0 10 Functional functional JJ 11 16 roles role NNS 17 19 of of IN 20 22 in in FW 23 27 vivo vivo FW 28 39 footprinted footprinte VBN 40 43 DNA dna NN 44 50 motifs motif NNS 51 57 within within IN 58 60 an an DT 61 73 alpha-globin alpha-globin NN 74 82 enhancer enhancer NN 82 83 . . . 84 93 Erythroid erythroid JJ 94 101 lineage lineage NN 102 105 and and CC 106 119 developmental developmental JJ 120 125 stage stage NN 126 139 specificities specificity NNS 139 140 . . . 142 157 Transcriptional transcriptional JJ 158 168 regulation regulation NN 169 171 of of IN 172 175 the the DT 176 181 human human JJ 182 192 alpha-like alpha-like JJ 193 199 globin globin NN 200 205 genes gene NNS 205 206 , , , 207 216 embryonic embryonic JJ 217 221 zeta zeta NN 222 223 2 2 CD 224 227 and and CC 228 233 adult adult JJ 234 239 alpha alpha NN 239 240 , , , 241 247 during during IN 248 257 erythroid erythroid JJ 258 269 development development NN 270 272 is be VBZ 273 281 mediated mediate VBN 282 284 by by IN 285 286 a a DT 287 293 distal distal JJ 294 302 enhancer enhancer NN 302 303 , , , 304 309 HS-40 HS-40 NNP 309 310 . . . 311 319 Previous previous JJ 320 331 protein-DNA protein-dna JJ 332 339 binding binding NN 340 347 studies study NNS 348 352 have have VBP 353 358 shown show VBN 359 363 that that IN 364 369 HS-40 HS-40 NNP 370 378 consists consist VBZ 379 381 of of IN 382 390 multiple multiple JJ 391 398 nuclear nuclear JJ 399 405 factor factor NN 406 413 binding binding NN 414 420 motifs motif NNS 421 425 that that WDT 426 429 are be VBP 430 438 occupied occupy VBN 439 441 in in FW 442 446 vivo vivo FW 447 449 in in IN 450 452 an an DT 453 462 erythroid erythroid JJ 463 471 lineage- lineage- NN 472 475 and and CC 476 489 developmental developmental JJ 490 504 stage-specific stage-specific JJ 505 511 manner manner NN 511 512 . . . 513 515 We we PRP 516 520 have have VBP 521 535 systematically systematically RB 536 544 analyzed analyze VBN 545 548 the the DT 549 559 functional functional JJ 560 565 roles role NNS 566 568 of of IN 569 574 these these DT 575 581 factor factor NN 582 589 binding binding NN 590 596 motifs motif NNS 597 599 of of IN 600 605 HS-40 hs-40 NN 606 608 by by IN 609 622 site-directed site-directed JJ 623 634 mutagenesis mutagenesis NN 635 638 and and CC 639 648 transient transient JJ 649 659 expression expression NN 660 665 assay assay NN 666 668 in in IN 669 678 erythroid erythroid JJ 679 683 cell cell NN 684 692 cultures culture NNS 692 693 . . . 694 699 Three three CD 700 702 of of IN 703 708 these these DT 709 714 HS-40 HS-40 NNP 715 723 enhancer enhancer NN 724 730 motifs motif NNS 730 731 , , , 732 743 5’NF-E2/AP1 5'nf-e2/ap1 NN 743 744 , , , 745 747 GT GT NNP 748 750 II ii CD 750 751 , , , 752 755 and and CC 756 765 GATA-1(c) gata-1(c) NN 765 766 , , , 767 777 positively positively RB 778 786 regulate regulate VBP 787 790 the the DT 791 795 zeta zeta NN 796 804 2-globin 2-globin NN 805 813 promoter promoter NN 814 822 activity activity NN 823 825 in in IN 826 841 embryonic/fetal embryonic/fetal JJ 842 851 erythroid erythroid JJ 852 856 K562 k562 NN 857 862 cells cell NNS 863 866 and and CC 867 870 the the DT 871 876 adult adult JJ 877 889 alpha-globin alpha-globin NN 890 898 promoter promoter NN 899 907 activity activity NN 908 910 in in IN 911 916 adult adult JJ 917 926 erythroid erythroid JJ 927 930 MEL MEL NNP 931 936 cells cell NNS 936 937 . . . 938 940 On on IN 941 944 the the DT 945 950 other other JJ 951 955 hand hand NN 955 956 , , , 957 960 the the DT 961 972 3’NF-E2/AP1 3'nf-e2/ap1 NN 973 978 motif motif NN 979 981 is be VBZ 982 986 able able JJ 987 989 to to TO 990 995 exert exert VB 996 1000 both both CC 1001 1009 positive positive JJ 1010 1013 and and CC 1014 1022 negative negative JJ 1023 1033 regulatory regulatory JJ 1034 1041 effects effect NNS 1042 1044 on on IN 1045 1048 the the DT 1049 1053 zeta zeta NN 1054 1062 2-globin 2-globin NN 1063 1071 promoter promoter NN 1072 1080 activity activity NN 1081 1083 in in IN 1084 1088 K562 k562 NN 1089 1094 cells cell NNS 1094 1095 , , , 1096 1099 and and CC 1100 1104 this this DT 1105 1109 dual dual JJ 1110 1118 function function NN 1119 1126 appears appear VBZ 1127 1129 to to TO 1130 1132 be be VB 1133 1142 modulated modulate VBN 1143 1150 through through IN 1151 1163 differential differential JJ 1164 1171 binding binding NN 1172 1174 of of IN 1175 1178 the the DT 1179 1189 ubiquitous ubiquitous JJ 1190 1193 AP1 ap1 NN 1194 1201 factors factor NNS 1202 1205 and and CC 1206 1209 the the DT 1210 1228 erythroid-enriched erythroid-enriched JJ 1229 1234 NF-E2 nf-e2 NN 1235 1241 factor factor NN 1241 1242 . . . 1243 1251 Mutation mutation NN 1252 1254 in in IN 1255 1258 the the DT 1259 1268 GATA-1(d) gata-1(d) NN 1269 1274 motif motif NN 1274 1275 , , , 1276 1281 which which WDT 1282 1290 exhibits exhibit VBZ 1291 1293 an an DT 1294 1299 adult adult JJ 1300 1318 erythroid-specific erythroid-specific JJ 1319 1326 genomic genomic JJ 1327 1336 footprint footprint NN 1336 1337 , , , 1338 1347 decreases decrease VBZ 1348 1351 the the DT 1352 1357 HS-40 hs-40 NN 1358 1366 enhancer enhancer NN 1367 1375 function function NN 1376 1378 in in IN 1379 1387 dimethyl dimethyl JJ 1388 1405 sulfoxide-induced sulfoxide-induced JJ 1406 1409 MEL MEL NNP 1410 1415 cells cell NNS 1416 1419 but but CC 1420 1423 not not RB 1424 1426 in in IN 1427 1431 K562 k562 NN 1432 1437 cells cell NNS 1437 1438 . . . 1439 1444 These these DT 1445 1452 studies study NNS 1453 1457 have have VBP 1458 1465 defined define VBN 1466 1469 the the DT 1470 1480 regulatory regulatory JJ 1481 1486 roles role NNS 1487 1489 of of IN 1490 1493 the the DT 1494 1503 different different JJ 1504 1509 HS-40 hs-40 NN 1510 1516 motifs motif NNS 1516 1517 . . . 1518 1521 The the DT 1522 1532 remarkable remarkable JJ 1533 1544 correlation correlation NN 1545 1552 between between IN 1553 1560 genomic genomic JJ 1561 1573 footprinting footprinting NN 1574 1578 data datum NNS 1579 1582 and and CC 1583 1586 the the DT 1587 1598 mutagenesis mutagenesis NN 1599 1606 results result VBZ 1607 1611 also also RB 1612 1620 suggests suggest VBZ 1621 1625 that that IN 1626 1629 the the DT 1630 1639 erythroid erythroid JJ 1640 1648 lineage- lineage- NN 1649 1652 and and CC 1653 1666 developmental developmental JJ 1667 1681 stage-specific stage-specific JJ 1682 1692 regulation regulation NN 1693 1695 of of IN 1696 1701 human human JJ 1702 1712 alpha-like alpha-like JJ 1713 1719 globin globin NN 1720 1729 promoters promoter NNS 1730 1732 is be VBZ 1733 1739 indeed indeed RB 1740 1749 modulated modulate VBN 1750 1752 by by IN 1753 1759 stable stable JJ 1760 1767 binding binding NN 1768 1770 of of IN 1771 1779 specific specific JJ 1780 1787 nuclear nuclear JJ 1788 1795 factors factor NNS 1796 1798 in in FW 1799 1803 vivo vivo FW 1803 1804 . . .