The genomes of humans, mice and many other species have been completely sequenced; nevertheless the knowledge of genome sequences as such does not shed light on questions concerning the functions of these sequences. Among the unanswered questions are those regarding the functions of most of the genes that encode proteins; the number of these genes is estimated to be 20 000. In order to describe biological functions of a gene, an informative modification (mutation, for instance a conditional deletion) must be inserted into the genes.
In order to annotate the function of human genes, functional (pheno)genomics needs to be combined with comparative genomics – the function of the human genome should be inferred from the function of an orthologous genetic product, e.g. from the mouse .
In recent years, mouse and rat models have been considered excellent models in the search for functions of genes within complex organisms as most of their physiological functions are very similar to those of humans and, also, their genetic differences are minimal (in comparison to other non-mammalian models). Although characterization of the function of a particular gene product (i.g. protein) in vitro delivers important information about molecular mechanisms, the verification of their real functions cannot be done without an intensive research at the level of complex organisms and their distinct physiological systems.
Functional genomics research program is based on previous work of a few research groups that have built up a substantial expertise and have already attained considerable scientific results in this field.
Research projects under research program of Functional Genomics / BIOCEV
- Phenogenomics: Systematic phenotypic analysis of mice and rat mutant models for the annotation of gene function
Radislav Sedlacek, PhDOpen or Close
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.
- Genetically defined mice models as a tool for studying human diseases
Jiri ForejtOpen or Close
While the analysis in human genomics is currently focusing on the genome-wide association studies (GWAS), the mouse model offers a complementary approach using genetic fractionation of polymorphisms on the model of the chromosome substitution strains (= consomic strains) and the recombinant inbred (RI) strains (see the current international project 1000 RI strains, Collaborative Cross). So far only 4 complete panels of the chromosome substitution strains exist in the world. Two of them were prepared in the USA, one in our laboratory and one in Japan.
We were first to use a powerful genetic polymorphism that occurred during evolution of two mouse sub-species; Mus m. musculus and Mus m. domesticus to create a panel of consomic strains. Mice from our consomic strains have been partly phenotyped in the PHENOME program in The Jackson Laboratory, Bar Harbor, USA.
These mice serve as a universal tool for studying wide range of complex human diseases as mentioned above. The consomic strains will find a special use in studying the modifier genes and for studying the genes controlling production of functional sperm.
Analysis of two mouse models of genetically controlled male infertility. To understand the principles of the epigenetic, parent-of-origin effects on meiosis of male-sterile chromosomal translocation carriers we are studying chromation modification in primary spermatocytes of the reciprocal +/T43H male hybrids which differ in the extent of meiotic pertubations. We will analyze the transcription profile changes associated with this type of transgenerational inheritance and we will study differential epigenetic marks established on a chromosomal domain of T43H translocation such as DNA methylation and histone modifications within germ cell lineage development.
The other model gains information of the role of individual genes in infertility by following the cellular, subcellular and molecular traits associated with male infertility in mouse sub-species Mus m. musculus and Mus m. domesticus hybrids the new knowledģe obtained from the mouse infertility model will be applied on data from human male infertility studies. As an example, our recent discovery of Prdm9 gene as mouse hybrid sterility prompted subsequent follow up of this gene in patiens of human infertility clinics.
- Rat models for metabolic disorders
(currently without PI)Open or Close
The goal of the current biomedical research is to reveal genetic determinants of common diseases, including cardiovascular and metabolic disorders which often occur together and are diagnosed as the metabolic syndrome, a complex trait arising from of intertwined network of genomic and environmental components.
The overall aim of our project is the systematic deconstruction of eco-genomic architecture of the metabolic syndrome. We are pursuing this goal using genetically designed rat models with modulated gene expression (single genes or metabolic/signaling pathways). We are particularly focusing on four major aspects of metabolic syndrome eco-genomic “landscape”.
Early environment and metabolic programming of metabolic syndrome. Our initial studies showed that the metabolic and hemodynamic outcomes of early environment challenges are to a certain extent dependent on the genomic background. We are set to determine major metabolic (biomarkers), transcriptomic (transcripts, pathways) and epigenomic (methylation) features related to developmental drivers of particular subsets of metabolic syndrome as seen in clinical setting.
Genetical genomics of metabolic syndrome. This approach involves identification of signaling and expression modules playing a role in pathogenesis of metabolic syndrome and its aspects. By combining the existing and newly generated –omics level data from designed rat models of metabolic syndrome we are seeking the sex, age and organ-specific expression and signaling networks and playing crucial roles in pathogenesis of the metabolic syndrome.
Nutrigenomics and pharmacogenomics of metabolic syndrome. Reflecting the complex network of dietary and pharmacological influences that only together with the genomic background make up the clinical presentation of metabolic syndrome in real life, we are systematically dissecting the diet-modulated, pharmacogenetic interactions we have previously described e.g. for the antidiabetic drug rosiglitazone. We are testing the effects of westernized diets and commonly used drugs known to affect features of metabolic syndrome when acting on specific genomic backgrounds in the designed rat models.
Towards integrative eco-genomics of metabolic syndrome. By integration of the results from the above project and the available date we aim to proceed beyond the identification of individual risk and protective variants using the tools of system biology (analysis and modeling of genetic and signaling networks). This effort should lead us towards identification of functional genomic signatures connected with manifestation of specific forms of metabolic syndrome.
- Mouse models for studying of physiology and pathophysiology of digestive epithelia
Martin GregorOpen or Close
Intermediate filament cytoskeletal proteins make up a large family of tissue and cell specific cytoplasmic and nuclear proteins. In epithelial cells, keratins are cytoplasmic intermediate filament (IF) proteins derived from more than 30 functional genes that are expressed in an epithelial cell-specific manner. In digestive simple-type epithelia, the major keratins are keratin polypeptide 8 and 18 (K8/K18) with variable levels of K7, K19 and K20 depending on the cell type. A major role of the keratin intermediate filaments in simple epithelia is to protect cells from mechanical and non-mechanical stresses. There is increasing evidence for the involvement of keratin-associated proteins with the modulation of these functions. One of these proteins is plectin, a multimodular cytolinker protein, belonging to the plakin protein family.
Plectins enormous versatility is based on several different plectin isoforms, which are generated by tissue and cell type–dependent alternative splicing of transcripts from a single gene with more than 40 exons.
The major goal of the project is the elucidating of mechanistic links mediating interplay between keratin IFs and integrins and its role in regulation of physiologic and pathologic processes in liver. Liver-specific plectin KO mice will be characterized and impact of plectin deficiency on keratin cytoarchitecture will be studied in primary hepatocytes. Further experiments will focus on characterization of integrin and plectin isoforms responsible for recruitment of keratin filaments to junctional complexes. We will further elucidate the role of plectin/keratin/integrin axis in regulation of TGF-b1 pro-fibrotic pathway with the regard to the contribution of particular integrin and plectin isoforms.
- Auditory Function in Mutant Mice
Jiří Popelář (prof. Josef Syka, guarantor)Open or Close
Hearing impairment is the most frequent inborn sensory defect. One out of every 500 children is born deaf. A large percentage of the hearing losses are monogenic. As the population is aging, the progressive hearing loss which attacks the majority of the population in old age is becoming an even more serious problem and it has its background in the pathological changes of the genome. In both cases, the mice models are of significant use when studying the genetic principles of the pathological conditions.
Recently, studies have begun on the hearing changes in some strains of laboratory rats. The defects are mostly related to the inner ear and are easily detectable by the examination of auditory stem potentials (ABR). But it is also possible to perform the screening with the help of the shock-induced reaction behavior (Startle Reflex) and its modification (PPI – Prepulse Inhibition) and to complete the examination with the observation of otoacoustic emission (DPOAE – distortion product oto-acoustic emissions) that inform about the function of receptor hair cells in the inner ear. For more detailed examination, confocal and electron microscopy, immunocytochemistry and other approaches are necessary.
In cooperation with the Transgenic and Archival Module of the Czech Centre for Phenogenomics (CCP), it will be possible to create targeted deletions of mouse mutants for certain genes suspected to play a role in inherited hearing loss and hearing impairment during the organism‘s aging. In case of suspected hearing loss in mice passing through the phenotypization in the mouse clinic, it will be possible to complete the „classic“ examinations (ABR, DPOAE, startle) even with the detailed morphological and histochemical studies.
- Conditional Mice Mutants as Tools for Studying Genetic Disorders and Eye Physiology
Zbyněk KozmíkOpen or Close
The mouse model represents an attractive alternative for studying the development and pathogenesis of the human eye. Currently, a systematic targeted mutagenesis of mice genes in the embryonic stem cells is being done (EUCOMM and KOMP consortia). Shortly, the conditional mutants for the majority of the genes will be available for phenotypic analysis. Their utility is, to a great extent, dependent on the availability of suitable strains of mice with the tissue or time limited activity of the Cre recombinase. For each area of interest, e.g. eye, brain, liver, it is necessary to have a panel of the Cre-transgenic mouse strains which enable a specific deletion of the given gene and a detailed study of its function in the context of the whole organism. The size of the Cre panel which it is necessary to create, is dependent primarily on the complexity of an organ (number of cellular types, development stages), but also by the current availability of Cre lines for the given organ.
For instance, the Cre expression in the very early embryonic retina, in certain types of retinal cells of an adult mouse and in the lens and the cornea of an adult mouse is not available for the functional genomics of the eye so far.
The project will be focused on the systematic preparation of new transgenic Cre lines with defined expressions in the eye tissues and the consecutive usage of the Cre lines for the systematic analysis of the conditional mutants in genes coding the transcriptional factors and components of the Wnt signaling pathway.
The new transgenic mice lines with the tissue and time limited activity of the Cre recombinase will be prepared in the presented project. Their utility will be verified by the deletion of genes which have the known pathogenesis. Then the suitable Cre lines will be used for the systematic gene deletions and the study of their function in eye tissues. The project will contribute to our understanding of eye diseases. Goal: 1/ To prepare the transgenic Cre constructs with the BAC recombineering technology and with their help to create the Cre lines for the functional genomic of the early retina and adult cornea, 2/ to prepare the transgenic constructs with the pharmacologically controlled form of the Cre recombinase (Cre-ERTM) and to create the Cre lines with time conditioned activity of the Cre recombinase, 3/ to map the function of the genes encoding transcription factors and components of the Wnt signaling pathway in the eye tissues.
- Lipocalins in Modulation of Mammalian Reproduction
Pavel StopkaOpen or Close
Over the past 10 years, a couple of tens of new genes belonging to the protein family of lipokalins (58 in mice – e.g. the main urinary, odorant binding proteins, probasin, Lcn and others) were discovered and described. These proteins participate in a whole series of important biological processes including detoxification (that is the extraction of metals, free radicals, etc.), pheromonal communication and transport of important molecules (e.g. steroids), in the process of the gamete ripening. All lipocalins which have so far been described have a similar size (approx. 18 – 25 kDa) and tertiary structure, but they differ on the level of the primary and secondary structure. Although this (monophyletic) family of proteins is one of the most numerous gene clusters of the mammals, it is at the same time the least investigated.
Our current research proved that this family of proteins is an extraordinarily universal communication medium by which the information transfer happens both between tissues and cells (i.e. by the hormone transfer) and between individuals (i.e. by the pheromone transfer).
Our latest research even shows that during the sequenation of the mouse genome, some sections were ‘omitted’. That’s why it is necessary to return to the original species from which the laboratory lines were derived and to focus on the sequenation of the transcriptome of the targeted tissues, in which the lipokalins are expressed. To study the phenotypic plasticity on the lipocalins level it is necessary to set the phenotypic variability on the qPCR level and to take advantage of the microarrays.
The goal of the project: 1/ to reconstruct the lipocalin evolution with the use of the interbreed comparison method on the genome level and on the level of the expression profiling, 2/ to create a systematic biological model of the function of the individual lipocalins on the level of biological processes (e.g. hormone transport, spermatogenesis, reproduction, pheromonal communication), 3/ to clarify the mechanism of the gene duplication within the individual gene clusters of lipocalins on the molecular level.
- Genetically modified mice as a tool to study renewal and neoplastic transformation of gastrointestinal epithelia
Vladimír KořínekOpen or Close
The single-layer epithelia of the small intestine and colon represent the most rapidly self-renewing adult tissue that completely regenerates approximately every five days. The proper maintenance of epithelial architecture is controlled by various signaling pathways that regulate the balance between the opposing processes of proliferation and differentiation. These pathways include Wnt/β-catenin, Hedgehog and Notch signaling, the ephrin type-B receptor (EphB)/ephrin-B cell communication system, the bone morphogenetic protein (BMP) signal transduction pathway and signaling downstream of the epidermal growth factor receptor (EGFR). Importantly, the majority of these pathways is deregulated in carcinoma of colon and rectum (colorectal cancer).
Colorectal cancer represents the third most common human malignancy worldwide. It is estimated that more than one million patients are clinically diagnosed each year; up to one third of the cases constitute metastatic settings resulting in a disease-related mortality rate exceeding 30%.
Colorectal cancers are characterized by a complex genomic “landscape”; individual tumors harbor nine rearranged loci on average and a median of 76 non-silent mutations. However, only a fraction of these changes is considered to be causative in tumor initiation and progression. The project will be focused on the on the analysis of crucial molecular mechanisms involved in physiological renewal and neoplastic transformation of cells of the gastrointestinal tract. The gene manipulation in mouse will be used as the main toll to accomplish the aims of the project.
The aims of the project are: (1) generation of novel transgenic mouse strains expressing DNA recombinases in cells of the gastrointestinal tissues; (2) identification and characterization of genes that encode tumor suppressors or oncogenes responsible for initiation or progression of gastrointestinal neoplasia; (3) generation and analysis of mice harboring conditional alleles of the selected gene(s); (4) alternatively, generation and analysis of mice harboring an activated oncogene in selected tissues.
- Molecular Mechanisms of Mammalian Germ Cell Development
Zdenek TrachtulecOpen or Close
The current fertility studies in model mammals move from the effects of single gene to genetic interactions important for human reproductive medicine that occur during spermatogenesis and oogenesis. The Prdm9 gene (also called Meisetz) is necessary for both male and female meiosis and fertility in the laboratory mouse. The biochemical function of the PRDM9 protein is to methylate histones. The mouse, bovine, and human PRDM9 proteins specify the sites of meiotic recombination. However, PRDM9 is dispensable for fertility in the dog. PRDM9 polymorphisms were revealed in sterile human patients and PRDM9 variation contributes to instability of the human genome. We have identified Prdm9 as the first vertebrate hybrid sterility gene. Different Prdm9 mutations display different stages and degrees of spermatogenetic arrest on various backgrounds, indicating that the resulting phenotype is dependent on genetic interactions of Prdm9.
The project aims are: analyses of genes regulating germ cell development in mouse and rat testes and ovaries, interspecific differences important for translation studies; analyses of interactions and incompatibilities of genes expressed in testes and ovaries; analyses of models of germ cell development defects that are affected by Prdm9, including complete meiotic arrest (azoospermia), limited fertility (reduced sperm count – oligospermia), sperm head malformations (teratozoospermia), and reproductive age defects (time-dependent arrest of germ cell development).
- Mechanisms involved in remodeling of chromatin structure during cell fate decisions
Tomáš StopkaOpen or Close
Chromatin is a nucleoprotein complex which is engaged in the transfer of biological information via mechanisms identifying DNA code and mechanisms detecting the sequence of posttranslationally modified histone proteins.
This so-called “histone code” determines, among others, the accessibility of chromatin to key molecules that are able to recognize the DNA code: for transcription factors. The accessibility of chromatin structure is accomplished by enzymes belonging to SWI/SNF2 family, which are able to regulate the interaction between DNA and histone proteins and enable a shift or complete revelation of DNA. Identification of protein partners within the SWI/SNF2 family and recognition of key mechanisms of chromatin remodeling – utilizing DNA template in vitro – have enabled to understand fundamental functional properties of chromatin-remodeling proteins.
Main ATPases of the SWI/SNF2 family are mammalian homologs of Brg1 and Smarca5, which play their roles in embryogenesis, cellular specification, replication control, organization of nucleolus, chromosome inactivation, and tumorigenesis.
Project focuses on studying the function that ISWI ATPase designated as Smarca5 (Snf2h, MommeD4, Iswi) in the development of haematopoiesis and in the regulation of leukaemogenesis, pursuant to preliminary results.
- Structures of the Cell Nucleus in Gene Expression
Pavel HozakOpen or Close
The cell nucleus as a carrier of the genetic information is a fascinating organelle, in which the complex principles of genetics are combined with a complicated spatial organization of DNA and all proteins cooperating with DNA during the life of the cell.
We use the methods of molecular biology together with advanced light and electron microscopy to describe processes taking place inside the cell nucleus.
OP RDE project
CZ.02.1.01/0.0/0.0/16_013/0001789 Upgrade of the Czech Centre for Phenogenomics: developing towards translation research
To keep pace with the current development of new technologies and knowledge to decipher complex biological processes linked to genetic base of human disorders, Czech Centre for Phenogenomics (CCP) strives to develop research activities advancing its expertise, service portfolio, and scientific impact.
Having established generation of genetically modified models and their standardized phenotyping, CCP aims to advance metabolic and aging analysis as well as data coming from models of rare and other genetic disease. Regarding the systemic approach and data usage, CCP aims to build a bioinformatics group that will analyse data coming from research activities of these grant projects, phenotyping tests of CCP, and later on also serve the user community.
A specific link to human medicine, especially to cancer development and its treatment, will be introduced by patient-derived xenographs (PDX) technology, which will include their standardized characterization, maintenance, biobanking, and distribution. Moreover, CCP strives to establish a National PDX reference centre and thus, not only to serve the medical community but also foster biomedical research. The outcome of this effort should be translated into future clinical application, in particular for the personalized medicine approach used in oncology.