is the interaction of the tumour cells under genotoxic stress and senescent cells with the immune system. One of the aims of this project is to uncover, how senescent cells, or cells in a genotoxic stress in general, contribute to the pro-tumorigenic and immunosuppressive microenvironment.