Синтез модифікованого гена транскрипційного фактора STAT5a

Abstract

Транскрипційні фактори родини STAT (Signal Transducer and Activator of Transcription) є ключовими регуляторами експресії генів стимульованих цитокінами, що впливають на різні клітинні процеси. Транскрипційний фактор STAT5a опосередковує стимуляцію клітинного росту, проліферації та ангіогенезу. Сучасні дослідження біологічної ролі білків STAT проводять шляхом створення експериментальних моделей мишей, що містять у геномі штучно синтезовані та модифіковані форми генів відповідних транскрипційних факторів. Метою даної роботи був синтез модифікованого гена STAT5a. Введення цього гена у геном миші дозволяє моделювати процеси активації STAT5a in vivo та досліджувати його роль у забезпеченні патологічних змін в організмі. Ключові слова: синтез ДНК, транскрипційні фактори STAT, модифікація генів.

Cytokines are small secreted proteins which mediate cell growth, proliferation, survival and apoptosis. They are crucial regulators of immunity and hematopoiesis. Cytokines generally act at very low concentration and short time spans. The JAK-STAT signaling pathway is one of the most important mechanisms of cell response to the cytokines. Binding of cytokine to the specific membrane receptor on the cell activates the JAK (Janus Kinase) kinase which adds a phosphate group to the STAT (Signal Transducer and Activator of Transcription) proteins. After posphorylation, STAT proteins form via their SH2 domain the active dimers. The dimers move into the cell nucleus via importin a/b and RanGDP complex and bind to DNA-recognition region called gamma activated sites (GAS) in the promoter region of cytokine inducible genes and activates transcription of these genes. Then, nuclear phosphatases dephosphorylate the STATs which leads to their inactivation and transportation from the nucleus by exportin crm1/RanGTP complex. There are seven members of STAT protein family: STAT1, STAT2, STAT3, STAT4, STAT5a STAT5b and STAT6. STAT1 and STAT2 become activated in response to the interferons and can form the homodimers or heterodimers with other STATs. STAT3 is activated by interleukin 5, interleukin-6, hepatocyte growth factor, interferons, epidermal growth factor and leukemia inhibitory factor (LIF). STAT3 regulates cell growth and apoptosis. Since it has anti-apoptotic and proliferative effects, hyperactivation of this transcription factor is associated with various tumors, such as chronic lymphocytic leukemia, Burkitt's lymphoma, cutaneous T cell lymphoma, Hodgkin desease, malignant melanoma, lung cancer, prostate carcinoma and renal cell carcinoma. STAT4 and STAT6 become phosphorylated in response to interleukin-4 and interleukin-12, respectively. They are involved in regulation of immune response and lymphocyte activation. The transcription factor STAT5b is activated by interleukin 2, interleikin 4 and CSF1. It mediates apoptosis, TCR signaling and liver gene expression. Our studies we focused on STAT5a, since it plays a crutial role in basic cell functions, like cell growth, proliferation, angiogenesis and additionally, serves as powerful anti-apoptotic factor through activation of BCL2L1/BCL-X(L) expression in the cell. JAK kinase phosphorylates STAT5a in response to interleukin 2, interleukin 3, interleukin 7 GM-CSF, erythropoietin and thrombopoietin. The high level of activated STAT5a was found in erythroleukemia, acute lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, megakaryocytic leukemia, sezary syndrome, anaplastic large T cell lymphoma, B cell lymphoma, head and neck cancer, cerebral menangiomas, neuroendocrine tumors, pancreatic carcinoma, ovarian carcinoma and colon cancer. Detection of hyperactivated STAT5a protein serves as one of the diagnotic markers of these tumors. Investigation of the role of STAT5a in tumor development is a very perspective direction of biology and medicine since it can help in designing a new effective diagnotic and therapeutic approaches. Modern investigations of the biological role of STATs in tumor formation are mainly performed by generation of transgenic animal models with modified genes of appropriate transcription factor. The main goal of this work was to synthesize modified STAT5a gene. We used classic methods for DNA synthesis and purification including polymerase chain reaction, site direct mutagenesis and electrophoresis in agarose gele. The cloning was performed with using various endonucleases and T4-ligase. All steps of DNA synthesis and cloning were analyzed by DNA sequencing. We also tested functional activity of modified STAT5a by transformation of NIH 3T3 cells. Our data confirmed synthesis of the modified STAT5a gene. The protein of this gene is expressed at high level and possesses a specific DNA binding activity. Introduction of modified gene of STAT5a into mouse genome allows to modulate the process of STAT5a hyperactivation in vivo and to investigate the role of this transcription factor in the mechanisms of tumor development. Key words: DNA syntesis, STAT transcription factors, gene modification

Транскрипционные факторы семейства STAT (Signal Transducer and Activator of Transcription) являются ключевыми регуляторами експрессии генов, стимулированных цитокинами, влияющими на разные клеточные процессы. Транскрипционный фактор STAT5a способствует стимуляции клеточного роста, пролиферации и ангиогенезу. Современные исследования биологической роли белков STAT проводят с использованием експериментальных мышиных моделей, имеющих в составе своего генома искусственно синтезированные и модифицированные гены транскрипционных факторов. Целью данной работы был синтез модифицированного гена STAT5a. Введение этого гена в геном мыши позволит моделировать процесс активации STAT5a in vivo и проводить исследование его роли в процессах развития патологических процессов в организме. Ключевые слова: синтез ДНК, транскрипционные факторы STAT, модификация генов.