E. S. Piruzian

Integrated network analysis of transcriptomic and proteomic data in psoriasis

BackgroundPsoriasis is complex inflammatory skin pathology of autoimmune origin. Several cell types are perturbed in this pathology, and underlying signaling events are complex and still poorly understood.ResultsIn order to gain insight into molecular machinery underlying the disease, we conducted a comprehensive meta-analysis of proteomics and transcriptomics of psoriatic lesions from independent studies. Network-based analysis revealed similarities in regulation at both proteomics and transcriptomics level. We identified a group of transcription factors responsible for overexpression of psoriasis genes and a number of previously unknown signaling pathways that may play a role in this process. We also evaluated functional synergy between transcriptomics and proteomics results.ConclusionsWe developed network-based methodology for integrative analysis of high throughput data sets of different types. Investigation of proteomics and transcriptomics data sets on psoriasis revealed versatility in regulatory machinery underlying pathology and showed complementarities between two levels of cellular organization.

[A thermostable Clostridium thermocellum lichenase-based reporter system for studying the gene expression regulation in prokaryotic and eukaryotic cells].

A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies.

Characterization of three Arabidopsis thaliana immunophilin genes involved in the plant defense response against Pseudomonas syringae.

Plant immunophilins are a broadly conserved family of proteins, which carry out a variety of cellular functions. In this study, we investigated three immunophilin genes involved in the Arabidopsis thaliana response to Pseudomonas syringae infection: a cytoplasmic localized AtCYP19, a cytoplasmic and nuclear localized AtCYP57, and one nucleus directed FKBP known as AtFKBP65. Arabidopsis knock-out mutations in these immunophilins result in an increased susceptibility to P. syringae, whereas overexpression of these genes alters the transcription profile of pathogen-related defense genes and led to enhanced resistance. Histochemical analysis revealed local gene expression of AtCYP19, AtCYP57, and AtFKBP65 in response to pathogen infection. AtCYP19 was shown to be involved in reactive oxygen species production, and both AtCYP57 and AtFKBP65 provided callose accumulation in plant cell wall. Identification of the involvement of these genes in biotic stress response brings a new set of data that will advance plant immune system research and can be widely used for further investigation in this area.

Comparative study of the expression of the native, modified, and hybrid cry3a genes of Bacillus thuringiensis in prokaryotic and eukaryotic cells

The cry3a gene of Bacillus thuringiensis was cloned. Based on sequence analysis of this gene, a modified gene, cry3aM, was constructed, which has the optimal codon composition for effective expression in eukaryotic cells. Hybrid genes cry3a-licBM2 and cry3aM-licBM2 were constructed, in which the sequences of the native and modified genes are fused with the reporter gene for thermostable lichenase in the reading frame. We have shown that the expression levels of hybrid genes cry3a-licBM2 and cry3aM-licBM2 in Escherichia coli are comparable, being 5% of those for reporter gene licBM2. In cells of a lower eukaryote Saccharomyces cerevisiae, the expression of hybrid gene cry3aM-licBM2, which contains the modified gene, considerably exceeded the level of expression of cry3a-licBM2 containing the native gene. The presence of lichenase in the composition of hybrid proteins was shown to facilitate selection and analysis of the expression level of hybrid proteins in transgenic organisms.

Preparation and properties of Clostridium thermocellum lichenase deletion variants and their use for construction of bifunctional hybrid proteins.

Major properties (pH and temperature optimum, stability) of lichenase (b-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.

Expression of a Thermostable Bacterial Cellulase in Transgenic Tobacco Plants

The bacterial gene of the thermostable endo-β-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.

Expression of hybrid cry3aM–licBM2 genes in transgenic potatoes (Solanum tuberosum)

The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.

[Bifunctional reporter genes: construction and expression in prokaryotic and eukaryotic cells].

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli β-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65°C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C or N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.

Transgenic Plants as a Tool for Plant Functional Genomics

Functional genomics aim to discover the biological function of particular genes and to uncover how sets of genes and their products work together. Transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis.

Integrated computational approach to the analysis of RNA-seq data reveals new transcriptional regulators of psoriasis

Psoriasis is a common inflammatory skin disease with complex etiology and chronic progression. To provide novel insights into the regulatory molecular mechanisms of the disease, we performed RNA sequencing analysis of 14 pairs of skin samples collected from patients with psoriasis. Subsequent pathway analysis and extraction of the transcriptional regulators governing psoriasis-associated pathways was executed using a combination of the MetaCore Interactome enrichment tool and the cisExpress algorithm, followed by comparison to a set of previously described psoriasis response elements. A comparative approach allowed us to identify 42 core transcriptional regulators of the disease associated with inflammation (NFκB, IRF9, JUN, FOS, SRF), the activity of T cells in psoriatic lesions (STAT6, FOXP3, NFATC2, GATA3, TCF7, RUNX1), the hyperproliferation and migration of keratinocytes (JUN, FOS, NFIB, TFAP2A, TFAP2C) and lipid metabolism (TFAP2, RARA, VDR). In addition to the core regulators, we identified 38 transcription factors previously not associated with the disease that can clarify the pathogenesis of psoriasis. To illustrate these findings, we analyzed the regulatory role of one of the identified transcription factors (TFs), FOXA1. Using ChIP-seq and RNA-seq data, we concluded that the atypical expression of the FOXA1 TF is an important player in the disease as it inhibits the maturation of naive T cells into the (CD4+FOXA1+CD47+CD69+PD-L1(hi)FOXP3−) regulatory T cell subpopulation, therefore contributing to the development of psoriatic skin lesions.