U3A cells stably over expressing STAT1 WT or sumoylation deficient STAT1 mutant were either left unstimulated or stimulated with human IFN. Immunoprecipitation of cross linked and scattered chromatin was performed with anti acetylated histone H4 antibody or anti rabbit IgG antibody as a control. http://www.selleckchem.com/products/MDV3100.html STAT1 K703R expressing cells showed increased acetylation of histone H4 when compared to STAT1 WT. This result suggests that the enhanced pro moter binding of sumoylation defective STAT1 results in enhanced association of histone acetyl transferases to the promoter leading to increased histone H4 acetylation. Sumoylation does not prevent STAT1 dimerization Dimerization mediated through the interactions between the Tyr701 phosphorylated tail segment of one STAT1 and the SH2 domain of an adjacent STAT1 is considered to be essential for proper DNA binding and transcrip tional activity of STAT1.
To investigate if sumoylation affects STAT1 dimerization and inhibits downstream DNA binding in this manner, cells were co transfected with HA and Flag tagged STAT1 together or without His tagged SUMO 1. After 48 hours, the cells were left unstimulated or stimulated with IFN and osmotic shock prior to cell lysis. Equal amounts of whole cell lysates were immunoprecipitated with anti Flag agarose beads and immunoblotting with anti HA antibody was used to detect if HA tagged STAT1 molecules interact with Flag tagged STAT1. As shown in Figure 4A, a slower migrating band corresponding to the size of SUMO 1 conjugated STAT1 was detected with anti HA antibody, suggesting that sumoylated HA tagged STAT1 interacts with STAT1 Flag.
Figure 4B shows as a control that anti Flag agarose does not pull down HA tagged STAT1. These results suggest that sumoylation of STAT1 does not prevent STAT1 dimerization and are consistent with the results that con jugated SUMO moiety affects the interaction between STAT1 and DNA through steric hindrance. Discussion Sumoylation is a common post translational modifica tion of transcription factors, but in several proteins the physiological functions and molecular mechanisms of this modification have remained enigmatic. Several lines of evidence support the concept that SUMO serves as a negative regulator of STAT1.
Furthermore, the results demonstrating that sumoylation also nega tively regulates STAT5 mediated signaling and the only STAT transcription factor in Drosophila melanogaster, Carfilzomib Stat92E, indicates that sumoylation is an evolutionary conserved post translational modification for some STAT transcription factors. Sumoylation is a highly reversible covalent modifica tion that is regulated through conjugating and deconju gating enzymes. Several studies support the importance of PIAS1 mediated sumoylation of the proteins. Recently, it was shown that PIAS1 regulates oncogenic signaling by sumoylating promyelocytic tumor suppressor that leads to its ubiquitination and proteosomal degrad ation.