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GENOMICS 20, 377- 385 (1994) The HNF-3 Gene Family of Transcription Factors in Mice: Gene Structure, cDNA Sequence, and mRNA Distribution KLAUS H. KAESTNER, HOLGER HIEMISCH, BRUNO lUCKOW,l AND GÜNTHER SCHÜTZ 2 Division of Molecular 8iology of the Celll, German Cancer Research Center, Im Neuenheimer Feld 280, 0·69120 Heidelberg, Germany Received Detober 26, 1993; revi sed January 1, 1994 quent isolation of the transcription factors important in The rat HNF-3 (hepatocyte nuclear factor 3) gene their regulation (for review see Lai and Darnell, 1991; family encodes three transcription raetors known to be Sladek and Darnell, 1992; De Simone and Cortese, important in the regulation or gene expression in liver 1991). and lung. We have eloned and cbaracterized the mouse The major liver-enriched transcription factors identigenes and cDNAs for HNF-3o:, {J, snd "y and analyzed fied so far include HNF-1, a POU-homeodomain protheir expression patterns in various adult tissues aod tein; C/ EBP" and ß, which are bZip proteins; HNF -4, a mouse embryonie stages. Tbe HNF -3 proteins are member of the thyroid-steroid hormone receptor superhighly conserved bctween mouse and rat, with the exfamily; and HNF-3, a representative ofthe HNF-3/ forkception oe the amino terminus or HNF-3'Y, which in head class of DNA-binding proteins (Lai and Darnell, mouse is more similar to those of HNF-30: and fJ than to the amino termini of tbe rat HNF-3"( protein. The 1991; Sladek and Darnell, 1992; De Simone and Cortese, mouse HNF-3 genes are small and contain only two or 1991, and references therein). The HNF -3 proteins were three (HNF -aß) exons with conserved intron-exon first identified by their ability to bind to important proboundaries. The proximal promoter of tbe mouse HNF- moter elements in the Ql-antitrypsin and transthyretin genes (Costa et al. , 1989). Other target sites for HNF-3 3{J gene is remarkably similar to that of the previously cloned rat HNF-3ß gene, but is different from tbe pro- have been described in the a-fetoprotein, albumin, tyro· moters ofthe HNF-3a and 'Y genes. The mRNA distribu- si ne aminotransferase, phosphoenolpyruvate kinase. tion ofthe mouse HNF-3 genes was analyzed by quanti- transferrin, and HNF-1a and HNF-3ß genes (Costa et tative RNase protection with gene-specific probes. al., 1989; Herbst et al. 1991; Nitsch et al., 1993; Ip et al., Wbile HNF-3a and ß are restricted mainly to endo- 1990; Auge-Gouillou et al., 1993; Kuo et al., 1992; Pani et derm-derived tissues (lung, liver, stomaeh. and small a/. , 1992b) as weil as the lung-specific gene CCI0 intestine), HNF-3"Y is more extensively expressed, be- (Sawaya et al., 1993). From comparison of the observed ing present additionally in ovary, tesUs, heart, and adi- HNF·3 binding sites, a consensus element, TATTGA c / pose tissue, but missing from lung. Transeripts for TTTA/ TG, has been derived (Costa et al., 1989), although HNF -3ß and a are detected most abundantly in midges- deviations from this consensus sequence have been tation embryos (Day 9.5), while HNF-3"( expression identified (Nitach et al., 1993; Pani et al., 1992b). The peaks around Day 15.5 of gestation. t;I 1994 Aeademtc: HNF -3 DNA-binding activities were purified from rat Presa,lne. liver extracts and the rat cDNAs subsequently cloned and sequenced (Lai et a/., 1990, 1991). The three binding activities observed in gel retardation assays were found INTRODucnON to correspond to three proteins encoded by independent genes, which were termed HNF ·30', ß, and 'Y (Lai et al., Much progress has been made toward understanding 1991). Deletion analysis of HNF -30' demonstrated that Iiver-specific gene expression through biochemical anal· the region between amino acids 124 and 288 is essential ysis of the promoters and enhancers of genes such as for DNA binding (Lai et al., 1990). Subsequent sequence those encoding albumin or transthyretin and the subse- comparison revealed striking similarities between HNF3 and the DrosophiltJ m elanogaster gene forkhead within the DNA binding domain (Lai et a/., 1991; Weigel and Tbe nucletide sequences reported in tbis paper have been deposited in the EMBL Date Bese under Accession Nos. X74936, X74937, Jäckle, 1990), for whieh 100 of 110 amino acids are idenX7493B, X76684, X76685, and X766B6. tieal between HNF -3ß and forkhead. The DrosophiltJ 1 Present address: Medizinische Poliklinik , Abt. Klinische Bioche gene forkhead has been described as a region-specific mie, Schillerstrasse 42, D -80336 Munieh, Germany. homeotic gene and is required for the proper formation 2 To whom correapondence should be addressed. Telephone: ++496221-423411. Fax: ++ 49-6221-423404. of the terminal structures of the Drosophila embryo 377 0888-7543/94 $6.00 Copyright © 1994 by Academic Press, Ine. All rights of reproduction in any form reserved. KAESTNER ET AL. 378 • • ~ 1 1 17 51 5 Y Y A D T 111 G N H T PAS C M 5 P M A IJ • • • • • • • • • • • • S V P V 5 N • M N 5 G L G • • 5 M N • S M N T • Y H T M N T H F N H 5 Y A • N T G L GAG • • L S P • G A VAG H P • • GAS AGA Y • • A P 5 N L G • • R 5 G G • G D A • • • !( I F !( B 5 X • • • • • • P H A K P P P 5 A E 5 P L • • • • H W G V • • H G K ASO • • • • LEG A • • • • • • • • • P A P G P • • • • PO' • L • , • • • • L • • • • • • • • • • • • • • • • • 1921 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Q Q • 5 P H K M L • T • r. 5 E • T Y Q • W I M D • TI r P • r X B • Q H Q • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • , • • • • • • • • • • • • • • B W • • • • • • • • • • • • • • • • • • • • GTGCTTTATTTATGGCTTATAAATGTGTGTTCTGGCTAGAAT 2082 120 203 840 243 960 283 • 1080 323 • 1200 363 • 1320 403 • 1440 443 • 1560 460 • 1680 • 1800 • GTTGTTGCAGAAAAGTCTGACTTTAAAAACAAACAAAC!AACAAAAAACTTTTGTGAGTGACTTGGTGTAAAACCATGTAGTTTTAACAGAAAACCAGAGGGTTGTACTGATGTTGAAAA , • • • • • • • • • • • GAGGAAAGAAAAATAAfGTAAGAGTCTGGTGTACCGGACCAGGAGAAAGGAGAAAAACACATCCCATTCTGGACATGGTGAAATCCAGGTCTCGGGTCTGATTTAATITATGGTTTCTGC • 600 163 • A1'GGAAATATATATGTATACATATAAACTTGTTTTAAAGGAGCCTTTGGTCTCCTCTATGTAGACTACTGCTTCTCAAGACATCTGCAGAGTTTGATTTTTGTTGTTGTTCTCTATTGCT • 480 123 , TTTGGCAGAGCTTTGAGGAAAAGTAGCCACCACACTTCAGGCCTCAAGGGAGCAGTCTCACCTGTCTGTGTCCCT!AATAGATGGGCCACAGTGltTCTGTCATTCTAAATAGGGAAGGGA • 360 83 • Q • 240 43 • • • 120 • ACTTCCTACTACCAAGGAGTGTACTCCAGGCCT1o.TTATGAACTCATCCTAAG1o.AGATGGCTT!CAGGCCCTGCTAGCTCTGGTCACTGGGGACAAGGGAAATGAGAGGC7GAGTGGAGAC T 5 Y Y 0 G V Y 5 R P IHN 5 S * • 1680 469 • • AAGGCCTACGAACAGGTCATGCACTACCCAGGGGGCTATGGTTCCCCCATGCCAGGCAGCTTGGCCATGGGCCCAGTCACGAACAAAGCGGGCCTGGATGCCTCGCCCCTGGCTGCAGAC K A Y E 0 V H H 'i P G G X G S P H P G 5 L A M G P V T N K A G L DAS P L A A D • 1560 456 • • CACCTGAAGCCCGAGCACCATTACGCCTTCAACCACCCCTTCTCTATCAACAACCTCATGTCGTCCGAGCAGCAACATCACCACAGCCACCACCACCATCAGCCCCACAAAATGGACCTC H L K P E H B Y A F H H P r 5 I N N L H 5 S E 0 0 H H H S H H H H 0 P H K M D L • 1440 416 3 AAGGGAGCACCTGCCTCTGCGCTGAGTCCTCCCGAGCCGGCGCCCTCGCCTGGGCAGCAGCAGCAGGCTGCACCCC/o.CCTGCTGGGCCCACCTCACCACCC!GGCCTGCCACCAGAGGCC K GAP A 5 ALS P P E P A P S P G 0 0 0 0 A A A H L L G P P H H P G L P P E A • 2041 • • G TCTCAGGCTCAGCTCGGGGAGGCCGCGGGCTCGGCCTCCGAGACTCCGGCGGGeACCGAGTCCCCCCATTCCAGCGCTTCTCCGTGTCAGGAGCACAAGCGAGGTGGCCTAAGCGAGeT1o. S 0 A 0 L G E A It G S ASE T P A G T E S P H 5 S A S P C 0 E H K P. G G L S E L • 1801 • L CAGAACGGCTGCTACCTGCGeCGCCAGAAGCGCTTCAAGTGTGAGAAGCAACTGGCACTGAAGGAAGCCGCGGGTGCGGCCAGTAGCGGAGGCAAGAAGACCGCTCCTGGGTCCCAGGCC E H G C Y T, B B Q K B f K C E K 0 L ALK E A AGA A S S G G K K I A P G S 0 A • 1681 • • • • • • MAI 1320 376 1166 CAGAACTCCATCCGCCACTCTCTCTCCTTCAACGACTGCTTTCTCAAGGTGCCCCGCTCGCCAGACAAGCCTGGCAAGGGCTCCTTCTGGACCCTGCACCCAGACTCGGGCAACATGTTC Q N S T P. !l S L $ f N Q C f 1, !S 'I p P. S P Q K P G lS Ci S r Ii I J, H P Q S G N M r • 1561 • • T 1200 336 • • I 1080 296 • • TACATCTCGCTCATCACCATGGCCATCCACC~~GCCCCAACAAGATGCTGACGCTGACCGAGATCT~TCAGTGGATCATGGACCTCTTOCCTTTCTACCCGCAGAAOCAGCAGeGeTGG S 960 256 • • CTTGeCCCCTACGCCAACATGAACTCGATGAGCCCCATGTACGGGCAGGCCGGCCTGAGCCGCGCTCGGGACCCCAAGACATACCGACGCAGCTACACACAOGCCAAACCTCCCTACTCG L A P Y A N H N 5 M 5 P M Y G 0 A G L 5 RAR D P K T Y B R S X T H 10. K P P Y S I 216 • GCCGGCGOCATGGCGGGCATGAGCGGCTCAGCCGGGGCGGCCGGCGTGGeGGGCATGGGACCTCACCTGAGTCCGAGTCTGAGCCCGCTCGGGGGACAGGCGGCCGGGGCCATGGGTGGC AGA MAG M 5 G SAG A A G VAG M G P H L 5 P 5 L S P L G G 0 A AGA M G G • 444 A ATGAGCATGTCCGCGGCTGOCATGGGCGGCGGTTCCGGCAACATGAGCGCGGGCTCCATGAACATGTCATCCTATGTGGGCGCTGGAATCAGCCCGTCGCTAGCTGGCATGTCCCCGGGC H 5 M S A /0. A M G G G 5 G N M SAG S H N M 5 S Y V G A G H 5 P 5 LAG M S P G • 1441 G GCCGTGA!GATGGAAGGGCTCGAGCCATCCGACTGGAGCAGCTACTACGCGGAGCCCGAGGGCTACtcTTCCGTGAGCAACATGAACGCCGGCCTGGGGATGAATGGCATGAACACATAC A V f( M E G L E P 5 D W 5 S Y Y A E P E G Y 5 S V S N H N A G L G M N G M N T Y Y 840 • DaS • • • • • • 120 116 • • • AAS • • • 600 136 • M • 1321 404 1 CTGACGACCAGGGCGGCCAGACCACGCGAGTCCTACGCGCCTCCTGAGGCCGCCCCGGGACTTAACTGTAACGGGGAGGGGCCTCCGGAGeAGCGGCCAGCGAGTTAAAGTATGCTGGGA • 1201 364 Y • • • • • • • • CACACACACACACAGACACACACACACCAAACCAACCACATAArA.·_~TTCCAAATAGTTTTATTTTTCACGCACAAAAAAAAAAA • 1081 324 s CCAAGCTGTGTATTCCAGACCCGTGCTAAATACTTCCTAGCTCCCAGAACTGAGGGTTTTGTCTGeATGGCCAACCTGGTAGCAGGAGAGAAAAACCAACAGCAAACAAACAAAACCACA 0 G V Y 5 R P V L N T 5 * • 961 284 • CGAGCAGGCGCTGCAGTACTCTCCTTATGGCGCTACCTTGCCCGCCAGTCTGCCCCTTGGCAGCGCCTCAGTGGCCACGAGGAGCCCCATCGAGCCCTCAGOCCTGGAGCCAGCCTACTA E 0 A L 0 X 5 P 'i G A T L P A S L P L G 5 A 5 V A T R S P I E P 5 ALE P A Y Y • 841 244 96 ACCCCACGAATCrCAGCTGCATCTGAAAGGGGATCCCCACTACTCCTTTAATCACCCCTTCTCCATCAACAACCTCATGTCCTCCTCCGAGCAACAGCACAAGCTGGACTTCAAGGCATA P H E S O L H L K G D P H X 5 r N H P F 5 I N N L M 5 5 S E 0 0 H K L D r K A 'i • 121 204 M CACGGCGACAGGGGGCGCTTCGGAGTTGAACTCTCCAGCGTCTTCATCTGCGCCCCCCATAAGCTCCGGGCCAGGGGCGCTGGCATCTGTACCCCCCTCTCACCCGGCTCACGGCCTCGC TAT G GAS E L K S P A S 5 S A P P I S 5 G P G A L A S V P P S R PAR G L A • 601 164 5 480 • • • • 360 56 • • • • GGGGAACCCC~GCCGAGTCN:COCTTCATTGGGGTCTGC~GG~GGCT~CAGCTAGAGGQCGCQOCGGCCCCCGGGCCCGCCGCCAGCOCCC~ACTCTGGN:C~AGCGGGGC G N • • • 481 124 S CGGCTGCTACTTGCGCCGCCAAAAGCGCTTCAAGTGTGAGAAGCAGCCGGGGGCCGGAGGTGGGAGTGGGGGCGGCGGCTCCAAAGGGGGCCCAGAAAGTCGCAAGGACCCCTCAGGCCC G C Y J, B B 9 !( B F K C E J( 0 P G A G G G 5 G G G G 5 K G G P E S B K D P S G P • 361 84 N X • • • • • 16 • • • RAG • • • • 241 44 T • • • 240 • H 1 121 4 T • • • 120 • CTCCATCCGCCACTCGCTGTCCTTCAACGATTGTTTCGTCAAGGTCGCACGATCCCCAGACAAGCCAGCCAAGCGCTCCTACrGGACGCTCCACCCGCACTCCGCCAACATGTTCCACAA S I B R 5 [, !j r N D C F V K V A B S P D K P G K G 5 Y ij T J, H P D S G N M r t N • 1 S • • • 1681 • CTCGCTCATCACGATGGCCATCCAGCAGGCGCCCAGCAAGATGCTCACGCTGAGCGAGATCTACCAGTGGATCATGGACCTCTTCCCCTATTACCGCCAGAACCAGCAGCGCTGGCAGAA S L I T M b I Q Q b P S !<j M L I J. S E ! Y Q 'd I 11 \) J, F P 'i 'i i\ Q H Q Q ß 'd Q B • 1561 451 • • • 't • • 1441 417 • • • • • • GTGCATGACTCccAtGGCGTACGeCCCGTCCAACCTGCGCCGCACCCGCGCGGGCGGCGGCCGCGACGCCAAGACATTCAAGCGCAGCTACCCTCACGCC~GCCGCCTTACTCCTACAT • 1321 317 A • • 1201 337 • • GACTGCGGCGGGCGTCACGGCCATGGGTACQGCGCTGAGCCCGGGAGGCATGGGCTCCATGGGCQCGCAGCCCGTCACCTCCATGAACGGCCTGGGTCCCTACGCCGCCGCCATGAACCC T A A G V T A H G TAL S f G G H G S M G A Q f V T S H N G L G P Y A A AHN f • 1081 297 E • • 961 257 • • CGGCAACATGACCCCGGCTTCCTTCAACATGTCCTACGCCAACACGGGCTTAGGGGCCGGCCTGAGTCCCGGTGCTGTGGCTGGCATGCCAGGGGCCTCTGCAGGCGCCATGAACAGCAT • 841 211 Q • • 121 • • CAGCTACTACGCGGACACGCAGGAGGCCTACTCCTCTGTCCCTGTCAGCAACATGAACtcCGGCCTGGGeTCTATGAACTCCATGAACACCTACATGACCATGAACACCATGACCACGAG • 601 131 • • • 481 91 • CCGCCCCGAGCAGCGCACCCGCCCGTCGCTCCGCACAGGGTTGGATGGTTGTGTCGGCCGGGCTGGCTCCAGGATGTTAGGGACTGTGAAGATGGAAGGGCATGAGAGCAACGACTGGAA H L G T V ~ H E G H E 5 N D W N • • 361 • • • 241 • CGCCGCGCCGCGCCGCCGCCGCCGCGCACGCCGCGCCCCGCAGCGCCGGGCTTCCTCCTCGCCCGGGTGGCGCTGGGCCCTCGAGCGCTCCGGTGACCGCAGCGGCTCCGCGCCCCTCCC • 121 • 1920 2040 379 THE MOUSE HNF-3 GENE FAMILY • • C • • • • • • 1 GCGGGACTCCCGGGCTGTGTGcctcAGGTCGGAACTCGGGGCTAGTGCCTGTAGAGAGACCGAAGCACTCGGTTCCCCCAGGGGGCCTCAGCCTGGGTGTGTGGGGGCGCAGGCCGGGGA 1 M , 121 • • • • • • TGCTGGGCTCAGTGAAGATGGAGGCTCATGACCTGGCCGAGTGGAGCTACTACCCGGAGGCGGGCGAGGTGTATTCTCCAGTGAATCCTGTGCCCACCATGGCCCCTCTCAActCCTACA ., L G S V K M E T L N P E V Y S P V • • G G L 0 ASP L P T G P S T P • • H A P L • N 5 Y M T A P L G P • • • • T F P 5 L • • • • • • • • • • • • • • • • • • • • • • • • • • • F S G L E L P GEL • • K n L • A P Y N F H • • H P F S I N • • H M S E • • 0 T K L n V G F G G Y • G A • E S G E P G V Y Y 0 S L Y • • S R S L L N • A S S T P S • • • • • • • • • • • • • • • • • • • • · . . ' " CCTTTTTTCTTTCTTTTTTCTTTTTTGGCAGACTTCTTGGTTCAGCAGATGCCAAATTGGCCACCATATCACATGGTGTCTTTTTTGACATtCTGGATGCATGGAAGGTCACTGTATTGG • • • • • • • • • • • • • • • • • • • 1440 1560 1680 • 1800 • 1920 • AAGCCCATCCCCACCCTGTCTGTGAGAAAGGGCTAGTGTGGGTGTCGGGAGTTCCTACTGAGGTCAAGTTCTTGTCTGGGGCTTGGGAATACTGeCTGTGTTTGGCCATrAI'IAAAGGCAC CATCTeCAT 1320 • CAAGGTGACATCTCAGCATGCTGCTATGCACCAAGATAGATCGTTACCACAGGCCTGCCATCTCCTTGGTGGAGGTTGGGtGAGAGGAAGAGGTGAGCAGACCTATCGAGTTTTTCTCTG • 1200 354 • CtGTGTTGGACATCTTGATTGACCtTtTGAGTATCTGACAGAACACATCTTCTTTGGCTCATTtTATCCTGGGATCGCCTCTTTTTTTTCCTCITCTTTTTCTTTTTCTTTTTTTCTTTT • 1080 321 • TGAAAGCTAGCCATATTAAGTGAAGCCATTGGGTGATTGATCCACTGGGTGCCTGATGGTCGTCATGTTGGATGACACATGTCTGGTCCTTTGGATGATCTGTTGGCAATCTTGATTGAC • 960 281 • • • 840 241 • AACTGCCATGGACTCTTTGGTAGGCCTAGGGTTGGGGTATTAGGAAGGCAGATGCGTTTGGAACTGCTGCGAAGGTGGTCATCTTGGACATAtTGTGI'IAGGCACTTAGACTGGTGTACTA • 120 201 • ATGATGGGCGTGGGCTGCAACGTTCTTGGGCTCTCATCTTTCTGGTTACACTTTGCTTGTCCCATTAATTAACATCTTATTTGGTCTATTACTGTGATATCACCCATTAGCTACTGTGGT • 600 161 • * • 121 • • L 480 • • • " • • • 360 • • • • • 240 41 • CCAAACTGGATGTGGGGTTTGGGGGCTACGGGGCTGAGI'IGTGGGGAGCCTGGAGTCTACTACCAGAGCCTCTATtCCCGCTCTCTGCTTAATGCATCCTAGCAGCGCAATTGGGAACGCC • 2041 A P • • • • • Y • 1921 T CGTCCACACCTTATTTCAGCGGCCtGGAGCTCCCGGGGGAACTAAAGTTGGATGCGCCCTATAACtTCAACCACCCTTTCTCTATCAACAACCTGATGTCAGAACAGACATCGACACCTT • 1801 V P • L A P P • 1681 N P CTGCCACCACTACAGCTGCCACTGCAGTCACCTCCCCGGCTCAGCCCCAGCCTACGCCATCtGAGCCCGAGGCCCAGAGTGCGGATGATGTGCGGGGTCTGGACTGCGCCTCACCTCCTT A T T T A A T A V T S P A 0 POP T P S E P E A 0 S G 0 0 V G G L 0 C A S P P S • 1561 • A G ACATGtTTGAGAACGGATGCTATCTCCGCCGGCAGAAGCGCTTCAAGCTGGAGGAGAAGGCAAAGAAAGGAAACAGCGCCACATCGGCCAGCAGGAATGGTACTGCGGGGTCAGCCACCT M F ENG C X J. B B Q J( B r J( J. f: f: KAK K G H S A T 5 A S R H G TAG 5 A T S • 1441 P • • • 1321 • E AACGTTCGCACAACTCCATCCCGCATTCGCTGTCCTTCAATGACtGCTTCGTCAAGGTGGCACGCTCCCCAGACAAGCCAGGCAAAGGCTCCTACTGGGCCTTGCATCCCAGCTCTGGGA B W Q N S I B H S L S F N D C r Y J( Y A R S P D J( P G J( G S X t! A L H P S S G N • 1201 X Y P CATATTCCTACATCTCTCTCATAACCATGGCTATTCAGCAGGCTCCAGGCAAGATGCTGACCCtGAGTGAAATCTACCAATGGATCATGGACCTCTTCCCGTACTACCGGGAGAACCAGC y S Y I S {. I T MAI Q Q A P G K M L T J. S E I Y Q W I Mn!. F P Y Y B f: H Q 0 • 1081 322 Y P • • 961 282 W S TGGGCACTGGTGGCAGCACCGGAGGCAGTGCTTCCGGGTATGTAGCCCCAGGGCCCGGGCTTGTACATGGAAAAGAGATGGCAAAGGGGTACCGGCGGCCACTGGCCCACGCCAAACCAC G T G G 5 T G G S A S G Y V A P G P G L V H G K E H A K G X B R P L AHA K P P • 841 242 P • • 121 202 • L S 5 • 601 162 A E TGACCTTGAACCCACTCAGCTCTCCCTACCCTCCCGGAGGGCTTCAGGCCTCCCCACTGCCTACAGGACCCCTGGCACCCCCAGCCCCCACTGCGCCCTTGGGGCCCACCTTCCCAAGeT • 481 122 A H D L • • 361 82 1 • • 241 120 2040 • AAM 2061 FIG.1. Nucleotide and translated amino acid sequences ofthe mouse HNF-3a, ß, and ')' cDNAs. Shown is the sequence afthe sense strand of composite cDNAs and the carresponding amino aeid sequence ofthe langest open reading frame for HNF -3a (A), HNF -3ß (B), and HNF -3')' (C). The boldfaee sequenees in A and C indieate potential polyadenylation signals. The sequenees ofthe IIO-amino-aeid forkhead DNA binding domains are underlined. (Jürgens and Weigel, 1988; Weigel et al., 1989). The forkhead gene is expressed in eetodermal as weil as endodermal portions of the gut, the yolk nuelei, the salivary glands, and eertain eells of the nervous system. Subsequently, sequenees closely related to the HNF -3/ forkhead DNA binding domain have been found in species ranging from yeast to man (Oliver et al., 1992; Knöchel et al., 1992; Dirksen and Jamrieh, 1992; Ruiz i Altaba and Jessell, 1992; Häeker et al., 1992: Tao and Lai, 1992; Li et al., 1991, 1992; Li and Vogt, 1993; Kaestner et al., 1993; Clevidenee et al., 1993). In mice, six HNF-3/forkhead homologues that have at least 57% amino acid identity within the DNA binding domain with forkhead have been deseribed (Kaestner et al., 1993). The rat HNF-3 genes have been shown to be expressed, in addition to liver, in stomaeh, intestine, and lung (Lai et al., 1990, 1991), whieh are all tissues derived, at least in part, from embryo nie endoderm. This fact, eombined with the high degree of similarity to the Drosophila gene forkhead, has led to the proposal that the HNF -3 genes are important in early endoderm and liver development in addition to their role in adult liver transcription (Lai and Darnell, 1991). This notion is supported by recent in situ hybridization studies on early postimplantation and midgestation mouse embryos (Sasaki and Hogan, 1993; Monaghan et al., 1993). The HNF -3 family members were found to be sequentially transeribed in the developing definite endoderm, HNF3ß being the first gene to be aetivated, followed by HNF3a and finally ')'. Interestingly, HNF-3ß and a mRNAs were also found in cells of the notochord and ventral neural epithelium, suggesting additional functions for these genes in mesoderm and neural axis formation (Monaghan et al., 1993). Reeent experiments suggest that the very early expression of the HNF -3 proteins in the liver primordium is responsible for the reorganization of the chromatin strueture, as the HNF -3 proteins are involved in the precise positioning of nucleosomes over the albumin enhancer only in those tissues where the enhaneer is aetive (MePherson et al., 1993). As a prerequisite to further our understanding of the importanee of the HNF -3 gene family in mouse development, through promoter analysis in trans genie mice and generation of null alleles via homologous recombination in embryonie stern cells, we have cloned and characterized cDNAs and genomic fragments encoding the mouse HNF -3 genes. In addition, we have quantitatively analyzed their expression pattern in adult mouse tissues and whole mouse embryos from midgestation to term. 380 KAESTNER ET AL. mHNF-3a B IH I ~ B B I ..,.I",. Xh B '''' B I IE IH I 11 Xh E IE >,AI> B, BamHI Bg, BgUi E. EcoRl H, Hindlll N,NoU S, Sall Xh, Xhol >,A' mHNF-3p E E I I IBg E Xh I I~ I I E Xh I I I • • Bg .. I >,A11 , )Al~ mHNF-3r .. FIG. 2. Res trietion map of the mouse HNF-3c:r. P, and l' genes. The restrietion map of each gene together wit h the extent of the lambda phage clones from which it was derived is shown. The exons of the three genes are shown as black hoxes, and the forkhead DNA binding domains are indicated as white baxes. MATERIALS AND METHODS Library construction und cDNA and genomic cloning. A 300-nt probe co rr~sponding to the forkhead domain of th c mouse HNF -3a gene originally obtained by peR (Kaestner et al., 1993) was used to sc reen two m ouse live r cDNA libraries (Ruppert e l 01., 1990; B. Luckow et al., 1994) using high -stringency hyhridization and w8shing conditions (Church and Gilbert, 1984), Fourteen hybridizing lambda phages were purified and the cDNAs were subcloned into Bluescript (Stratagene) aod classified by dideo):.y sequencing (Sanger et al., 1977). The complete coding 88 well as pan of the untranslated regions of t he mOuse HNF -3a:, ß. and 'Y cDNAs were sequenced after generating nested deletions of the cDNAs with exonuclease III (Henikoff, 19M). Genomic clones containing the HNF -3a and f3 genes weTe obtained through screening 9f an amplified m ouse 129/018 library in '\GEM-12 TABLE 1 Intron-Exon Boundaries or tbe HNF -3 Genes in Mice HNF-3a Exon 1 Exon 2 AACGCAGGAG~gagaggcggg ,.,."" T Q ., .•. , ., • ....... 2.5- kb intron ..... ', . . ... , •.. .. .. .... tctccgcccc~GCCTA CTCCT EZ4 AZSY S HNF-3ß Exon 1 Exon 2 GCGAGTTAAAgt t t t t ctaaat ., ... • ' , , ... , •. . , .•..... 1.5-kb intron " . .. ..•.. . , .. . . . . .. .... c t cggc t tc cagTATGCTGGGA - - MI L G Exon '2 Exon 3 GGAGCCCGAGgtaa gc tctt gc .. .... ....• . . ..•... .. ... O.5-kb int ron., .....•... ' .' .. ........ ctc tgt cc gcagGGCTACTCTT E P E - C Y S 24 n HNF -3~ Exon 1 Exon 2 GGCGGGCGAGgtgtgt cct cgg ...... .. ... .. ... , .. , .. .. 8-kb intron .. . , .. .... , .... . ... , .. .. ctctccctc tagGTGTATTCTC A G E -V y S 23 24 Note. The intrQn-exon bollndaries were obtained by comparison of cDNA and genomic sequences. The consensus dinucleotides gt and ag at the intron borders are underlined. THE MOUSE HNF-3 GENE FAMILY FIG.3. Mapping ofthe transcription initiation sites ofthe mouse HNF-3a, ß. snd 'Y genes. Synthetic oligonucleotide primers complementary to the sense strand ofthe cDNAs were end-labeled with 32p to a specific activity of 108 cpm!p.g. Primer extension analysis was carried out with 5 /.Lg of poly(A+) RNA from stomach or 5 /lg tRNA. Marker lanes contain the dideoxy nucleotide sequencing reactions of the first exon of each gene using the same primers. Arrows indicate the major start sites of transcription. (Promega) probe and screen. An embryonie kindly provided by Anton Berns (Amsterdam) using the hybridization conditions as used for the cDNA library additional genomic Iibrary was constructed from murine stern cells. Genomic DNA was isolated from the murine 381 embryonie stern celliine E14TG2a (Hooper et al., 1987), parlially digested with the restriction endonuclease Sau3AI. size fractionated (16 to 23 kb fragments), and ligated into .\Dash 11 (Stratagene) according to Frischauf (1987). Two million primary phages of this library were screened with various portions ofthe mouse HNF-31' cDNA to assemble the complete HNF -31' contig. The genomic lambda phages were mapped. and exon-containing fragments subcloned and sequenced to obtain the intron-exon boundaries. Primer extension analysis. Primer extension analysis was carried out as deseribed previously (Kaestner et al., 1989) using synthetie oligonucleotides and 5 ~g tRNA or poly(A +) RNA purified from stornach using oligo(dT) Dynabeads (Dynal). The primers used were complementary to nucleotides 64 to 85 of the HNF -3a cDNA, nucleotides 61 to 83 ofthe HNF-3ß eDNA, and nucleotides 61 to 83 ofthe HNF-31' cDNA (see Fig. 1). To map the endpoint of the primer-extended product, dideoxy sequeneing (Sanger et al., 1977) of genomic subclones eontaining the first exon of eaeh gene was performed using the eorresponding oligonucleotide as a primer. RNA isolation and RNase protection analysis. Total RNA from a variety of mouse tissues or whole mouse embryos was isolated by eentrifugation through a CsCI eushion after bomogenization in guanidinium thioeyanate (Chirgwin et aL, 1979). The quality ofthe RNA preparations was eontrolled by ethidium bromide staining of tbe 18S and 288 rRNAs after electrophoretie separation ofthe RNA in denaturing agarose gels. RNase protection analysis was performed as described previously (Kaestner et al., 1989) using (a - 32P1UTP-Iabeled antisense RNA probes derived from Bluescript (Stratagene) subclones containing 191 bp (HNF-3a, position 1165 to 1355), 221 bp (HNF-3ß, position 1169 to 1389), or 338 bp (HNF-3')', position 993 to 1330) of tbe mouse HNF-3 cDNAs. The probes chosen were from regions outside the conserved domains ofthe HNF -3 family to avoid the possibility of cross-hybridization. The antisense probes were hybridized against 50,ug (adult tissues) or 20,ug (embryonie sampies) total RNA A ACGATCCTTG CGCTGTAGCT CTGATGCCAC CACCCAGGTC CTTCTCCCGC AGCACAGCTC TTTGGGTCCA GAGCCCTGGC CTGTCCTCCA -396 SP! AGGCACCGCC TATTTTTCCT TTTCTTCTTT TTTTTCCTTT TCTTTTCTTT CTCTTTTTTT CCTTTTCCAA AGGGGGTCAC ACACACACCC -306 CCAAT-BOX GCCCTATTTT CCTCTTTCCC TAGCCGCGAG GCTTAAACCA AITATACTGC TTTGTAAACA AAGTGAGGGC CAGGTTTGGG GGAGGGGATG -216 SP! SP! SP! GCGGGAGGGG CGCGGGGGGC GCGCAGGCGC TGGCGCGGCG GGGCGGGAGG CGCGGCGGCT GGACTGGCGG GCGGCCGCCT CACAGGTGCA -126 CCTCGGGCTT TGTAGGTGCG AGCGTCTTTG TGCGGCGGAC AAATGGGGAG AGGACGAGGA GGTGGGCACT CCGGCGACGT AAGATCCACA -36 SP1 +1 TCAGCTCAAC TGCACTCGCT TCGCACAGGC CGCCCGCTCA CTTCCCGCGG AGGCGCTGCC GGGCGCCGGC TCCGCGGCCG CCTCCTGTCC +56 CCGGCGCTGC CCCCTCCcgc cgcgccg B HNF-3 CCTAGTCTCG GTCTTGGTAG CTAACAATAT AAATGACATA CTCTGTTGTT TTCATGTITG TTTTGTTTGG GGCAGACAAG GTTTCTCTCT -365 GTAGCCGGAT GTCTTGAAAC TCACTCTATA GACTAGGTCA GCCTAGACTT CTCTGAGATC CTCCTGCCTC TGCCTCAAAG GCAGAAGACA -275 ACACTTCAAA TGACACTTTT TAACACCTAG AAGCTAAAGA GAAAAGAAAC TGAAATTTTC AATTTTTATA AGACAGTCCT GGTCTCTGCA -195 UFl-H3P HNF-3 GGCAGAGAAC ACAGATCCTC CTGAAGTCAT CCCACAAGGC CCATTATTGA TTTTTTCTCC TGCCCIACCC CCC~CCTACT GCCCTGTTTG -95 LF-H3P UF2-H3ß TTTTAGTTAC GAAATGCTTT GGGCACCTTG GATTTAACTG AAAAGTAACC TTGAAACACC GAGGCCCTCA TGCCAGAGGC AAATCGCTGC -5 +1 CTCCCGGGTA TTGGCTGCAG CTAAACGGGT CTCTCCAGGC CGACTGAGGT GGGTAGCCAG AAAGAGGACT GAGGTAActg acga C TCTAGAGTTT CCCTCCTTTA GCTGGAGATG ATGAAACACT TAGAGGCAGA AAGATTCCCC GGCTCCCGCT CTGTTGCTCT AGGGCTTTTG -230 GGATCAGCAG GGTGTGGCTC TCTACCCACG CCTTGTAGTC CCCGACTCTT CATCCATCGC AAGCTTCCAG GTGCCAGGAC CGAGGCTCCC -140 SP! AGTGCCTGAG GTCTCTCTTC TTGCGATCCC GCAGGGGGCG CCCCAATCCG AGGGCGCCGC GCTCGGGGAG GCGCGCGGGG AGCCCGGGGC -50 TFIID +1 G!iG.CCCGGGA GGGGGTG'I'CC CGGCIATAAll. AAGTGGCTGC CTCCCAAGGC GCCTGGGCCA gcgggactcc caggctgtgt gcctcaggt FIG. 4. Nucleotide sequenee ofthe 5' ftanking regions ofthe mouse HNF -3a (A), ß (B), and ')' (e) genes. The transcription initiation sites determined by primer extension analysis (Fig. 3) are shown in boldface, and the 5' most were designated as + 1. Potential binding sites for known transcription faetors are underlined. The sequenees present in the cDNAs are shown in lower ease letters. 382 KAESTNER ET AL. B u e:v < Z B a: CI> '"-.. ~ ::0 - HNF-3y HNF-3~ HNF-3a u - < z a: .!! o ~ >-" ,. <Ii g D ~ i' ~ ,;'" <; .t ~ i- " ;}o b'" ~ ",<> .j' .; v ,- " .f ~ •• ~ i5' ~ i>! ,f .$' <f ~ ". f J$' ,l ri- /' ..- ~ if ~ o· tl v FIG. 5. Quantitat ive analysis of the tissue distribution of the mouse HNF -3a, ß. snd 'Y mRNAs. (A) Fifty m icrograms oftotal RNA from the t is8ues indicated WIIS hybridized to a cocktail of excess 32P_labeled antisense RNA prohes specific for H N F -3a, ß. and 'Y. and the level orthe mRNAs for 811 three genes was determined by R Nasc protection analysis 8 5 described under Materials snd Met hods. The lane marked " Prohes" contains 1000 dpm each of the three prabes used and served w standardize the values obtained in the qua nHtation (see below) . The arrows indica te t he positions ofthe probes (a, ß. and y) and the protected fragments (HNF-3a, ß. and y). (B) The signals obtained in A were quantified using a phosphoimager and converted to Cmol-specific mRNA per milligram total tissue RNA. The signals were corrected Cor the specific activit.y 01 the diffe rent probes and reHect the true ratios of the three mRNAs. 8t 54<) C in 80% formaruide overnight. Excess probes were re moved by digestion with RNases A and Tl and, the protectcd probe fragme nts analyzed on denaturing 6% polyacrylamide gels. The signals obtained were quantified on a Molecular Dynamics phosphoimager and con verted to fm ol RNA/ mg total RNA assuming 100% hybridization of the target RNAs and rlormalizing the signals for thc number oeUMP res i.duCos incorporated in the hybridizing portion of Coach probe . RESULTS AND DlSCUSSION Using a 300-nt probe spanning the forkhead DNA bin ding domain of the mouse HNF -3a gene (Kaestner et al., 1993), we screened two m ouse liver cD NA libraries. A total of 15 phages (10 for HNF-3a, 1 for HNF-3ß. and 4 THE MOUSE HNF-3 GENE FAMILY 383 tain genomic fragments containing a11 three genes. The resulting phages were mapped and the exon-containing regions identified by Southern blotting and subcloning. The restriction patterns ofthe cloned regions were compared to those obtained in mouse genomic Southern blots to exclude possible rearrangements of the lambda phages (data not shown). The resulting gene structures for HNF -3", ß, and '1 are shown in Fig. 2. All three genes span less than 10 kb and contain two (HNF -3a and '1) or three exons (HNF -3ß). The intron-exon boundaries were determined by comparing the cDNA and genomic sequences and are summarized in Table 1. Interestingly, the intron within the coding region is at tbe same relative position in all three genes, that is, at -1 with respect to a conserved YS dipeptide, indicating a Common ancestor gene. In addition, the above-mentioned diveI~ FIG.6. Temporal expression of the HNF-3a, ß, and 'Y mRNAs gence of the amino terminus of the mouse HNF -3'1 gene during mouse emhryogenesis. Twenty micrograms oftotal RNA from the developmental stages indicated (in days post coitum) were anafrom its rat counterpart cannot be explained by alterlyzed by RN ase protection as described in the legend to Fig. 5. The nate exon usage, as tbis region covers tbe first 55 amino antisense probes and protected fragments are indicated by arrows. acids, while the intron is located at position 24. In addition, no evidence for alternate splice variants of HNF -3'1 for HNF -3'1) were purified from L3 million phages was found in RN ase protection experiments using under high-stringency conditions_ The phage inserts probes covering the first 452 nucleotides of the HNF -3'1 ranged from 1 to 2.1 kb and contained the complete cod· cDNA (data not shown). The intron-exon boundaries of ing regions of all three genes. As demonstrated by se- the rat HNF-3ß gene, the only other HNF-3 gene anaquence analysis and transcriptional start site mapping, Iyzed so far (Pani et al., 1992b), are identical to the ones the cDNA lack less than 50 bp at the 5' end. The cDNAs for the mouse gene described here. To determine the for HNF -3ß and '1 are almost full length, as the corre- start site of transcription for HNF -3a, ß, and ,)" we persponding mRNAs were shown to be 2.3 and 2.1 kb in size formed primer extension experiments with gene-specific (data not shown). The cDNAs for HNF-3" are lacking primers. The results of these experiments using 1.6 kb of untranslated sequence at the 3' end, as its poly(A+) RNA from stornach (where the HNF -3 gene mRNA was found to be 3.4 kb in length (data not family is expressed most strongly, see below) or tRNA a8 shown). The sequence of the mouse HNF-3 cDNAs was negative contra 1 are shown in Fig. 3 alongside a sequencdetermined and is shown in Fig. 1 along with the amino ing ladder using the same primer. In a11 three cases, mulacid sequence for the longest open reading frame. The tiple, but closely spaced, start sites are apparent. The cDNAs encode proteins of 468 (HNF-3,,), 459 (HNFmost 5' of these transcription initiation sites was desig3ß), and 353 (HNF-3'Y) amino acids with calculated monated as position +1 (see Fig. 4). The transcriptional lecular weights of 48.9, 48.5, and 34.6 kDa, respectively. start site ofthe rat HNF -3ß gene (Pani et al., 1992b) has As expected, sequence comparison demonstrated that been mapped to within 5 nt of the one observed in the mouse and rat sequences (Lai et al., 1990, 1991) are mouse, again underscoring the elose relationship behighly conserved, with amino acid similarity ranging tween the two genes. from 93% (for HNF-3'Y) to 99% (for HNF-3ß). A noteAs an initial step toward the understanding of the regworthy exception is the amino terminus of the HNF -3 '1 ulatory elements governing the expression of the HNF-3 gene, at which the mouse sequence is more similar to the gene family, we subcloned and sequenced the proximal mouse HNF -3" and ß genes (16 of the first 55 amino acids are conserved) than to its rat counterpart (6 of 55 promoters of the three mouse genes and searched them conserved amino acids). This is espeeially interesting in for potential bin ding sites of known transcription faclight of the finding that the amino-terminal 52 amino tors (Fig. 4). Despite the relatedness ofthe three cDNAs acids of the HNF -3ß protein were shown to be important and the similarity ofthe mRNA distribution ofthe three in transcriptional aetivation through testing of deletion genes, the promoters are dissimilar. The promoters of mutants in transfection assays (region IV; Pani et al., HNF -3" and '1 have in common only binding sites for 1992a). This domain is rieh in serine and tyrosine resi- the general transcription factor Sp1 (Fig. 4), while only dues and contains two putative casein kinase I phos- HNF-3'Y contains a canonical TATA-box at the approphorylation sites. Therefore, it seems likely that the priate position relative to the start site of transcription. amino terminus of the mouse HNF -3')' gene ean function The proximal promoters of the rat and mouse HNF -3ß in transcriptional activation as weIl. This region of the genes are remarkably weil conserved, with 116 of the mouse HNF -3')' sequence was also confirmed by se- first 120 nucleotides being identical. Especially imporquencing the corresponding genomic region (see below). tant is the fact that the two bin ding sites for a liver-speUsing the mouse cDNA probes for HNF-3a, ß, and '1, cific protein, termed LF - H3ß, and a binding site for we screened two mouse genomic phage libraries to ob- HNF -3 itself are conserved in sequence as we11 as spac- 384 KAESTNER ET AL. ing (Fig. 4; and Pani et al., 1992b). Pani et al. (1992b) transient dip in the mRNA levels of HNF-3a and ß have shown through transfeetion of rat HNF -3ß pro- corresponds to the disappearance of the two mRNAs moter mutants in HepG2 cells that both bin ding sites from the embryo nie liver as revealed by in situ hybridizaare important for strang expression of chimeric HNF- tion (Monaghan et al., 1993). HNF-3y, while hardly de3ß/reporter gene constructs. They proposed a model for tectable on Day 9.5, is being activated on Day 12.5 and cell-specific transcription of the HNF -3 gene in liver peaks on Days 15.5 and 16.5. This peak in mRNA levels cells involving activation by the cell-specific LF-H3ß coincides with the increased expression of HNF -31' in protein and maintenance ofthis expression through posi- endoderm-derived structures such as the liver, stomaeh, tive autoregulation by HNF-3ß. Interestingly, a second intestine, and pancreas observed by in situ hybridization HNF -3 binding site of identical sequence is found far- (Monaghan et al., 1993). ther upstream (position -400 to -390) in the mouse As an initial step toward understanding the role of the HNF -3ß promoter, which might also take part in this HNF -3 gene family of transcription factors, we bave proposed auto regulation. A second possibility is the regu- cloned and characterized the cDNAs and genes of its lation of the HNF -3ß promoter by HNF -3« and y, wbich three members. The information obtained will aUow are also present in liver and have similar DNA binding identification of important regulatory elements in the properties (Lai et al., 1991). Most likely, similar regula- promoters of HNF -3", ß, and y through transfeetion of tory pathways are operative in the regulation of the chimeric promoter/reporter gene constructs into tissue mouse and rat HNF -3ß genes. This notion is supported culture ceUs and by analysis of these constructs in transby the finding that the proximal mouse HNF -3ß pro- genie mice. Furthermore, gene targeting experiments in moter confers cell-type-specific expression on a ß-galac- mouse embryo nie stern ceUs that will allow us to detertosidase reporter gene (data not shown). A detailed pro- mine the precise role of these genes in mouse embryonie moter analysis of the divergent HNF -3« and y genes development and tissue-specific transcriptional regulathrough transfeetion as weH as transgenie mouse experi- tion in the adult are in progress. ments is in progress to understand which regulatory pathways control these genes, as their proximal proACKNOWLEDGMENTS moters lack binding sites for HNF -3 and LF - H3ß. As a first step toward understanding the full potential We thank Drs. A. P. Monaghan and J. Blendy for critically reading of the HNF -3 gene family as transcription factors in the manuseript, B. Lupp and H. Kern for technieal assistanee, and W. mice, we sturlied the expression patterns of its three Fleischer for oligonuc1eotide synthesis as weil as photographie work. members through quantitative RNase protection using This work was supported by the Deutsche Forschungsgemeinschaft gene-specific probes (see Materials and Methods for de- through Sonderforsehungsbereieh 229, the Leibniz-Programm, and Fonds der Chemischen Industrie. tails), allowing for a first direct comparison of the expression levels of the three genes. The results of our survey of adult mouse tissues are shown in Fig. 5. While REFERENCES HNF -3« is expressed strongly in large intestine and stomaeh, and weaker in liver, lung, and smaH intestine, Auge-Gouillou, C., Petropoulos, 1., and Zakin, M. M. (1993). Liverenriehed HNF -3 alpha and ubiquitous faetors interact with the huHNF-3ß mRNA is present in the same tissues at more man transferrin gene enhancer. FEBS Lett. 323: 4-10. balanced levels. HNF -3y is expressed strongly in large and smaH intestine, stomaeh, and liver, but is absent Chirgwin, J. M., Przybyla, A. E., MaeDonald, R. J., and Rutter, W. J. (1979). Isolation ofbiologically active ribonuc1eic acid from sourees from lung. This is consistent with the observation of enriched in ribonuclease. Biochemistry 18: 5294-5299. Monaghan et al. (1993) that HNF -3y expression in mid- Chureh, G. M., and Gilbert, W. (1984). Genomic sequencing. Proc. gestation mouse embryos is restricted to derivatives of Natl. Aead. Sei. USA 81: 1991-1995. the endoderm posterior to the liver. The expression of Clevidence, D. E., Overdier, D. G., Tao, W., Qian, X., Pani, L., Lai, E., and Costa, R. H. (1993). Identification of nine tissue-specific tranHNF -3« and ß in the adult mouse is restricted to derivascription factors ofthe HNF -3/forkhead DNA binding domain famtives ofthe embryonie endoderm, while HNF-3y mRNA ily. Proc. Natl. Aead. Sei. USA 90: 3948-3952. was also found in heart, adipose tissue, ovary, and testis Costa, RH., Grayson, D. R, and DamelI, J. E. (1989). Multiple hepaas weil as embryonic stern cells. The tissue distribution tocyte-enriched nuclear faetors function in the regulation of ofthe HNF-3 mRNAs in mice is qualitatively similar to transthyretin and (1'-1 antitrypsin genes. MoL Gell. Biol. 9: 1415- . that observed in the rat (Sladek and DamelI, 1992; Lai et 1425. al., 1991). The expression of HNF-3y in heart, adipose De Siroone, V., and Cortese, R (1991). Transcriptional regulation of liver-specific gene expression. Curr. Opin. Cel! Biol. 3: 960-965. tissue and embryonic stern cells has not been observed before. We extended our analysis ofthe expression ofthe Dirksen, M. L., and Jamrieh, M. (1992). A novel, activin-indueible, blastopore lip-specific gene of Xenopus laevis contains a forkhead HNF -3 gene family to RNAs obtained from mouse emDNA binding domain. Genes Deu. 6: 599-608. bryos from Day 9.5 post coitum onward using the same Frisehauf, A. M. (1987). Construction and characterization of a genogene-specific probes (Fig. 6). HNF -3ß is expressed mie library in A. Methods Enzymol. 152: 190~ 199. strongly on Days 9.5 and 10.5, weakly detected during Häcker, V., Grossniklaus, V., Gehring, W. J., and Jäckle, H. (1992). the next 2 days, and returns to an intermediate level of Developroentally regulated Drosophila gene family eneoding the fork head domain. Proe. Natl. Aead. Sei. USA 89: B754-B75B. expression on Day 15.5. HNF -3« follows a similar pattern as HNF -3ß, but is expressed at lower levels. The Henikoff, S. (1984). Unidireetional digestion with exonuclease 111 THE MOUSE HNF-3 GENE FAMILY creates targeted breakpoints for DNA sequencing. Gene 28: 351~ 355. Herbst, R. S., Nielsch, U., Sladek, F., Lai, E., Babiss, L. E., and DarneU, J. E. (1991). Differential regulation of hepatocyte-enriched transcription factars explains changes in albumin and transthyreUn gene expression among hepatoma cells. New Biol. 3: 289-296. Hooper, M., Hardy, K., Handyside, A., Hunter, S., and Mank, M. (1987). HPRT-deticient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326: 292-295. Ip, Y. T., POOD, D., Stone, n., Granner, D. K., andChalkley, R. (1990). Interaction of a liver-specific factar with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene. Mol. Cello Biol. 10: 3770-3781. Jürgens, G., and Weigel, D. (1988). Terminal versus segmental development in the Drosophila embryo: The role of the homeotic gene lorkhead. Roux's Areh. Deu. Biol. 197: 345-354. Kaestner, K. H., Lee, K.-H., Schlöndorff, J., Hiemisch, H., Monaghan, A. P., and Schütz, G. (1993). Six members of the mouse forkhead gene family are developmentally regulated. Proe. Natl. Aead. Sei. USA 90: 7628-7631. Kaestner, K. H., Ntambi, J. M., Kelly, T. J., and Lane, M. D. (1989). Differentiation-induced gene expression in 3T3-L1 preadipoeytes: A second differentially expressed gene encoding stearoyl-CoA desaturase. J. Biol. Chern. 264: 14755-14761. Knöchel, S., Lef, J., Clement, J., Klacke, B., Hille, S., Köster, M., and Knöchel, W. (1992). Activin A induced expression of a lork head related gene in posterior chardamesoderm (natochard) of Xenopus laevis embryos. Mech. Deu. 38: 157-165. Kua, C. J., Conley, P. B., Chen, L., Sladek, F. M., DamelI, J. E., and Ccabtree, G. R. (1992). A transcriptional hierarchy involved in mammalian cell-type specifieation. Nature 355: 458-460. Lai, E., snd Damell, J. E. (1991). Transcriptional control in hepatocytes: A window on development. Trends Bioehem. Sei. 16: 427430. Lai, E., Prezioso, V. R, Smith, E., Litvin, 0., Costa, H. R., and Darnell, J. E., Jr. (1990). HNF3A, a hepatocyte-enriched transcription faetor of novel strueture is regulated transcriptionally. Genes Deu. 4: 1427-1436. Lai, E., Prezioso, V. R., Tao, W., Chen, W. S., and DamelI, J. E., Jr. (1991). Hepatocyte nuclear factor 3a belongs to a family in mammals that is hamolagous to the Drosophila homeotic gene lorkhead. Genes Deu. 5: 416-427. Li, C., Lai, C., Sigman, D. S., and Gaynor, R. B. (1991). Cloning of a cellular factor, interleukin binding factor, that binds to NFAT-like motifs in the human immunodeficiency virus long terminal repeats. Proc. Natl. Aead. Sei. USA 88: 7739-7743. Li, C., Lusis, A. J., Sparkes, R., Tran, S.-M., and Gaynor, R. (1992). Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein HTLF. Genomics 13: 658-664. Li, J., and Vogt, P. K. (1993). The retroviral oncogene qin belongs to the transcription factor family that includes the homeotic gene fork head. Proc. Natl. Aead. Sei. USA 90: 4490-4494. Luckow, B., Bunz, F., Stillman, B., Lichter, P., and Schütz, G. (1994). 385 Cloning, expression and chromosomal loealization of the 140 kDa subunit of replication faetor C from mouse and man. Mol. Cello Bool., • m press. McPherson, C. E., Shim, E.-Y, Friedman, D. S., and Zaret, K. S. (1993). An active tissue-specific enhancer and bound transcription factors existing in a precisely positioned nucleosomal array. Cell 75: 387-398. Monaghan, A. P., Kaestner, K. H., Grau, E., and Schütz, G. (1993). Post-implantation expression patterns indicate a role for the mouse forkhead/HNF -3 a, ß and "y genes in determination of the definite endoderm, chordamesoderm and neuroeetoderm. Deuelopment 119: 567-578. Nitsch, D., Boshart, M., and Schütz, G. (1993). Activation ofthe tyrosine aminotransferase gene is dependent on synergy between liverspecific and hormone-responsive elements. Proe. Nat!. Acad. Sei. USA 90: 5479-5483. Oliver S. G., et al. (1992). The camplete DNA sequence of yeast chromosome III. Nature 357: 38-46. Pani, L., Overdier, D. G., Porcella, A., Qian, X., Lai, E., and eosta, R. H. (1992a). Hepatocyte nuclear faetor 3ß contains two transcriptional activation domains, one of which is novel and conserved with the Drosophila fork head protein. MoI- Cello Biol. 12: 3723-3732. Pani, L., Qian, X., Clevidence, D., and Costa, R H. (1992b). The restricted promoter activity of the liver transcription {actor hepatocyte nuclear factor 3ß involves a cell-specific faetor and positive autoregulation. Mol. Cello Biol. 12: 552-562. Ruiz i Altaba, A., and Jessell, T. M. (1992). Pintallauis, a gene expressed in the organizer and midline cells of frog embryos: Involvement in the development ofthe neural axis. Deuelopment 116: 8193. Ruppert, S., Boshart, M., Bosch, F. X., Schmid, W., Fournier, R. E. K., and Schütz, G. (1990). Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes. Ce1l61: 895-904. Sanger, F., Nickien, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Aead. Sei. USA 74: 5463-5467. Sasaki, H., and Hogan, B. L. M. (1993). Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Deuelopment 118: 47-59. Sawaya, P. L., Stripp, B. R, Whitsett, J. A., and Luse. D. S. (1993). The lung-specific CClO gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families. Mol. Cell. Biol. 13: 3860-3871. Sladek, F. M., and Damell, J. E. (1992). Mechanisms ofliver-specific gene expression. Curr. Opin. Gen.et. Deu. 2: 256-259. Tao, W., and Lai, E. (1992). Telencephalon-restricted expression of BF-1, a new member af the HNF-3/lork head gene family in the developing rat brain. Neuron 8: 957-966. Weigel, D., and Jäckle, H. (1990). The lork head domain: A novel DNA binding motif of eucariotic transcription factors. Cell63: 455-456. Weigel, D., Jürgens, G., Küttner, F., Seifert, E., and Jäckle, H. (1989). The homeotic gene lork head encodes a nuclear protein and is expressed in the terminal regions ofthe drosophila embryo. Ce1l57: 645-658.