The Mechanisms By Which Nuclear Matrix-Enriched SUMOylated Proteins And SUMO E3 Ligase PIAS1 Interact With And Regulate Chromatin

As an important post-translational modification,SUMOylation plays important roles in DNA damage repair,cell cycle,gene expression regulation,chromatin structure and other biological processes.Aberrant SUMOylation is closely related to cancer and diabetes.Accumulative evidence indicates that most of the identified SUMOylated proteins are enriched in nucleus.In addition,numerous studies have shown association of SUMOylation machinery with chromatin.However,it is unclear whether SUMOylated proteins are directly enriched at chromatin and how SUMOylated proteins interact with chromatin.In this Ph.D.thesis,we first confirmed through different biochemical fractionation methods that SUMOylated proteins were enriched in nucleus.Furthermore,our fractionation experiments demonstrated that SUMOylated proteins are highly enriched in the insoluble nuclear matrix and chromatin fraction.Importantly,SUMOylated proteins were enriched in nuclear matrix rather than chromatin,because SUMOylated proteins remained in the nuclear matrix after complete removal of chromatin by different DNA hydrolases.Consistent with enrichment of SUMOylated proteins in nuclear matrix,we found that the SUMOylation machinery are also enriched in nuclear matrix,in agreement with some previous publications.To investigate how nuclear matrixenriched SUMOylation machinery regulates chromatin,we focused our study on SUMO E3 ligase PIAS1.To this end,we decided to identify PIAS1 direct interacting proteins via a chemical crosslinking followed by immune-affinity purification at denatural condition and protein identification by mass spectrometry.Through this approach,we successfully identified several highly reliable PIAS1 binding proteins,including histone proteins H3,H2 A.Z and H1 and nonhistone nuclear matrix proteins DDX17 and hn RNPU.Furthermore,by a series of in vitro and in vivo experiments we confirmed the interaction between PIAS1 and H3,H2 A.Z and DDX17,and demonstrated that PIAS1 mainly bound to H3,H2 A.Z and DDX17 through its SAP domain.Meanwhile,we showed that PIAS1 is capable of catalyzing SUMOylation of H3,H2 A and H2 B,but H2 A.Z and H4.Collectively,our results indicate that PIAS1,although mainly located in the nuclear matrix,can directly interact with and catalyze SUMOylation of nuclear matrix and chromatin proteins.In effort to understand how SUMOylated proteins involve in chromatin function,we uncovered that SUMO proteins have the ability to bind nucleosomes.Through in vitro pulldown assays,we found that GST-SUMO-1/2 but not ubiquitin could bind nucleosomes and histone octamers.Furthermore,tandemly fused SUMO proteins exhibited an enhanced binding activity for nucleosomes,and covalent cross-linking coupled mass spectrometry identified direct binding of GST-SUMO-1 to nucleosomal H2 A.We also determined the SUMO regions required for binding of nucleosomes and demonstrated that the middle region of SUMO-1/2 is required and this interaction is to large extent independent of histone tails.By chemical cross-linking coupled mass spectrometry,the 67 of SUMO-1 was identified as the key site for binding of nucleosomes.We also analyzed the effect of methylation status on the binding of SUMO with nucleosomes through in vitro pulldown assays.The results showed that methionine restriction enhances the interaction between SUMO and nucleosomes.Taken together,our study indicates that although SUMOylated proteins are enriched in nuclear matrix,SUMO may promote the binding of SUMOylated proteins to chromatin and participate in chromatin-related biological processes through direct binding of nucleosomes.In summary,the major findings uncovered in our study are as follows: 1)SUMOylated proteins are mainly enriched in nuclear matrix;2)The SUMOylation machinery,such as PIAS1,is also enriched in the nuclear matrix.PIAS1 not only interacts with the nuclear matrix proteins,but also binds histones and promotes histone SUMOylation;3)SUMO itself has the ability to bind histones,which may mediate the interaction between SUMOylated proteins and chromatin.Taken together,our work provides novel insights into how nuclear matrix-enriched SUMOylated proteins and machinery interact with and regulate chromatin.