Primary Studies On Fuctions Of BRD2 Nuclear Localization Signal And Initiation Fuction Of Cellular Apoptosis Of BRD2

A new member of the bromodomain protein family independentlycloned through eDNA representational difference analysis in ourlaboratory, BRD7 has the function of reversing malignant phenotype ofnasopharyngeal carcinoma cells and inhibiting the growth ofnasopharyngeal carcinoma cells, hence a powerful candidate as anasopharyngeal carcinoma inhibiting gene. BRD2 is a BRD7-interactingprotein screened out from eDNA library of embryonic brain by the yeasttwo-hybrid system, which also finds evidence by performingco-immuno-preeipitation and subcellular co-localization. BRD2, alsoknown as Ring 3, a member of the classⅡhistocompatibility antigenfamily, is a cyclin-dependent protein kinase with a significant activity ofprotein kinase and plays an important role in the regulation of drosophiladevelopment and human growth. Both BRD2 and BRD7 belong to thebromodomain protein family. Most members of this family participate inthe acetylization of histone, and it has been proved that somebromodomain proteins are nuclear transcriptional factors participating inthe transcriptional regulation of gene and closely related to thepathogenesis of various tumors.In the mouse fibroblast BALB, BRD2 is mainly located in the cell nucleus, with its C-terminal ends carrying with a typical nuclearlocalization sequence (NLS) abundant of basic amino acid residuesequence, namely KKKKRKAEKHR. As the structural foundation forthe translocation of transcriptional factors and other nuclear proteincytoplasm to the nucleus, NLS plays a crucial role in protein's nuclearentry and translocation as well as functional regulation. At the same time,by GFP-mediated subcellular localization and flow cytometry, Dr.ZHOU Ming of our laboratory has proved that BRD2 takes an initiatingrole in cell apoptosis.This study aims to examine the mode of BRD2's subcelluar localizationon one hand, and the structure foundation as well as the mechanism onthe molecular level of its cell apoptosis initiating role on the other.Our detailed experimental results are as follows:Ⅰ. Mode of subcelluar localization of BRD21. Mode of subcelluar localization of BRD2 in different cell linesBRD2 is a BRD7-interacting protein, and it has been proved ofclose relationship with BRD7 by various evidences. Subcelluarlocalization mode of BRD2 in three kinds of cell lines, namely COS7,Hela and HNE1, have been examined separately by constructing the green fluorescent protein (GFP)-labeled BRD2 eukaryotic expressionvector pEGFP-C3/BRD2 and applying GFP-mediated subcellularlocalization method. The results show that in all the three, BRD2 islocated in the cell nucleus, with relatively even distribution in eitherdiffused or dotted pattem, without showing obvious cell type specificity.In the BRD2-transfected COS7, HeLa and HNE1, however, phenomenaof cell apoptosis as shown by the irregularities, condensation andbreaking of cell nucleus are observed in many cells, with that in COS7and HeLa being the most obvious, which supports the conclusion thatBRD2 takes the apoptosis initiating role in these cell lines.2. Mode of subcelluar localization of various truncated proteins ofBRD2The subcelluar localization mode of the truncated proteins of BRD2N-terminal ends (aa 1-435) and C-terminal ends (aa 428-798) in threekinds of cell lines, namely COS7, Hela and HNE1, have been examinedseparately by applying the green fluorescent protein (GFP)-mediatedsubcellular localization method. The results show that in all of the three,BRD2 N-terminal ends and C-terminal ends are located in the cellnucleus, with relatively even distribution in either diffused or dottedpattern, without showing obvious cell type specificity. At the sametime, cells showing traces of apoptosis as is demonstrated by irregularities, condensation and breaking of cell nucleus are significantlymore in the over-expressed cells of BRD2 C-terminal ends (aa 428-798)than in the over-expressed cells of BRD2 N-terminal ends (aa 1-435).3. Separation and identification of BRD2's new non-typical NLSAs a nuclear transcriptional protein, BRD2 needs to enter the nucleusafter being synthesized in cytoplasm. In the above mentioned study onthe subcellular localization of BRD2's various truncated proteins, it hasbeen observed that both BRD2's N-terminal ends and C-terminal endsare located in the cell nucleus, which suggests that there is at least oneNLS in each of the two truncated proteins. It is reported that a classicalNLS, namely KKKKRKAEKHR, is present in BRD2's amino acidsequence (aa 552-560), which is the structure foundation for thetruncated proteins of BRD2's C-terminal ends to enter cell nucleus. Onthe other hand, so far, no NLS has been reported to be present in thetruncated proteins of BRD2's N-terminal ends. After structural searchfor BRD2 N-terminal ends on www.expasy.ch, we have found that its275-282 amino acids take the characteristic of NLS and named it aspNLS. Through constructing the pEGFP-C2/pNLS expression vector,however, we have found that this hypothetical nuclear localization signalpNLS takes no role of mediating heterogenic protein GFP for nucleiclocalization, suggesting that pNLS is not the NLS of BRD2 N-terminal ends.Further through "walk sequencing", we separated BRD2 N-terminalends into different domains and sections in accordance with its structuralfeatures, then linked them separately to the pEGFP vector according tothe open-reading frames. By GFP-mediated subcellular localization, wehave found that 283-335 amino acids of BRD2 N-terminal ends take therole of mediating heterogenic protein GFP for nuclear localization. Thisconclusion has also been further confirmed by cytoplasm and nucleusprotein distribution extraction and Westem blotting test, suggesting thatthe 283-335 amino acids of BRD2 N-terminal ends are the structuralfoundation for the truncated proteins of BRD2 N-terminal ends' entry intothe nucleus. Furthermore, bioinformatics analysis has shown that thisdomain does not have the characteristic of classical NLS, hence anon-typical NLS of BRD2.Ⅱ. Structure foundation and molecular mechanism for initiationfuction of cell apoptosis of the BRD21. Structure foundation for initiation fuction of cell apoptosis of theBRD2Results of earlier researches have shown that BRD2 takes the role ofinitiating cell apoptosis, while the structural foundation of this role is not yet known. This study, while examining the subcelluar localization ofBRD2's various truncated proteins, has found that though both theBRD2 N-terminal ends (aa 1-435) and BRD2 C-terminal ends (aa428-798) are located in the cell nucleus, cells that show traces ofapoptosis as demonstrated by irregularities, condensation and breakingof cell nucleus are significantly more in the over-expressed cells ofBRD2 C-terminal ends (aa 428-798) than in the over-expressed cells ofBRD2N-terminalends(aa 1-435). Further through Hochest 33258mediated cell apoptosis test, we have found that the apoptotic index (AI)reaches to 43%±8% in over-expressed COS7 of BRD2 C-terminal ends,while that in the HNE1 is 4%±2%, with significant difference (P＜0.05)as compared with that of vacant vector GFP and of BRD2 N-terminalends. At the same time, we have applied transient transfection to COS7and HNE1 cell lines with pEGFP-C3/BRD2 C-terminal end expressionvector, and applied apoptosis test to GFP-positive cells by flowcytometry, getting apoptosis percentages of 38.5% and 13.5%,respectively. This further demonstrates that BRD2 C-terminal ends takethe cell apoptosis initiating role in both COS7 and HNE1 cell lines, andare the structural foundation of the BRD2's cell apoptosis initiating role.No bromodomain has been observed in this domain, which suggests thatthe BRD2's cell apoptosis initiating role does not depend on thebomodomain. 2. Influence of BRD2 on the expression of key target molecules inapoptosis pathwaysBy separately transfecting HNE1 cell lines with pEGFP-C3/BRD2,pEGFP-C3/BRD2 C-terminal ends and pEGFP-C3/BRD2 N-terminalends, and testing the expression of key target molecules in apoptosispathways by Western Blotting after they are transiently expressed, wehave found that in HNE1 cell lines where the truncated proteins ofBRD2 and BRD2 C-terminal ends are over-expressed, the expression ofp53, caspase 8 and capase 3 has been significantly up-regulated, whilethat of bcl-2 significantly down-regulated. On the other hand, thetruncated protein of BRD2's N-terminal ends has no significantinfluence on the expression of these key target molecules. Thisdemonstrates that the over-expression of BRD2 and BRD2 C-terminalends can influence at the protein level the expression of key targetmolecules in apoptosis pathways which are mediated by Fas, thusinitiating cell apoptosis.To sum up, on the foundation laid by previous studies which haveidentified BRD2 as an important BRD7-interacting protein, the currentstudy has identified the subcellular localization mode of BRD2 indifferent tumor cell lines or primarily cultivated cell lines; observed that both the truncated proteins of BRD2 N-terminal ends and C-terminalends are located in cell nucleus without showing significant cell typespecificity; found that the 283-335 amino acids of BRD2 take the role ofmediating heterogenic protein GFP for nuclear localization, hence a newnon-typical NLS of BRD2; proved that BRD2 takes the role of cellapoptosis initiating, while its C-terminal ends (aa 428-798) are thestructural foundation of this role, which works without dependence onBRD2's bromodomain; Furthermore, BRD2 and its C-terminal ends canseparately influence the expression level of the key target moleculesp53, bcl-2, caspase-8 and caspase-3 in apoptosis pathways, suggestingthat BRD2 may participate in the cell apoptosis process via theFas-mediated apoptosis pathway.