0 20 Activation-dependent activation-dependent JJ 21 36 transcriptional transcriptional JJ 37 47 regulation regulation NN 48 50 of of IN 51 54 the the DT 55 60 human human JJ 61 64 Fas Fas NNP 65 73 promoter promoter NN 74 82 requires require VBZ 83 92 NF-kappaB NF-kappaB NNP 93 100 p50-p65 p50-p65 NN 101 112 recruitment recruitment NN 112 113 . . . 115 118 Fas Fas NNP 119 120 ( ( ( 120 124 CD95 CD95 NNP 124 125 ) ) ) 126 129 and and CC 130 133 Fas Fas NNP 134 140 ligand ligand NN 141 142 ( ( ( 142 147 CD95L CD95L NNP 147 148 ) ) ) 149 152 are be VBP 153 155 an an DT 156 167 interacting interact VBG 168 183 receptor-ligand receptor-ligand JJ 184 188 pair pair NN 189 197 required require VBN 198 201 for for IN 202 208 immune immune JJ 209 220 homeostasis homeostasis NN 220 221 . . . 222 232 Lymphocyte Lymphocyte NNP 233 243 activation activation NN 244 251 results result VBZ 252 254 in in IN 255 258 the the DT 259 271 upregulation upregulation NN 272 274 of of IN 275 278 Fas Fas NNP 279 289 expression expression NN 290 293 and and CC 294 297 the the DT 298 309 acquisition acquisition NN 310 312 of of IN 313 324 sensitivity sensitivity NN 325 327 to to TO 328 341 FasL-mediated fasl-mediated JJ 342 351 apoptosis apoptosis NN 351 352 . . . 353 361 Although although IN 362 365 Fas Fas NNP 366 378 upregulation upregulation NN 379 381 is be VBZ 382 389 central central JJ 390 392 to to TO 393 396 the the DT 397 409 preservation preservation NN 410 412 of of IN 413 424 immunologic immunologic JJ 425 434 tolerance tolerance NN 434 435 , , , 436 442 little little JJ 443 445 is be VBZ 446 451 known know VBN 452 457 about about IN 458 461 the the DT 462 471 molecular molecular JJ 472 481 machinery machinery NN 482 492 underlying underlie VBG 493 497 this this DT 498 505 process process NN 505 506 . . . 507 509 To to TO 510 521 investigate investigate VB 522 525 the the DT 526 532 events event NNS 533 541 involved involve VBN 542 544 in in IN 545 563 activation-induced activation-induced JJ 564 567 Fas Fas NNP 568 580 upregulation upregulation NN 580 581 , , , 582 584 we we PRP 585 589 have have VBP 590 598 examined examine VBN 599 603 mRNA mrna NN 604 616 accumulation accumulation NN 616 617 , , , 618 621 fas fas NN 622 630 promoter promoter NN 631 639 activity activity NN 639 640 , , , 641 644 and and CC 645 652 protein protein NN 653 663 expression expression NN 664 666 in in IN 667 670 the the DT 671 677 Jurkat Jurkat NNP 678 684 T-cell T-cell NNP 685 689 line line NN 690 697 treated treat VBN 698 702 with with IN 703 710 phorbol phorbol NN 711 720 myristate myristate NN 721 728 acetate acetate NN 729 732 and and CC 733 742 ionomycin ionomycin NN 743 744 ( ( ( 744 747 P/I P/I NNP 747 748 ) ) ) 748 749 , , , 750 765 pharmacological pharmacological JJ 766 772 mimics mimic NNS 773 775 of of IN 776 782 T-cell t-cell NN 783 791 receptor receptor NN 792 802 activation activation NN 802 803 . . . 804 812 Although although IN 813 820 resting rest VBG 821 827 Jurkat Jurkat NNP 828 833 cells cell NNS 834 841 express express VBP 842 845 Fas Fas NNP 845 846 , , , 847 850 Fas Fas NNP 851 855 mRNA mRNA NNP 856 859 was be VBD 860 867 induced induce VBN 868 881 approximately approximately RB 882 889 10-fold 10-fold RB 890 892 in in IN 893 894 2 2 CD 895 896 h h NN 897 901 upon upon IN 902 905 P/I P/I NNP 906 917 stimulation stimulation NN 917 918 . . . 919 924 Using use VBG 925 935 sequential sequential JJ 936 944 deletion deletion NN 945 952 mutants mutant NNS 953 955 of of IN 956 959 the the DT 960 965 human human JJ 966 969 fas fas NN 970 978 promoter promoter NN 979 981 in in IN 982 991 transient transient JJ 992 1004 transfection transfection NN 1005 1011 assays assay NNS 1011 1012 , , , 1013 1015 we we PRP 1016 1026 identified identify VBD 1027 1028 a a DT 1029 1034 47-bp 47-bp JJ 1035 1043 sequence sequence NN 1044 1045 ( ( ( 1045 1054 positions position NNS 1055 1059 -306 -306 CD 1060 1062 to to TO 1063 1067 -260 -260 CD 1068 1076 relative relative JJ 1077 1079 to to TO 1080 1083 the the DT 1084 1087 ATG ATG NNP 1087 1088 ) ) ) 1089 1097 required require VBN 1098 1101 for for IN 1102 1119 activation-driven activation-driven JJ 1120 1123 fas fas NN 1124 1136 upregulation upregulation NN 1136 1137 . . . 1138 1146 Sequence sequence NN 1147 1155 analysis analysis NN 1156 1164 revealed reveal VBD 1165 1168 the the DT 1169 1177 presence presence NN 1178 1180 of of IN 1181 1182 a a DT 1183 1193 previously previously RB 1194 1206 unrecognized unrecognized JJ 1207 1216 composite composite JJ 1217 1224 binding binding NN 1225 1229 site site NN 1230 1233 for for IN 1234 1238 both both CC 1239 1242 the the DT 1243 1246 Sp1 sp1 NN 1247 1250 and and CC 1251 1260 NF-kappaB NF-kappaB NNP 1261 1274 transcription transcription NN 1275 1282 factors factor NNS 1283 1285 at at IN 1286 1295 positions position NNS 1296 1300 -295 -295 CD 1301 1303 to to TO 1304 1308 -286 -286 CD 1308 1309 . . . 1310 1325 Electrophoretic electrophoretic JJ 1326 1334 mobility mobility NN 1335 1340 shift shift NN 1341 1346 assay assay NN 1347 1348 ( ( ( 1348 1352 EMSA EMSA NNP 1352 1353 ) ) ) 1354 1357 and and CC 1358 1368 supershift supershift NN 1369 1377 analyses analysis NNS 1378 1380 of of IN 1381 1385 this this DT 1386 1392 region region NN 1393 1403 documented document VBD 1404 1416 constitutive constitutive JJ 1417 1424 binding binding NN 1425 1427 of of IN 1428 1431 Sp1 sp1 NN 1432 1434 in in IN 1435 1446 unactivated unactivated JJ 1447 1454 nuclear nuclear JJ 1455 1463 extracts extract NNS 1464 1467 and and CC 1468 1477 inducible inducible JJ 1478 1485 binding binding NN 1486 1488 of of IN 1489 1496 p50-p65 p50-p65 NN 1497 1506 NF-kappaB NF-kappaB NNP 1507 1519 heterodimers heterodimer NNS 1520 1525 after after IN 1526 1529 P/I P/I NNP 1530 1540 activation activation NN 1540 1541 . . . 1542 1545 Sp1 Sp1 NNP 1546 1549 and and CC 1550 1559 NF-kappaB NF-kappaB NNP 1560 1573 transcription transcription NN 1574 1580 factor factor NN 1581 1588 binding binding NN 1589 1592 was be VBD 1593 1598 shown show VBN 1599 1601 to to TO 1602 1604 be be VB 1605 1613 mutually mutually RB 1614 1623 exclusive exclusive JJ 1624 1626 by by IN 1627 1631 EMSA EMSA NNP 1632 1644 displacement displacement NN 1645 1652 studies study NNS 1653 1657 with with IN 1658 1666 purified purify VBN 1667 1678 recombinant recombinant JJ 1679 1682 Sp1 sp1 NN 1683 1686 and and CC 1687 1698 recombinant recombinant JJ 1699 1702 p50 p50 NN 1702 1703 . . . 1704 1707 The the DT 1708 1718 functional functional JJ 1719 1731 contribution contribution NN 1732 1734 of of IN 1735 1738 the the DT 1739 1749 kappaB-Sp1 kappab-sp1 NN 1750 1759 composite composite JJ 1760 1764 site site NN 1765 1767 in in IN 1768 1781 P/I-inducible p/i-inducible JJ 1782 1785 fas fas NN 1786 1794 promoter promoter NN 1795 1805 activation activation NN 1806 1809 was be VBD 1810 1818 verified verify VBN 1819 1821 by by IN 1822 1827 using use VBG 1828 1838 kappaB-Sp1 kappab-sp1 NN 1839 1850 concatamers concatamer NNS 1851 1852 ( ( ( 1852 1856 -295 -295 CD 1857 1859 to to TO 1860 1864 -286 -286 CD 1864 1865 ) ) ) 1866 1868 in in IN 1869 1870 a a DT 1871 1880 thymidine thymidine NN 1881 1887 kinase kinase NNP 1888 1903 promoter-driven promoter-driven JJ 1904 1912 reporter reporter NN 1913 1922 construct construct NN 1923 1926 and and CC 1927 1933 native native JJ 1934 1942 promoter promoter NN 1943 1953 constructs construct NNS 1954 1956 in in IN 1957 1963 Jurkat Jurkat NNP 1964 1969 cells cell NNS 1970 1984 overexpressing overexpresse VBG 1985 1998 IkappaB-alpha IkappaB-alpha NNP 1998 1999 . . . 2000 2013 Site-directed site-directed JJ 2014 2025 mutagenesis mutagenesis NN 2026 2028 of of IN 2029 2032 the the DT 2033 2041 critical critical JJ 2042 2049 guanine guanine NN 2050 2061 nucleotides nucleotide NNS 2062 2064 in in IN 2065 2068 the the DT 2069 2079 kappaB-Sp1 kappab-sp1 NN 2080 2087 element element NN 2088 2098 documented document VBD 2099 2102 the the DT 2103 2112 essential essential JJ 2113 2117 role role NN 2118 2120 of of IN 2121 2125 this this DT 2126 2130 site site NN 2131 2133 in in IN 2134 2154 activation-dependent activation-dependent JJ 2155 2158 fas fas NN 2159 2167 promoter promoter NN 2168 2177 induction induction NN 2177 2178 . . .