0 1 A a DT 2 10 critical critical JJ 11 15 role role NN 16 18 of of IN 19 23 Sp1- sp1- NN 24 27 and and CC 28 39 Ets-related ets-related JJ 40 53 transcription transcription NN 54 61 factors factor NNS 62 64 in in IN 65 76 maintaining maintain VBG 77 89 CTL-specific ctl-specific JJ 90 100 expression expression NN 101 103 of of IN 104 107 the the DT 108 113 mouse mouse NN 114 122 perforin perforin NN 123 127 gene gene NN 127 128 . . . 130 134 This this DT 135 140 study study NN 141 144 was be VBD 145 153 designed design VBN 154 156 to to TO 157 166 determine determine VB 167 170 the the DT 171 180 potential potential JJ 181 193 cis-elements cis-element NNS 194 202 involved involve VBN 203 205 in in IN 206 221 transcriptional transcriptional JJ 222 232 regulation regulation NN 233 235 of of IN 236 239 the the DT 240 245 mouse mouse NN 246 254 perforin perforin NN 255 259 gene gene NN 259 260 . . . 261 266 DNase dnase NN 267 268 I i CD 269 283 hypersensitive hypersensitive JJ 284 288 site site NN 289 290 ( ( ( 290 293 DHS DHS NNP 293 294 ) ) ) 295 302 mapping mapping NN 303 311 revealed reveal VBD 312 316 that that IN 317 320 the the DT 321 329 perforin perforin NN 330 335 locus locus NN 336 345 contained contain VBD 346 349 six six CD 350 353 DHS dhs NN 354 360 within within IN 361 364 7.0 7.0 CD 365 367 kb kb NN 368 370 of of IN 371 374 the the DT 375 376 5 5 CD 376 377 ’ ' SYM 378 386 upstream upstream JJ 387 395 sequence sequence NN 396 397 ( ( ( 397 401 -7.0 -7.0 CD 402 404 kb kb NN 404 405 ) ) ) 406 409 and and CC 410 413 two two CD 414 417 DHS dhs NN 418 420 in in IN 421 427 intron intron NN 428 429 2 2 CD 429 430 . . . 431 434 The the DT 435 438 six six CD 439 440 5 5 CD 440 441 ’ ' SYM 442 450 upstream upstream JJ 451 454 and and CC 455 458 one one CD 459 467 intronic intronic JJ 468 471 DHS dhs NN 472 476 were be VBD 477 485 detected detect VBN 486 488 in in IN 489 493 only only RB 494 513 perforin-expressing perforin-expressing JJ 514 525 lymphocytes lymphocyte NNS 525 526 . . . 527 542 Chloramphenicol Chloramphenicol NNP 543 560 acetyltransferase acetyltransferase NNP 561 562 ( ( ( 562 565 CAT CAT NNP 565 566 ) ) ) 567 577 activities activity NNS 578 586 directed direct VBN 587 589 by by IN 590 591 5 5 CD 591 592 ’ ' SYM 593 601 upstream upstream JJ 602 610 promoter promoter NN 611 615 were be VBD 616 624 detected detect VBN 625 639 preferentially preferentially RB 640 642 in in IN 643 662 perforin-expressing perforin-expressing JJ 663 667 cell cell NN 668 673 lines line NNS 673 674 . . . 675 676 A a DT 677 686 construct construct NN 687 693 termed term VBN 694 699 PFP5a PFP5a NNP 700 710 containing contain VBG 711 715 -795 -795 CD 716 718 bp bp NN 719 728 exhibited exhibit VBD 729 732 the the DT 733 740 highest high JJS 741 744 CAT CAT NNP 745 753 activity activity NN 753 754 , , , 755 758 and and CC 759 766 PFP9a20 PFP9a20 NNP 767 777 containing contain VBG 778 782 only only RB 783 786 -73 -73 CD 787 789 bp bp NN 790 794 also also RB 795 803 produced produce VBD 804 817 significantly significantly RB 818 822 high high JJ 823 826 CAT CAT NNP 827 835 activity activity NN 836 838 in in IN 839 846 CTLL-R8 ctll-r8 NN 847 852 cells cell NNS 852 853 . . . 854 857 The the DT 858 866 proximal proximal JJ 867 873 region region NN 874 876 in in IN 877 884 PFP9a20 PFP9a20 NNP 885 894 contained contain VBD 895 898 two two CD 899 908 potential potential JJ 909 912 Sp1 sp1 NN 913 920 binding binding NN 921 926 sites site NNS 927 928 ( ( ( 928 930 GC GC NNP 931 934 box box NN 935 938 and and CC 939 941 GT gt NN 942 945 box box NN 945 946 ) ) ) 947 950 and and CC 951 954 one one CD 955 958 Ets ets NN 959 966 binding binding NN 967 971 site site NN 972 973 ( ( ( 973 976 EBS EBS NNP 976 977 ) ) ) 977 978 . . . 979 994 Electrophoretic electrophoretic JJ 995 1003 mobility mobility NN 1004 1009 shift shift NN 1010 1015 assay assay NN 1016 1022 showed show VBD 1023 1027 that that IN 1028 1032 each each DT 1033 1035 of of IN 1036 1039 the the DT 1040 1052 cis-elements cis-element NNS 1053 1058 bound bind VBD 1059 1067 specific specific JJ 1068 1075 protein protein NN 1076 1083 factors factor NNS 1083 1084 . . . 1085 1089 When when WRB 1090 1102 single-point single-point JJ 1103 1111 mutation mutation NN 1112 1115 was be VBD 1116 1126 introduced introduce VBN 1127 1129 to to TO 1130 1134 each each DT 1135 1137 GC GC NNP 1138 1141 box box NN 1141 1142 , , , 1143 1146 EBS EBS NNP 1146 1147 , , , 1148 1151 and and CC 1152 1154 GT GT NNP 1155 1158 box box NN 1159 1161 in in IN 1162 1169 PFP9a20 PFP9a20 NNP 1169 1170 , , , 1171 1173 at at IN 1174 1179 least least JJS 1180 1186 3-fold 3-fold RB 1187 1191 less less JJR 1192 1195 CAT CAT NNP 1196 1204 activity activity NN 1205 1208 was be VBD 1209 1217 observed observe VBN 1218 1220 in in IN 1221 1228 CTLL-R8 ctll-r8 NN 1229 1234 cells cell NNS 1234 1235 . . . 1236 1238 To to TO 1239 1246 confirm confirm VB 1247 1250 the the DT 1251 1261 importance importance NN 1262 1264 of of IN 1265 1268 the the DT 1269 1274 three three CD 1275 1285 cis-acting cis-acting JJ 1286 1294 elements element NNS 1295 1297 in in IN 1298 1301 the the DT 1302 1310 perforin perforin NN 1311 1315 gene gene NN 1316 1326 expression expression NN 1326 1327 , , , 1328 1333 point point NN 1334 1342 mutation mutation NN 1343 1346 was be VBD 1347 1357 introduced introduce VBN 1358 1363 again again RB 1364 1366 to to TO 1367 1371 each each DT 1372 1380 proximal proximal JJ 1381 1383 GC GC NNP 1384 1387 box box NN 1387 1388 , , , 1389 1392 EBS EBS NNP 1392 1393 , , , 1394 1397 and and CC 1398 1400 GT GT NNP 1401 1404 box box NN 1405 1407 of of IN 1408 1413 PFP5a PFP5a NNP 1413 1414 . . . 1415 1418 The the DT 1419 1424 point point NN 1425 1434 mutations mutation NNS 1435 1443 resulted result VBD 1444 1446 in in IN 1447 1448 a a DT 1449 1453 2.5- 2.5- CD 1454 1456 to to TO 1457 1463 3-fold 3-fold JJ 1464 1473 reduction reduction NN 1474 1476 of of IN 1477 1480 CAT CAT NNP 1481 1489 activity activity NN 1489 1490 . . . 1491 1494 The the DT 1495 1502 results result NNS 1503 1510 suggest suggest VBP 1511 1515 that that IN 1516 1517 a a DT 1518 1529 combination combination NN 1530 1532 of of IN 1533 1536 the the DT 1537 1542 three three CD 1543 1551 proximal proximal JJ 1552 1562 cis-acting cis-acting JJ 1563 1571 elements element NNS 1572 1575 may may MD 1576 1586 constitute constitute VB 1587 1588 a a DT 1589 1596 minimal minimal JJ 1597 1603 region region NN 1604 1615 responsible responsible JJ 1616 1619 for for IN 1620 1632 CTL-specific ctl-specific JJ 1633 1643 expression expression NN 1644 1646 of of IN 1647 1655 perforin perforin NN 1655 1656 . . .