Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in vivo in activated T-cells

IL-2 gene expression in activated T-cells is initiated by chromatin remodeling at the IL-2 proximal promoter and conversion of a transcriptional repressor into a potent transcriptional activator. A purine-box regulator complex was purified from activated Jurkat T-cell nuclei based on sequence-specific DNA binding to the antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target DNA sequence in the proximal IL-2 promoter. ARRE DNA-binding subunits were identified as NF90, NF45 and systemic lupus erythematosis autoantigens, Ku80 and Ku70. Monoclonal antibodies to Ku80, Ku70 and NF90 specifically inhibit constitutive and inducible ARRE DNA-binding activity in Jurkat T-cells. Ku80, Ku70 and NF90 bind specifically to the IL-2 gene promoter in vivo, as demonstrated by chromatin immunoprecipitation. Activation of Jurkat T-cells and mouse primary spleen cells induces binding of Ku80 and NF90 to the IL-2 promoter in vivo, and decreases binding of Ku70 to the IL-2 promoter in vivo, and these dynamic changes are inhibited by immunosuppressants cyclosporin A and triptolide. Dynamic changes in binding of Ku80, Ku70 and NF90 to the IL-2 proximal promoter in vivo correlate with chromatin remodeling and transcriptional initiation in activated T-cells

Empirical Analysis of Sustainable Trade Effects of FTAs Based on Augmented Gravity Model: A Case Study of China

China has developed many strategies to promote sustainable trade, including accelerating the signing of free-trade agreements (FTAs). However, there is a lack of studies examining the impact of FTAs on the sustainable growth of foreign trade from a holistic perspective. This paper applies an augmented gravity model to verify the trade creation and diversion effects of China’s FTAs that are currently in effect. A panel data analysis over the period of 1995–2019 is conducted on 34 countries and regions, including China, and Poisson pseudo-maximum likelihood (PPML) is employed to test three different fixed-effects models. The results show that the trade effects differ across FTAs, with nine FTAs generally making positive contributions to China’s trade and social welfare growth, and the remaining five FTAs exhibiting varying degrees of trade diversion or contraction effects. Empirically, it is clear that participation in FTAs has contributed to the sustainable trade of China, but China still needs to take prudent action to pursue further trade liberalization in order to counter the potential threat of trade diversion

Model for dynamic binding of Ku80, Ku70, NF90, NF45 and DNA-PKcs to the IL-2 proximal promoter in resting and activated T-cells

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> () In resting T-cells, Ku70 and Ku80 bind to the ARRE sequence in the IL-2 proximal promoter and contribute to a closed chromatin conformation and transcriptional repression. () T-cell activation induces chromatin remodeling associated with decreased binding of Ku70 to the IL-2 promoter and increased binding of NF90 and Ku80 to the IL-2 promoter. Associations between NF45/NF90 heterodimer and Ku70. Ku80 and DNA-PKcs are weakened. IL-2 chromatin remodeling is linked to the conversion of the purine-box regulator from a transcriptional repressor into a potent transcriptional activator, indicated by new interactions of transactivation (TA) domains on NF90 and NF45 with RNA Pol II

Purine-box regulator in T-cell nuclear extracts contains ARRE–DNA-binding subunits Ku80, Ku70 and NF90

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> EMSA of nuclear proteins from NS and PMA + ionomycin (P/I)-stimulated Jurkat T-cells. () Monoclonal antibodies against Ku were preincubated with nuclear proteins before addition of P-labeled oligonucleotide probe: 162 recognizes the native Ku70/Ku80 heterodimer, 111 recognizes Ku80 and N3H10 recognizes Ku70. Polyclonal antibodies against NF-ATp or cytoplasmic phospholipase A2 (cPLA2) did not inhibit binding of purine-box regulator complex, while cyclosporin A (CsA) caused partial inhibition. () Ku monoclonal antibodies did not inhibit induced NF-κB DNA-binding. () Monoclonal antibodies to NF90 (mAB anti-DRBP76), Ku80 and Ku70, but not NFATc1 or NFATc2, specifically inhibited purine-box regulator ARRE–DNA-binding activity in nuclear extracts of nonstimulated and P/I-stimulated Jurkat T-cells

Ku80, Ku70 and NF90 bind specifically and dynamically to the IL-2 promoter

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> Jurkat T-cells or mouse primary spleen cells were NS or stimulated for 4 hr with PMA + ionomycin (S) then nuclear proteins were crosslinked to chromatin with 1% formaldehyde. () Sheared and restricted chromatin was used as template for PCR amplifications (35 cycles) of IL-2 intron 3 (negative control), IL-2 proximal promoter, myc1 (negative control) and myc11 (origin of DNA replication, positive control for Ku binding) sequences. () Ku80 chromatin immunoprecipitation (ChIP) was performed using mAB 111. () Ku70 ChIP was performed using polyclonal antibody. () NF90 ChIP was performed using mAB DRBP76. () Input chromatin from mouse spleen cells was used as template for amplification of IL-2 proximal promoter, IL-2 intron 3 (negative control) and adenosine deaminase origin of DNA replication (positive control for Ku binding). () Ku80 ChIP. () Ku70 ChIP. () NF90 ChIP. () Jurkat T-cells were NS or stimulated for 4 hr with PMA + ionomycin (P/I) or stimulated in the presence of immunosuppressants cyclosporin A (P/I/CsA) or triptolide (P/I/Trip), then IL-2 promoter ChIP was performed, using specific antibodies against Ku80, Ku70 and NF90 and 30 cycles of PCR amplification

Purification of purine-box regulator complexes reveals ARRE–DNA-binding subunits NF90, NF45, Ku80 and Ku70

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> EMSA were used to monitor enrichment of ARRE–DNA-binding complexes from nuclear extracts of Jurkat T-cells stimulated with PMA + Ionomycin. () Diethylaminoethyl (DEAE) elution. Fractions enriched in complex I (and small amounts of complexes II and III) were pooled and dialyzed. () Carboxymethyl (CM) elution. Note the substantial conversion of complex I into complexes II and III. Fractions containing complex I only were pooled and dialyzed. () Octylamine elutions with steps of KCl. Right, oligonucleotide competitions demonstrate greater inhibition of complex I in elution 0.2 with 50 ng of wild type (W) compared to mutant (M) ARRE, yet 50 ng of M does produce substantial inhibition of complex I. () DNA-affinity elution. Octylamine elutions 0.2 M KCl/pool A, enriched in complex I only, were diluted and loaded onto a mutant ARRE–DNA-affinity column and eluted with the indicated steps of KCl. Note the presence of complexes I and III in eluted fraction 0.2, associated with principal protein subunits at 45, 80, 90, 130 and 350 kDa shown by SDS–PAGE with silver staining. Ten nanograms of wild-type (W) ARRE oligonucleotide inhibits complex I and promotes conversion to complex III more potently than mutant (M) oligonucleotide. () DNA-affinity elution. Octylamine elutions enriched in complexes II and III, pool B, were diluted and loaded onto a wild-type ARRE–DNA-affinity column, and maximal DNA-binding activity was eluted at 0.4 M KCl, associated with protein subunits at 70, 80 and 350 kDa shown by SDS–PAGE with silver staining. Internal tryptic peptide sequences of the 70 and 80 kDa proteins identified them as Ku70 and Ku80. Western immunoblot with human JM anti-Ku sera (right) confirms the identity of Ku70 and Ku80 (lane 4), and suggests the slowest migrating protein to be DNA-dependent protein kinase (DPK) catalytic subunit

Association of NF45 and NF90 with Ku80, Ku70 and DNA-dependent protein kinase is altered during T-cell activation

<p><b>Copyright information:</b></p><p>Taken from "Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in activated T-cells"</p><p></p><p>Nucleic Acids Research 2007;35(7):2302-2310.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874627.</p><p>© 2007 The Author(s)</p> Jurkat T-cells were either NS, stimulated with PMA + ionomycin (P/I) for 4 hr or stimulated in the presence of CsA (P/I/CsA, 1000 ng/ml) or triptolide (P/I/Trip, 1000 ng/ml). Extracted nuclear proteins (lane 1) were co-immunoprecipitated with protein A beads (control, lane 2) or immobilized antibody against NF45 (lanes 3–6) and the immunoprecipitates (IP) were washed at moderate stringency and analyzed by Western immunoblotting. The observation of decreased association of Ku70 and Ku80 with NF45 in activated T-cells was reproduced more than 6×