Transcription Regulation Mechanisms Of HTLV-1Virally Encoded Tax Protein On HMGB1Gene In T Lymphocytes

Objective:To observe human HMGB1mRNA and protein expression levels in different T lymphocytes (HTLV-1+, HTLV-1-or Tax+, Tax" T cells). This study also aimed to elucidate whether HTLV-1virally encoded Tax protein inflences HMGB1expression in the T lymphocytes.Methods:Total RNA and protein were extracted from HTLV-1+T cells (MT-4, MT-2), Tax+T cells (TaxP) and Tax" T cells (Jurkat, TaxN), as well as Jurkat cells transfected transiently with expression vector pCMV-Tax or its mutants (M22, M47) for24h. To evaluate whether Tax protein inflences HMGB1expression levels in T lymphocytes, total RNA was then reversely transcribed into cDNA to amplify human HMGB1mRNA by reverse transcription PCR (RT-PCR) and real time quantitative PCR (RT-qPCR), and HMGB1protein levels were also detected by western blot assays in different T lymphocytes and Jurkat cells transfected transiently with pCMV-Tax or its mutants (M22, M47).Results:HMGB1transcriptional activity and protein expression levels in Tax+T cells (TaxP) were significantly higher than that in Tax-T cells (Jurkat, TaxN), and HMGB1mRNA expression levels was slightly lower in HTLV-1+T cells (MT-4, MT-2) than that in HTLV-1-T cells (TaxP, Jurkat), however, the HMGB1protein did not change significantly between Jurkat cells and HTLV-1+T cells (MT-4, MT-2). Additionally, the results of transient transfection with Tax or its mutants (M22, M47) also showed that Tax protein enhanced HMGB1mRNA and protein expression in Jurkat cells.Conclusions:HTLV-1virally encoded Tax protein probably functions as a transcriptional activator through the interaction between Tax and a transcriptional regulator to enhance HMGB1gene transcription and protein expression. The other proteins of the HTLV-1virus discover a phenomenon of slight inhibition on HMGB1gene expression. These findings would provide a foundation for further research about regulation mechanisms of Tax protein on HMGB1gene. Objective:To construct respectively different luciferase reporter plasmids of human HMGB1regulation genes and their stable sublines of Jurkat cells. This study aimed to conduct transcriptional regulation mechanisms of Tax protein on HMGB1gene in the T cells.Methods:HMGB1gene regulatory sequences (-83to+83,-383to+83,-688to+83,-975to+83,-1163to+83,-1327to+83,-1520to+83) were amplified by PCR with DNA as template from Jurkat cells, in which3(?)-flanking regions were fixed. HMGB1regulatory genes were ligated into pMD18-T vectors respectively and then transformed into E.coli strain DH5a. The single ampicillin-selected DH5a clone was picked for culturing overnight and plasmid extraction. The extracted plasmids were digested with the desired restriction enzymes of Kpn Ⅰ/HindⅢ or BamH Ⅰ/HindⅢ for double digestion identification. The correctness of HMGB1regulatory sequence was confirmed with DNA sequencing. The positive clones were then ligated into reporter vector pGL3-neo-luc to construct a series of deletion reporter plasmids (pGL3-HMGB1-luc). Similarly,-1520-+83HMGB1gene was digested with Xho Ⅰ/HindⅢ to obtain-504to+83region, and this region was ligated into reporter vector pGL3-neo-luc to form a pGL3-HMGB1-luc plasmid containing-504to+83HMGB1region. Furthermore, different reporter plasmids (pGL3-HMGB1-luc and pGL3-neo-luc) were transfected transiently into Jurkat cells for48h and then were screened with600μg/ml G418for20d, and a single positive clone was sustained screening with300μg/ml for2～3months. Following, stable strains were confirmed by luciferase activity. Results:Seven of HMGB1regulatory genes (1603bp,1410bp,1246bp,1058bp,771bp,466bp,166bp) were cloned with DNA from Jurkat cells successfully, and a587bp HMGB1frgment (-504to+83) was obtained by Xho I/HindⅢ from1603bp frgment, in which3(?)-flanking regions were fixed, and ligated into reporter vector pGL3-neo-luc to form pGL3-HMGB1-luc plasmids, which were designated pHLuc1to pHLuc8according to the length of HMGB1gene from short to long, respectively corresponding to-83to+83,-383to+83,-504to+83,-688to+83,-975to+83,-1163to+83,-1327to+83,-1520to+83HMGB1region. Jurkat cells transfected transiently with reporter plasmids (pHLucl-pHLuc8or pGL3-neo-luc) were screened respectively by drug G418to construct successfully NEO cells and HJ1-HJ8cells, according to reporter plasmids pGL3-neo-luc and pHLucl-pHLuc8.Conclusions:We successfully constructed HMGB1reporter gene plasmids and their HJ stable cell lines. This will lay the foundation for finding HMGB1promoter region and exploring the roles or mechanisms of HMGB1gene regulatory genes used in adult T-cell leukemia. Objective:To observe human HMGB1promoter activity in different T lymphocytes and explore the effect of HTLV-1virally encoded Tax protein on HMGB1gene transcription.Methods:Various pGL3-HMGB1-luc reporter plasmids and pGL3-neo-luc (control group) were transfected transiently into THP1, Hela, Jurkat, TaxP, TaxN, MT-4and MT-2cells for24h, additionally, pGL3-HMGB1-luc reporter plasmids and pCMV-Tax or its mutants (M22, M47) were co-transfected transiently into Jurkat cells for24h, as well as pCMV-Neo as the control. Then, luciferase activity was detected and the relative luciferase activity (the ratio of the luciferase activity, HMGB1/neo in different cells or Tax/Neo in Tax+and Tax-T cells) was compared. Additionally, Tax or its mutants (M22, M47) were transfected transiently into HJ stable cell lines (HJ1-HJ8cells), together with pCMV-Neo as the control, and luciferase activity was detected and the relative luciferase activity (Tax/Neo) in different HJ sublines was calculated. Furthermore, by retroviral transfection in contact with cell adhesion, we developed respectively an in vitro co-culture system comprising HTLV-1+T cells (MT-2) and HJ stable cells, at1×105and4×105per well in24-well plates for24h, together with Hut-78cells (uninfected T cell line) as a negative control. Followingly, all cells were harvested to assay the relative luciferase activity of different HJ stable lines [(MT-2)/(Hut-78)]. Taken together, we obversed whether there was regulation differences of Tax protein on transient HMGB1genes and stable HMGB1genes. Finally, by the mediation of BAY11-7082(NF-κB inhibitor), we treated Jurkat cells transfected with Tax or its mutants (M22, M47) together with pGL3-HMGB1-luc reporter plasmids and TaxP cells transfected with pGL3-HMGB1-luc reporter plasmids. Results:The regulation trend of HMGB1gene was slightly similar in different types of cells, but not identical and the maximal promoter activity in plasmid pHLuc3containing-504to+83HMGB1fragment. Interestingly, transcriptional activity of HMGB1gene in HTLV-1+T cells (MT-4, MT-2) was slightly lower than HTLV-1-T cells (Jurkat and TaxP). Tax protein affected the transcriptional regulation of HMGB1gene probably on-1520～-975region in Tax+T cells (TaxP) in comparison with Tax" T cells (TaxN). Additionally, the results of co-transfection with pGL3-HMGB1-luc reporter plasmids and pCMV-Tax into Jurkat cells also showed that Tax protein promoted the transcriptional expression of HMGB1gene from plasmid pHLuc6-pHLuc8, but not on the-975～+83HMGB1gene from plasmid pHLuc1-pHLuc5. And the co-expression of Tax significantly increased HMGB1promoter activity on plasmid pHLuc6in a dose-dependent manner. Furthermore, the results of Tax or its mutants (M22, M47) with pHLuc3or pHLuc6into Jurkat cells also discovered that Tax protein did not affect transcription of-504～+83HMGB1gene on the pHLuc3group, however, enhanced HMGB1transcription on the pHLuc6group. But, BAY11-7082could not inhibit HMGB1transcriptional activity up-regulated by Tax protein.Conclusions:The region between nt-504and-383is essential for basal promoter activity, Tax protein probably enriches on-1163～975HMGB1region to enhance its transcription, but via non-NF-KB pathway. Objective:To investigate Tax protein enriching on HMGB1sites, cis-elements and being in interaction with transcription factors in T lymphocytes.Methods:TaxP cells were fixed and broken open, and sonication was performed to shear the chromatin to a manageable size. By chromatin immunoprecipitation (ChIP), purified DNA was obtained. Using the specific primers, we conducted real time quantitative PCR to quantify the fold enrichment levels of Tax protein in two separate portions of the HMGB1gene centered at-1103(the fragment encompassing-1163to-1043) and-797(the fragment spanning-848to-746) with respect to the site of Tax-enhancing HMGB1transcription. By bioinformatics, cis-elements for transcription factors between-1163to-975on human HMGB1gene were analyzed, a deletion mutation was each performed for transcription factors by using wt pHLuc6to generate mut reporter plasmids, each deleting the corresponding cis-element. PCMV-Tax and pHLuc6or its mutants were co-transfected transiently into Jurkat cells, as well as a significant mut pHLuc6together with pCMV-Tax (pCMV-Neo as the control) into Jurkat cells. After24h, luciferase activity was detected, transcription regulation differences of Tax protein on HMGB1gene were compared between mut pHLuc6and wt pHLuc6for a key transcription factor.Results:ChIPed DNA was amplified for the regions centered at-1103and-797by RT-qPCR, with respect to the site of Tax-enhancing HMGB1transcription. We found that stronger Tax enrichment at the-1103site than that at the-797site. In a word, Tax protein was recruited in the-1163to-975HMGB1region. By bioinformatics, we found that-1163to-975HMGB1region revealed cis-elements for the following transcription factors:CdxA, AP-1, AML-la, USF, C/EBP and v-Myb. Then, using wt pHLuc6, we each performed a deletion mutation for the corresponding cis-element to generate six mut reporter plasmids, designated as mLuc6-AM for AML-la, mLuc6-AP for AP-1, mLuc6-Cd for CdxA, mLuc6-U for USF, mLuc6-M for v-Myb, and mLuc6-C for C/EBP, respectively. As shown by the luciferase activity assays, the mutation in the C/EBP-motif (mLuc6-C) reduced the transcription activity of-1163-+83HMGB1gene (pHLuc6) to52%in Jurkat cells transfected with pCMV-Tax and wt pHLuc6or its mumants (mut pHLuc6). Tax protein did not affect mut HMGB1(mLuc6-C) transcription in Jurkat cells.Conclusions:Tax protein may be recruited in the-1163to-975HMGB1region to enhance transcription by interaction with C/EBP. Transcriptional regulation mechanisms of Tax protein on HMGB1gene will provide a new approach for the clinical therapy of ATL.