Study On The Relationship Between STAT1 And Nuclear Matrix

Background and objective: Signal transducer and activator of transcription 1 (STAT1) , one of important nuclear transcription factors, is mainly cytoplasmic. When the cells are stimulated by sigals from outside, STAT1 is phosphorylated and translocates into the nucleus taking part in target genes transcription. In resting cells, a relatively small quantity of STAT1 resides in nucleus and is involved in the constitutive expression of certain genes. The function of mediating transcriptional regulation is controlled tightly through interaction with other transcriptin factors and coactivators, but how they assemble in the transcription initiation complex remains unknown. The nuclear matrix is an insoluble protein structure, which plays an important role in higher-order organization of chromatin and mediates the control of gene expression. Recent studies have revealed that nuclear matrix is intimately related with many transcription factors: it provids the architecture for transcriptions; furthermore, many nuclear matrix proteins have been identified as transcription factors. As yet, no studies on the relationship between STAT1 and the nuclear matrix have been reported. This paper was aimed to clarify their relationship in the hope of understanding the regulation mechanism of STAT1-mediated transcription.Materials and methods: In this paper, the Hela cells were used as the experimental model and two different approaches were employed: (1)Whole-cell extracts, purified nuclear extracts and nuclear matrix protein from Hela cells stimulated or unstimulated with IFN-γ were prepared as described by Wen's method were subjected to Western blot analysis to test the expression of STAT1 and pSTAT1. (2)The Hela cells treated or untreated with IFN-γ were extracted as described byStaufenbiel with some modifications. After extraction, the cells and the residual structures (nucleus and nuclear matrix) in situ obtained during fractionation were fixed and subjected to immunofluorescent analysis to confirm the location and disribution of STAT1 and pSTATl.Results: The Western blot analysis revealed: In whole-cell extracts, nuclear extracts and nuclear matrix protein from cells untreated, there were positive bands of STAT1 but not pSTATl at 91KD. While there were positive bands of both STAT1 and pSTATl in all three kinds of extracts either treated or untreated with IFN-y. Moreover in the nuclear matrix protein extracted from treated cells, the band density of STAT1 and pSTATl decreased with the dosage increasement of DNase I. The in situ immunofluorescent studies showed: The green positive signals of STAT1 were cytoplasmic in untreated cells. After stimulation, the positive signals of STAT1 also appeared in the nucleus and localized in the nuclear matrix and nonnuclear matrix. There were no positive signals of pSTATl in untreated cells, but appeared in the cytoplasm, nucleus and nuclear matrix of the treated cells. The positive signals of STAT1 and pSTATl scattered evenly in the nucleus in situ but localized in speckles or formed an apparent boundary.Conclusions: In this study, we unequivocally establish that STAT1 is associated with the nuclear matrix: 1. An experiment model of Hela cells was developed for study on the interaction between STAT1 and nuclear matrix. 2. STAT1 is a constitutive component of nuclear matrix and nuclear matrix-associated protein; 3. STAT1 is recruited in the nuclear matrix after IFN-y treatment, so nuclear matrix is involved in STAT1 signal parthway in an inducible fashion; 4. The activated STAT1 is associated with nuclear matrix in a DNA-depended fashion. Besides the DNA-binding domain of STAT1, the other candidates linking STAT1 to nuclear matrix is appealing. 5. STAT1 is still tested positively in the nuclear matrix after excessive DNase I digestion. Therefore STAT1 binds nuclear matrix principally but not exclusively mediated by DNA.Both STAT1 and nuclear matrix are involved in gene transcription. Our findingthat STATl is associated with the nuclear matrix suggests that the biological effects of STATl is possibly resulted from the interaction between STATl and nuclear matrix.