0 4 PU.1 PU.1 NNP 5 6 ( ( ( 6 11 Spi-1 Spi-1 NNP 11 12 ) ) ) 13 16 and and CC 17 22 C/EBP C/EBP NNP 23 28 alpha alpha NN 29 37 regulate regulate VBP 38 48 expression expression NN 49 51 of of IN 52 55 the the DT 56 78 granulocyte-macrophage granulocyte-macrophage JJ 79 97 colony-stimulating colony-stimulating JJ 98 104 factor factor NN 105 113 receptor receptor NN 114 119 alpha alpha NN 120 124 gene gene NN 124 125 . . . 127 133 Growth growth NN 134 140 factor factor NN 141 150 receptors receptor NNS 151 155 play play VBP 156 158 an an DT 159 168 important important JJ 169 173 role role NN 174 176 in in IN 177 190 hematopoiesis hematopoiesis NN 190 191 . . . 192 194 In in IN 195 200 order order NN 201 203 to to TO 204 211 further further RB 212 222 understand understand VB 223 226 the the DT 227 237 mechanisms mechanism NNS 238 247 directing direct VBG 248 251 the the DT 252 262 expression expression NN 263 265 of of IN 266 271 these these DT 272 275 key key JJ 276 286 regulators regulator NNS 287 289 of of IN 290 303 hematopoiesis hematopoiesis NN 303 304 , , , 305 307 we we PRP 308 317 initiated initiate VBD 318 319 a a DT 320 325 study study NN 326 339 investigating investigate VBG 340 343 the the DT 344 357 transcription transcription NN 358 365 factors factor NNS 366 376 activating activate VBG 377 380 the the DT 381 391 expression expression NN 392 394 of of IN 395 398 the the DT 399 421 granulocyte-macrophage granulocyte-macrophage JJ 422 440 colony-stimulating colony-stimulating JJ 441 447 factor factor NN 448 449 ( ( ( 449 455 GM-CSF GM-CSF NNP 455 456 ) ) ) 457 465 receptor receptor NN 466 471 alpha alpha NN 472 476 gene gene NN 476 477 . . . 478 482 Here here RB 482 483 , , , 484 486 we we PRP 487 498 demonstrate demonstrate VBP 499 503 that that IN 504 507 the the DT 508 513 human human JJ 514 520 GM-CSF GM-CSF NNP 521 529 receptor receptor NN 530 535 alpha alpha NN 536 544 promoter promoter NN 545 552 directs direct VBZ 553 561 reporter reporter NN 562 566 gene gene NN 567 575 activity activity NN 576 578 in in IN 579 580 a a DT 581 596 tissue-specific tissue-specific JJ 597 604 fashion fashion NN 605 607 in in IN 608 622 myelomonocytic myelomonocytic JJ 623 628 cells cell NNS 628 629 , , , 630 635 which which WDT 636 646 correlates correlate VBZ 647 651 with with IN 652 655 its its PRP$ 656 666 expression expression NN 667 674 pattern pattern NN 675 677 as as IN 678 686 analyzed analyze VBN 687 689 by by IN 690 697 reverse reverse JJ 698 711 transcription transcription NN 712 715 PCR pcr NN 715 716 . . . 717 720 The the DT 721 727 GM-CSF GM-CSF NNP 728 736 receptor receptor NN 737 742 alpha alpha NN 743 751 promoter promoter NN 752 760 contains contain VBZ 761 763 an an DT 764 773 important important JJ 774 784 functional functional JJ 785 789 site site NN 790 797 between between IN 798 807 positions position NNS 808 811 -53 -53 CD 812 815 and and CC 816 819 -41 -41 CD 820 822 as as IN 823 833 identified identify VBN 834 836 by by IN 837 845 deletion deletion NN 846 854 analysis analysis NN 855 857 of of IN 858 866 reporter reporter NN 867 877 constructs construct NNS 877 878 . . . 879 881 We we PRP 882 886 show show VBP 887 891 that that IN 892 895 the the DT 896 903 myeloid myeloid JJ 904 907 and and CC 908 909 B B NNP 910 914 cell cell NN 915 928 transcription transcription NN 929 935 factor factor NN 936 940 PU.1 pu.1 NN 941 946 binds bind VBZ 947 959 specifically specifically RB 960 962 to to TO 963 967 this this DT 968 972 site site NN 972 973 . . . 974 985 Furthermore furthermore RB 985 986 , , , 987 989 we we PRP 990 1001 demonstrate demonstrate VBP 1002 1006 that that IN 1007 1008 a a DT 1009 1014 CCAAT CCAAT NNP 1015 1019 site site NN 1020 1027 located located JJ 1028 1036 upstream upstream RB 1037 1039 of of IN 1040 1043 the the DT 1044 1048 PU.1 pu.1 NN 1049 1053 site site NN 1054 1061 between between IN 1062 1071 positions position NNS 1072 1075 -70 -70 CD 1076 1079 and and CC 1080 1083 -54 -54 CD 1084 1086 is be VBZ 1087 1095 involved involve VBN 1096 1098 in in IN 1099 1116 positive-negative positive-negative JJ 1117 1127 regulation regulation NN 1128 1130 of of IN 1131 1134 the the DT 1135 1141 GM-CSF GM-CSF NNP 1142 1150 receptor receptor NN 1151 1156 alpha alpha NN 1157 1165 promoter promoter NN 1166 1174 activity activity NN 1174 1175 . . . 1176 1181 C/EBP C/EBP NNP 1182 1187 alpha alpha NN 1188 1190 is be VBZ 1191 1194 the the DT 1195 1200 major major JJ 1201 1223 CCAAT/enhancer-binding ccaat/enhancer-binding JJ 1224 1231 protein protein NN 1232 1233 ( ( ( 1233 1238 C/EBP C/EBP NNP 1238 1239 ) ) ) 1240 1244 form form NN 1245 1252 binding binding NN 1253 1255 to to TO 1256 1260 this this DT 1261 1265 site site NN 1266 1268 in in IN 1269 1276 nuclear nuclear JJ 1277 1285 extracts extract NNS 1286 1288 of of IN 1289 1293 U937 u937 NN 1294 1299 cells cell NNS 1299 1300 . . . 1301 1306 Point Point NNP 1307 1316 mutations mutation NNS 1317 1319 of of IN 1320 1326 either either CC 1327 1330 the the DT 1331 1335 PU.1 pu.1 NN 1336 1340 site site NN 1341 1343 or or CC 1344 1347 the the DT 1348 1353 C/EBP C/EBP NNP 1354 1358 site site NN 1359 1363 that that WDT 1364 1371 abolish abolish VBP 1372 1375 the the DT 1376 1383 binding binding NN 1384 1386 of of IN 1387 1390 the the DT 1391 1401 respective respective JJ 1402 1409 factors factor NNS 1410 1416 result result VBP 1417 1419 in in IN 1420 1421 a a DT 1422 1433 significant significant JJ 1434 1442 decrease decrease NN 1443 1445 of of IN 1446 1452 GM-CSF GM-CSF NNP 1453 1461 receptor receptor NN 1462 1467 alpha alpha NN 1468 1476 promoter promoter NN 1477 1485 activity activity NN 1486 1488 in in IN 1489 1503 myelomonocytic myelomonocytic JJ 1504 1509 cells cell NNS 1510 1514 only only RB 1514 1515 . . . 1516 1527 Furthermore furthermore RB 1527 1528 , , , 1529 1531 we we PRP 1532 1543 demonstrate demonstrate VBP 1544 1548 that that IN 1549 1551 in in IN 1552 1559 myeloid myeloid JJ 1560 1563 and and CC 1564 1565 B B NNP 1566 1570 cell cell NN 1571 1579 extracts extract NNS 1579 1580 , , , 1581 1585 PU.1 pu.1 NN 1586 1591 forms form VBZ 1592 1593 a a DT 1594 1599 novel novel JJ 1599 1600 , , , 1601 1609 specific specific JJ 1609 1610 , , , 1611 1615 more more RBR 1616 1622 slowly slowly RB 1623 1632 migrating migrate VBG 1633 1640 complex complex NN 1641 1642 ( ( ( 1642 1647 PU-SF PU-SF NNP 1647 1648 ) ) ) 1649 1653 when when WRB 1654 1661 binding bind VBG 1662 1665 the the DT 1666 1672 GM-CSF GM-CSF NNP 1673 1681 receptor receptor NN 1682 1687 alpha alpha NN 1688 1696 promoter promoter NN 1697 1701 PU.1 pu.1 NN 1702 1706 site site NN 1706 1707 . . . 1708 1712 This this DT 1713 1715 is be VBZ 1716 1719 the the DT 1720 1725 first first JJ 1726 1739 demonstration demonstration NN 1740 1742 of of IN 1743 1744 a a DT 1745 1753 specific specific JJ 1754 1765 interaction interaction NN 1766 1770 with with IN 1771 1775 PU.1 pu.1 NN 1776 1778 on on IN 1779 1780 a a DT 1781 1788 myeloid myeloid JJ 1789 1793 PU.1 pu.1 NN 1794 1801 binding binding NN 1802 1806 site site NN 1806 1807 . . . 1808 1811 The the DT 1812 1817 novel novel JJ 1818 1825 complex complex NN 1826 1828 is be VBZ 1829 1837 distinct distinct JJ 1838 1842 from from IN 1843 1847 that that DT 1848 1857 described describe VBN 1858 1868 previously previously RB 1869 1871 as as IN 1872 1879 binding binding NN 1880 1882 to to TO 1883 1884 B B NNP 1885 1889 cell cell NN 1890 1898 enhancer enhancer NN 1899 1904 sites site NNS 1905 1908 and and CC 1909 1912 can can MD 1913 1915 be be VB 1916 1922 formed form VBN 1923 1925 by by IN 1926 1934 addition addition NN 1935 1937 of of IN 1938 1942 PU.1 pu.1 NN 1943 1945 to to TO 1946 1954 extracts extract NNS 1955 1959 from from IN 1960 1967 certain certain JJ 1968 1978 nonmyeloid nonmyeloid JJ 1979 1983 cell cell NN 1984 1989 types type NNS 1990 1995 which which WDT 1996 1998 do do VBP 1999 2002 not not RB 2003 2010 express express VB 2011 2015 PU.1 pu.1 NN 2015 2016 , , , 2017 2026 including include VBG 2027 2028 T t NN 2029 2034 cells cell NNS 2035 2038 and and CC 2039 2049 epithelial epithelial JJ 2050 2055 cells cell NNS 2055 2056 , , , 2057 2060 but but CC 2061 2064 not not RB 2065 2069 from from IN 2070 2079 erythroid erythroid JJ 2080 2085 cells cell NNS 2085 2086 . . . 2087 2098 Furthermore furthermore RB 2098 2099 , , , 2100 2102 we we PRP 2103 2114 demonstrate demonstrate VBP 2115 2119 that that IN 2120 2123 the the DT 2124 2129 PU-SF PU-SF NNP 2130 2137 complex complex NN 2138 2143 binds bind VBZ 2144 2146 to to TO 2147 2151 PU.1 pu.1 NN 2152 2157 sites site NNS 2158 2163 found find VBN 2164 2166 on on IN 2167 2168 a a DT 2169 2175 number number NN 2176 2178 of of IN 2179 2186 myeloid myeloid JJ 2187 2196 promoters promoter NNS 2196 2197 , , , 2198 2201 and and CC 2202 2205 its its PRP$ 2206 2215 formation formation NN 2216 2224 requires require VBZ 2225 2227 an an DT 2228 2234 intact intact JJ 2235 2239 PU.1 pu.1 NN 2240 2244 site site NN 2245 2253 adjacent adjacent JJ 2254 2256 to to TO 2257 2258 a a DT 2259 2274 single-stranded single-stranded JJ 2275 2281 region region NN 2281 2282 . . . 2283 2293 Expression expression NN 2294 2296 of of IN 2297 2301 PU.1 pu.1 NN 2302 2304 in in IN 2305 2315 nonmyeloid nonmyeloid JJ 2316 2321 cells cell NNS 2322 2325 can can MD 2326 2334 activate activate VB 2335 2338 the the DT 2339 2345 GM-CSF GM-CSF NNP 2346 2354 receptor receptor NN 2355 2360 alpha alpha NN 2361 2369 promoter promoter NN 2369 2370 . . . 2371 2379 Deletion deletion NN 2380 2382 of of IN 2383 2386 the the DT 2387 2401 amino-terminal amino-terminal JJ 2402 2408 region region NN 2409 2411 of of IN 2412 2416 PU.1 pu.1 NN 2417 2424 results result VBZ 2425 2427 in in IN 2428 2429 a a DT 2430 2437 failure failure NN 2438 2440 to to TO 2441 2445 form form VB 2446 2449 the the DT 2450 2455 PU-SF PU-SF NNP 2456 2463 complex complex NN 2464 2467 and and CC 2468 2470 in in IN 2471 2472 a a DT 2473 2484 concomitant concomitant JJ 2485 2489 loss loss NN 2490 2492 of of IN 2493 2508 transactivation transactivation NN 2508 2509 , , , 2510 2520 suggesting suggest VBG 2521 2525 that that IN 2526 2535 formation formation NN 2536 2538 of of IN 2539 2542 the the DT 2543 2548 PU-SF PU-SF NNP 2549 2556 complex complex NN 2557 2559 is be VBZ 2560 2562 of of IN 2563 2573 functional functional JJ 2574 2584 importance importance NN 2585 2588 for for IN 2589 2592 the the DT 2593 2601 activity activity NN 2602 2604 of of IN 2605 2608 the the DT 2609 2615 GM-CSF GM-CSF NNP 2616 2624 receptor receptor NN 2625 2630 alpha alpha NN 2631 2639 promoter promoter NN 2639 2640 . . . 2641 2648 Finally finally RB 2648 2649 , , , 2650 2652 we we PRP 2653 2664 demonstrate demonstrate VBP 2665 2669 that that IN 2670 2675 C/EBP C/EBP NNP 2676 2681 alpha alpha NN 2682 2685 can can MD 2686 2690 also also RB 2691 2697 active active VB 2698 2701 the the DT 2702 2708 GM-CSF GM-CSF NNP 2709 2717 receptor receptor NN 2718 2723 alpha alpha NN 2724 2732 promoter promoter NN 2733 2735 in in IN 2736 2746 nonmyeloid nonmyeloid JJ 2747 2752 cells cell NNS 2752 2753 . . . 2754 2755 ( ( ( 2755 2763 ABSTRACT abstract NN 2764 2773 TRUNCATED truncated VBN 2774 2776 AT at IN 2777 2780 400 400 CD 2781 2786 WORDS words NNS 2786 2787 ) ) )