Chromatin Remodeling Complex Subunit Hsnf5 Combination A Protein Hnop17 Identification And Function Of A Preliminary Study

Chromatin structure is an interesting paradox.How do you maintain a compacted genome that will fit in the eukaryotic nucleus while still maintaining a DNA template that is readily accessible for replication,transcription and DNA damage repair? The answer lies in the fact that chromatin is a dynamic structure with many layers of complexity and regulation. The basic unit of chromatin is nucleosomes which contain a histone octamer and is wrapped with 147 base pairs of DNA.At the next layer,nucleosomes are arranged into long linear arrays with a width of～10nm.Then,through intra- and inter-nucleosomal interactions,the linear arrays are futher compacted to chromatin fibers with a width of～30nm.Finally,long-range interactions,either direct or mediated through other nohistone proteins increase the level of chromatin compaction and form fiber with a width of 100-400nm.ATP-dependent chromatin remodling enzyme is one kind of the major contributors to the dynamic nature of chromatin.It uses the energy derived from the hydrolysis of ATP to alter chromatin structure by disrupting or mobilizing nucleosomes.This kind of enzyme is subdivided into three subfamilies:SWI/SNF,Mi-2/CHD and ISWI families.SWI/SNF complex is the first discovered chromatin remodling enzyme from S.cerevisiae. In mammals,homologs of SWI/SNF have highly conserved subunits including BRG1/BRM and INI1/SNF5.SNF5 has been found to be crucial in early steps of fetal development.It functions at turner suppression and regulates cell cycle progression. The coordinated expression of the genome in response to extracellular cues is ensured by enzymatic cascades signaling to the nucleus.These pathways generate chromatin modifications at specific loci controlling the transcription of signal-dependent and tissue-specific genes.The SWI/SNF complex offers the ideal surface for integrating these signals through interaction with various proteins which is involved in different cellular signaling pathway.INI1/SNF5/BAF47 interacts with c-Myc and p53,play essential role in G1 phase progression and apoptosis.We need to know if there are other proteins interact with SNF5 and hope to get more clues about how SWI/SNF regulates gene expression and how INI1/SNF5 acts as turner suppresser and cell cycle regulater.So we use hSNF5 as bait, screen library of human fetal brain cDNA,got several candidates of hSNF5 binding protein. One of them is hNOP17,which still is a function unknown protein.Here,we confirmed the interaction between hNOP17 and hSNF5,begun to explore the function of hNOP17.hSNF5 interacted with hNOP17 in VitroWe performed a GST pull down assay using GST-hNOP17 fusion protein and the lyates of 293T cells which were transient transfected with pcDNA4-hSNF5-c-Myc,hSNF5-c-Myc interacted with GST-hNOP17,compared with a GST control and a control of the 293T cells lyates containing hSNF5 with Glutathione Sepharose 4B.hSNF5 interacted with hNOP17 in VivoEukaryotic expression vectors of Flag-hNOP17 and hSNF5-c-Myc were co-transfected into 293T cells,and the cell lyates were immunoprecipitated with the c-Myc antibody. Western blots show that Flag-hNOP17 interacted specifically with hSNF5-c-Myc. Compared with control:lysates of 293T cells transfected with empty vectors were immunoprecipitated by anti-c-Myc antibody;lysates of 293T cells transfected with pcDNA6-flag-hnop17 and pcDNA4-hsnf5-c-Myc incubated with proteinG-agarose and without anti-c-Myc antibody.The leusine zipper domain and N terminal 1-56aa residues of hNOP17 were important for the interaction between hNOP17 and hSNF5 in Vivo.To determine the binding domain of hNOP17 and hSNF5,we co-expressed different deletion hNOP17 and hSNF5 in 293T cells.The cell lyates were immunoprecipitated with the anti-c-Myc antibody.Western blots showed that hSNF5 only interacted specifically with hNOP17 fragments which remain the N termination 193-290 amino acid residues and one part of N terminal,such as FLAG-hNOP17△57-68 and hNOP17△85-193,hSNF5 didn't interact with deleted hNOP17 which only have N,C terminal or the isolated middle domain.These indicated that the N termination 1-57 and C termination leusine zipper domain(193-290aa) were important for the binding of hNOP17 with hSNF5.The C terminal of hSNF5 was essential for its interaction with hNOP17 We deleted the coil-coiled domain and two repeat domains of hSNF5 step by step,which all located in the C terminal of hSNF5.Then we co-expressed different deletion hSNF5-Myc and Flag-hNOP17 in 293T cells.The results of immunoprecipitation assay indicated that no delected hSNF5 could interact with hNOP17.The C terminal of hSNF5 was essential for its binding with hNOP17 and we still need more data for finding the minimal domain of hSNF5 for this interaction.hNOP17 and hSNF5 colocated at cell nuclearFirst,co-transfected pDsRed-hNOP17 and pcDNA4-hsnf5-c-Myc to 293 cells and stained hSNF5 by anti- c-Myc and FITC-anti- IgG.The images shew that the fluorescence of hNOP 17 and hSNF5 overlaped at the same position of cells.Second,co-transfected pcDNA6-hNOP17 and pcDNA4-hsnf5-c-Myc to 293 cells,we used anti-hNOP17 rabbit serum and TRITC-antirabbit-IgG stained the hNOP17,used anti-c -Myc mouse antibody and FITC-anti-mouse IgG stained the hSNF5.The red and green fluorescence mostly located in nucleus,and there were some overlapped red and green dots. These indicated that hNOP17 and hSNF5 co-located in nucleus.Third,we stained the endogenous hNOP17 in SH-Sy5y cells and RD cells by anti-hNOP17 rabbit serum and TRITC-antirabbit-IgG,the red fluorescence located mainly in the nucleus, confirmed that endogenous hNOP17 mainly located in nucleues.hNOP17 expressed broadly in multiple tissues.hNOP17 was a function unknown protein.To determine the distribution of hNOP17 in multi-tissues,we hybridized Mutiple Tissue Northern(MTN) Blots membranes by 32P-labeled hnop17 cDNA probe.The x-ray film shew that hNOP17 was expressed broadly,there were higher expression levels especially in muscle,brain,live,placenta and kidney.The effects of hNOP17 and hSNF5 on p21-lucAs a tumer suppresser and cell cycle regulater,hSNF5 has been reported binding to the promoter area of several genes,such as p21CIP/WAF1 and p16INK4a gene.To investigate whether the p21 promoter was affected by the hNOP17 and hSNF5,we measured the transcriptional activation activity of hNOP17 and hSNF5 on p21-luc.Dose depended, hSNF5 activated p21 promoter and hNOP17 suppressed the activation of p21 promoter. Overexpressed hNOP17 inhibited the effect of hSNF5 on p21 promoter.More investigation is needed for the detailed mechanism.In conclusion,hNOP17 is a novel protein interacting with hSNF5.Multi-evidences have proved this interaction.We began to emplore the function of this novel protein,p21 is a good begaining. Part two:MEKK3 interacting protein in multi-cellular signaling pathwayMitogen-activated protein kinase kinase kinase 3(MEKK3) is a member of mitogen activated protein kinase(MAPK) family.It is invovled in muti-cellular signaling transduction pathway including tumor necrosis factor receptor(TNFR),interleukin-1 receptor(IL-1R) and Toll-like receptor(TLR) induced pathway.MEKK3 knockout mice die in early embryonic day.MEKK3 deficency in cells lead to impaired NFκB,JNK, ERK5 and p38 activity.These indicate the essential role of MEKK3 in eukaryotic cellular signaling pathway,but the mechanism still need to be explored.In this work,we got several endogenous proteins candidates which interacted with MEKK3 through tandem affinity purification including TRAF7,ERK5 and TGFβ-activating kinase(TAK1), furtherly we confirmed the direct interaction between MEKK3 and TAK1 by FRET assay (Fluorescence Resonance Energy Transfer).We confirmed that TAK1 and TAB1 regulated the phosphorylation of MEKK3 and then affected the function of MEKK3 in NF-κB pathway,but MEKK3 could not affect the phosphorylation of TAK1 and might not be the upstream regulator of TAK1.It was TAB1 who played important role in regulating TAK1 phosphorylation.We also found that TAB1 regulated the interaction between MEKK3 and TAK1.Later,we confirmed that MEKK3 interacted with IKKβ.All the datas provided clues for the position of MEKK3 in multi-signaling pathway,especially in NF-κB pathway.IntroductionNuclear factor kappa B(NF-κB) is a crucial and well defined cellular signaling pathway and displays multi-physiological roles in eukaryotic cells such as cellular differentiation, survival,cell proliferation and immune responses.In most mammalian cells,NF-κB complexes are inactive,residing primarily in the cytoplasm interacting with any of the...