Scand. J. Immunol. 31, 257-267, 1990

Structural Analyses of Human Developmentally Regulated Vh3 Genes p. p. C H E N Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California. USA

Chen, P.P. Structural Analyses of Human Developmentally Regulated Vh3 Genes. Scand. J. Immunol. 31,257-261, 1990 In mice, a restricted set of the Jh-proximal Vh genes arc preferentially expressed during early ontogeny. Recently, analyses of human Ig cDN A from a fetal liver revealed a restricted set of Vh genes which belong to ihe Vhl. 3. 4. and 6 families. Although ihe Vh6 and some Vh5 genes are proximal to ihe Jh region, no Vh5 gene was found in the fetal liver, suggesting that the dislance between the Jh genes and some early-expressed Vh genes may not be the only faclor responsible for Vh gene expression during early development. As an initial step in searching for other underlying mechanisms, we characterized iwo human germline Vh3 genes which belong to the developmentally restricted Vh repertoire, and found ihat they contain many enhancer-like sequences which are identical, or highly homologous to, various iranscriplional enhancer motifs. Hence, it is conceivable Ihat. in addition lo the established positional effects, cis regulatory elements may be important in the programmed expression of some Vh genes during early Blymphocyte development. Pojen P. Chen. PhD. Departmeni oJ Molecular and Experimental Medicine. Research Insliiuie of Scripps Clinic, 10666 North Torrey Pines Road. La Jolla. Cciliforniii 92037. USA

Immnunoglobulin (Ig) variable regions are encoded by the variable region genes (V), diversity (D) gene segments, and joining (J) region genes [26. 59]. Through independent assortments of these gene segments, coupled with imprecise joining and generation of the N regions at the junctions, and the combinations of heavy and light chain V regions, an enormous antibody repertoire is generated. Furthermore, the antibody repertoire is enlarged tremendously by somatie diversification of rearranged Ig gene segments [28, 36, 39, 52, 59]. In mice, the Vh genes of different families are typically clustered together, with minimal intcrspersion [1.8, 9]. The Vh genes of the 7183 family are located most closely to the Jh locus. Yancopoulos et al. [62] and Perlmutter et al. [48] found that many Vh genes of the 7183 family were preferentially rearranged in pre-B-cell lines established by transformation with Abelson murine leukaemia virus and in pre-B-cell hybridomas derived from fetal liver [1, 32, 48, 62]. Subsequently. Northern blot analysis of mRNA from newborn liver and adult spleen showed that the

more Jh-proximal Vh genes, particularly the Vh8lx gene and other Vh7l83-Vh genes, are expressed more frequently in the fetal liver than in adult spleen [2, 47, 63]. These observations prompted the authors to suggest that the recombinational machinery may work, at least in part, by tracking one-dimensionally upstream from the DJh eomplex in searching for Vh genes [2]. In humans, Vh genes from different families are interspersed extensively, and the single Vh6 gene and at least one Vh gene of the Vh5 family are most proximal to the Jh locus [6, 10. 27, 30. 56]. Interestingly, similar to mice. Schroeder et al. [55] recently found that a restrieted set of human Vh genes was preferentially expressed in a fetal liver. Among the VhcDNA clones isolated from a 130day-old fetal liver, two (51P1. 20P3), five (56P1, 30PI, 38P1, 60P2. and 20PI), one (58P2) and one (15P1) cDNA belonged, respectively, to the Vhl, 3, 4, and 6 families. In particular. 56PI was isolated three times, while 5IP1. 58P2. and 60P2 were each isolated twice. By sequence eomparison, we noted that 38P1 and the 13-2 Vh gene encoded an identical amino acid sequence and

257

258

P. P. Chen

differed from each other by only one nucleotide in thecoding region, and that 30P1 differed from the VH26 gene by two nueleotides and one amino aeid residue [6, 41]. However, we recently found VH26 to be actually identical to 30PI at both protein and DNA levels [1 Ij. In addition, excepting a few bases near the VDJ junction, the 20PI/ M26 sequence is identieal to the 9-1 Vh gene, and 60P2 differs from the 8-1B gene by only one amino acid residue and by five bases at the DNA level [6,37]. Accordingly, to delineate the possible structural basis for the restricted expression of some Vh3 genes during early ontogenie development, we decided to isolate the germline Vh3 gene forthe56Pl cDNA. Here, we report a new human Vh gene which encodes an identical amino acid sequence as that of 56PI. and the additonal DNA sequence of VH26 over a 2-kb region. An analysis of the genes suggests new potential mechanisms that, together with the established positional effects, may contribute to the preferential Vh rearrangement in early stages. MATERIALS AND M E T H O D S Library, probes and .screening. A human genomic DNA library was generated by cloning partially digested DNA frotn the Y79 retinobla:itoma cell line into EMBL-3 [33]. The library was kindly provided by Dr Wen-Hwa Lee. A Vh.l cDNA clone (56P1: from positions - 124 to 408 in the Cu region) was kindly provided by Drs H.W. Schroeder and R.M. Perlmuller [55]. and was used to screen one million recombinant clones in 2 x SSC ( l x S S C = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) at 65 C. followed by washing twice in I x SSC at 65 C [38]. DNA .sequencing. Appropriate fragments L-ontaining interesting regions of Ihe isolated EMBL-3 recombinant clones and the VH26 gene (pVH26-8; a 2--kb fragmenl in pUC19 was kindly provided by Dr T. Rabbitts) were stibcloncd into MI3mp8 [42]. The desired single-stranded recombinant phages were isolated and sequcnced by the dideoxynucleotide chaintermination melhod. In addition to the universal sequencing primer, additional oligonucleotides corresponding to different regions of ihe Vh.l genes were synthesized and used in sequencing. The computer programs of the Genetics Computer Group were used to assemble, edit, and analyse all sequence dala [14].

radiolabelled 56P1. and 335 positive clones were identified after washing with I x SSC at 65 C. Among them. 41 clones gave very strong signals. Of these 41. two (i.e. clones 5 and 33) were chosen for further characterization. The results showed that they contained human Vh3 genes that were designated Humhv3OO5 and Humhv3O33. Humhv3005 was initially sequenccd from positions l87-393(Eig. 1). It contains the Vh octamer promoter, the splicing signals (i.e. GT and AG sequences immediately after and before the leader regions respectively), and the heptamer/23 spacer/nonamer for gene rearrangement [6.59]. Thus, hv3OO5 is likely to be a functional Vh3 gene. Significantly, hv3OO5 is almost identical to the probe 56P1. Among the overlapped region of 379 bases, they differ from each other by only three bases, at positions - 182 (the intron before the leader region). 135 and 168 (Fig. 1). The last two differences are at the third codon positon and do not lead to amino acid changes. Accordingly. hv3OO5 is likely to be the corresponding germline gene for the rearranged Vh3 gene 56P1. Subsequently, we sequenced this gene over a 2.7-kb region,from - 2 k b to -1-0.7 kb(Eig. I). Regarding the three base differenees between hv3OO5 and 56PI. the possibility exists that they may be due to Vh gene polymorphism, somatic changes in 56P1. and/or a distinct different Vh gene for ,'i6Pl. Because of the limited data about human Vh gene repertoire and Vh gene sequences, the relationship between hv3OO5 and 56PI cannot be defined precisely. Fig. 2 shows the genomic structure of the Humhv3033. from positions - 7 8 to 753. The deduced amino acid sequence of this gene eontains a stop codon at the amino acid position 59 in the second complementarity-determining region (CDR), and thus is a pseudogene. Except for two ambiguous nueleotides in the first CDR of the 2-3 Vh gene, hv3033 is completely identical to the 2-3 Vh3 gene in an overlapped region of 422 bases, from positions - 7 8 to 344 [6],

Further characterization of VH26

RESULTS holation and characterization of Humhr3005 and Humlw3033 genes One million recombinant clones from a human genomic DNA library were screened with the

By sequence comparison, we noted that the reported VH26 gene is highly homologous to 30PL deviating from it by only 3/351 nueleotides in the overlapped region. To obtain a highly specific probe for the putative germline gene for 30PI, we undertook to sequence a large segment

Human Earty-E.xpressed Vh3 Genes

259

AGATCTTTTATATTGCGCATGGGTTTTTGTTTCTTCTGGGCTGTAGCAGAGATCATTGATTGGCGGTCAGGAATAAGCAGAGTTAGTGTAAAATGCACGC AAATAGTTAAACAACTGAGGAGATTAGAATTTAAAGAGAAGTGTATGATATGTTTTGAAATATAATGTTTGTCTTICCACTTTTGGTTTTTGTGAGCAGG

-1840 -1740

AAATAATGATAAGACTGAGGTGTTTGGAAAATAAAGTTTAGTGTTAAAGTTGGCGTGATTATTTGGATAAAGTGGAGCAAGAATATTAATAATAATTGTG TAGGAAAAGGGTGCTAGGAGCAGGAGTTTGAGAGTGTAATACGATGAGGAGGTGGATGGTGAGGCAACTCACTGAATATGTGGAAGGCAGGCTATTGGAA

-1640 -IS40

TCTTAAATGCGATGCAGTGGTATCTGGCGCAGGTACACTAATATATGGGTGCTGGTTGTCTGTGGAGGTCGTGTCTGCTCAGATTTCAGGTTTTGTTTAT TGTTTGTTTTGTGTCTGATATAAAGTCAGATATGTTGAAGGTTTTGTTTTTTGTATTTGTAGTAGTTCAGCTTTGTTGTTACTGAGGTGAGAATAAGGTC

-1440 -1340

-1339 -1239

ATAGTTTAGACATTTTTACATTCGCATGTGGAGTAGGTGCTTTTCTCTATCAAATGCATTAAGTGAGAGAAGAATCACATTTGGTTAGAGGTGAAGAATT AAATAGTTTGGGATATATTTATGTAGTGGAATCTAATGCAGCTTGAAATGAAGTGATGCCTGAGTGATTGAAAAAAACATGCCTAAATTCTCAAAGAACT

-1240 -1140

-1139 -1039

GTGCTGAGTGAAAGAAACTAACGAATTAAGAGTAAATTTTACGTGATAGATTTGTAGAAATTTTAGAAGATCCCACTATTATAAATTAAGATGGAGAAGA TTTAAATATTTGTGAGAATATGGTATTGGGAGTAATGGGGATGTGAGTTAAATTTCAGAGGAATAAGAGAAAGATTTAGGGATTAATTTATTGAAAGCTT

-1040 -940

GATTGAAGTGCTGAGTAAATGGTTGCAAACATAGGTGTAGGTTTTTGAAATCATTCACGATAAATTTGAATTATTTATTAATTAGAGTGGAATAAAGCAA TAAGAAAGAAAGTGATGAGATAATATTTGAGTGAATTGGAGGAATAAATAGATGGATATTAACAGAAGGAATATAAGTGAGTTCCAAAAACATAGAGATG

-840 -740

AACCGTGGTTCACTGTGGATATTTAGATAAATTAGAGAAAGTGGTGATAAGAGATGGGGAATGGTGGAGAGTTGAGTAGGGATGGGGCATGGTGGGGTGG AGTTGTGTGAGGGGAGCTGGCTCGTCGAGTGGTTAGAGGAGAGGGGCAGATAATAGGACTGATTGTTTTTAGATGTGTTATGTTAGACACGCTGGAGAAC

-640 -540

-539 -439

TGGTGTGTTGTGTATGTAAATTATGTCGTGTAAAATATAAGATTGAAGGCTGGGTTAAATATATTGTGTAAATATGTAAGAATAAAATAAAGTTATGAGA GGTAAGTGTTAATGAAGGCACAATGATATAATAATATATTTTCCTGAATGATGGAATTATTAGCAATCTGCGCGAGGGCACTTCATCTGCGCTGAGGGCA

-440 -340

-339

GGCTGTGCTCAGATCTCGGAGCGCAGAGCTTGCTATATAGTGGGGGACATGGAAATAGGGCGGTCGCTGTACTGATGAAAAGCAGGCGAGCGCTGAGGCT

- lt,3'i

-739 -639

M

•19

-239

G

L

S

-240 W

V

F

t. V

A

L

I. R

G

lyV

0

Q

G

L

V

E

S

G

G

G

V

V

Q

P

G

R

S

L

R

L

S

TCTGATAAGGGTGTCGTTGTGTTTGCAGGTGTGCACTGTCAGCTGGAGGTGCTGGftCTGTGGGGGAGGGGTGGTCCAGGCTGGGAGGTGGCTGAGAGTGT C

A

A

S

G

F

T

F

S

S

Y

A

M

H

W

V

R

Q

A

P

G

K

G

L

E

W

V

A

V

I

S

Y

D

G D R . l . . . CDR2 GDRl .. . . CD2 CCTGTGCAGCCTGTGGATTCACGTTGAGTAGGTATGGTATGGAGTGGGTGCGCGAGGCTGCAGGGAAGGGGGTAGAGTGGGTGGGAGTTATATCATATCA G

S

N

K

V

V

A

D

S

-13 -140 -^

GGGTTTTGCTCGTTGGTCTTTTAAGAGGTGATTGATGGAGAAATAGAGAGAGTGAGTGTGAGTGAACATGAGTGAGAAAAACTGGATTTGTGTGCCATTT

-4 -39

F

GGAGCTGTGGGAGAGGAGCCCAGCAGTGGAAGTGGGGGGTGTTTGCATTGGGTGATGAGGAGTGAAGACAGAGGACTCACGATGGAGTTTGGGGTGAGGT

-12 -139

E

V

K

G

R

F

T

I

S

R

D

N

S

K

N

T

L

V

L

Q

H

N

S

L

R

CDH2_ . . . . . . . TGGAAGTAATAAATACTAGGGAGACTGCGTGAAGGGCGGATTCACCATCTGGAGAGACAATTGGAAGAACAGGGTGTATCTGGAAATGAACAGCGTGAGA

-40

21 61 54 161 87 2 61

A

E D T A V Y V G A R *7-iner* . . **9-iner** CGTGAGGACACGGGTGTGTATTAGTGTGCGAGAGACACAGTGAGGGGAAGTGATTGTGGGCGCAGAGAGAAAGGTCGCTGGAGGAAGGGTGGGGGGAAAT

361

362 462

rA^CnfTGAGGCCGCGCTGAGGAGGCAGTGATCAGAGTCAGGCGTGGAGGCAGGTGCAGATGGAGGGTGTTTGGTGTGAGGATGTGGGAGTTTGTGTTCTT cTGAGAGTTCGCGAGGGAAGGTGTTAAATTTAGAAAAGTGTGCCTAACAATGTGTTGTGTATGCATATGAGGAGCTTTTCTGCGTGGCACAAAATOTiGA

461 561

562 662

-[-rcAGGGTGAGACGGATGAAAATTGCTGAAGGATGGTCACAAGGATCAGAGTCGTGAGTAAGGTGAGGGCTTCGTGGTGATTGTTGTGAAATCAGAGCCA GGAGAGGGAGCTGGGfGAGATTCCGTGACTGGAACAGTGTTTATGGATGC

661 /11

262

FIG. I. Genomic structure of Ihe ]iuman VhJl gene, Huinhv3OO5. The nucleotide sequence begins at thic nucleotide position — 1939, based on the + I assignment given to the tirst base for Ihe ftrsl amino acid residtie. The deduced amino acid sequence of hv3OO5 is shown iibove the nucleotide sequence. The complementarity-determining regions (CDR) and the conserved nucleotide seqtietices for promoter, splicing, and rearrangement (i.e. GT/AG, heptamer, and nonamer) are marked. The enhancer-like sequences are underlined.

of the VH26 gene in order to identify the region in which VH26 deviates most significantly from other human Vh3 genes. The results showed that our sequence of the VH26 gene differs from the reported sequence of VH 26 by Tour bases [11] and is actually identical to the 30P1 sequence. Sinee the pVH26-8 plasmid includes a 1.3-kb upstream region, which may contain regulatory elements for gene expression, we sequenecd the entire 2-kb insert (Fig. 3).

DISCUSSION Programmed expression of Vh genes during development of the B-cell repertoire has been clearly documented in mice [4S, 62, 63]. Interestingly, most developmentally regulated Vh genes are close to the DhJh locus, suggesting that recombinational machinery may reach Vh genes by the most efficient one-dimensional tracking during early B-ccll development [1, 2]. However,

260

P. P. Chen hv3O33 2-3

GGATAAGAGTGAGAGAAACAGTGGATACGTGTGGCAGTTTCTGACCAGGGTTTCTTTTTG

G

hv3033 2-3

E V Q L V E S G G G L V Q P 1. . . . TTTGCAGGTGTCCAGTGTGAGaTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCT

G

hv3O33 2-3

G

S

V

L

Q

-19

R

C

L

S

C

A

A

S

G

F

T

F

S

14 't2

Y

Y Y M 34 31 CDRl GGGGGGTCCGTGAGACTCTCGTGTGCAGCCTCTGGATTCACCTTCAGTTACTACTACATG 1 0 2 _

S G V R Q A P G K G L E W V G F I R N K 54 CDRl . . . . 50^ _CDR2 hv3033 AGCGGGGTCCGCCAGGCTCCCGGGAAGGGGCTGGAATGGGTAGGTTTCATTAGAAACAAA 1 6 2 2-3 -NN _.

hv3033 2-3

A N G G T T E * T T S V K G R F T I S R CDR2 62 68 GCTAATGGTGGGACAACAGAATAGACCACGTCTGTGAAAGGCAGATTCACAATCTCAAGA -__ D

hv3033 2-3

D

S

K

S

I

T

Y

L

Q

M

K

S

L

K

T

E

D

T

222

A

GATGATTCCAAAAGCATCACCTATCTGCAAATGAAGAGCCTGAAAACCGAGGACACGGGC V Y Y C S

74

94 282

R

hv3O33 2-3

100 .*7-mer* . 23 bp spacer .**9-mer**. GTGTATTACTGTTCGAGAGACAGAGTGAGGGGAGGTCAGTGTGAGCCCGGACACAAACCT 3 4 2 --

hv3033 2-3

CCCTGCAGGGGCGCGCGGGGCCACCAGGGGGCGGTAGGGACCGACTGAGGGGGGGAGAGG --/// end of the published sequence

402

hv3O33 hv3033

AGCAGGTGCCQGGAGAGGTTTCCTTTGTCCTCAGCTGGAAAAGTGAGGTTTGTGTTTGCG GGACTCTGGAGCTTTCTAGGCTGTGACGTTTTATTACTTGTGTTTATTATGAATTTATTA

462 522

hv3O33 hv3033

TCGTTAGTATTTAAATTTTAGTAATTTAAAAATTATATATATATGTTTACACTTTAATGA AATAAGCATTCCTATTTGCACCGATTCTTGCAGAGTTTTATTAACATTTGTTGACGTCAG

582 642

hv3033 hv3O33

CAGCTACGAAGGTATAGGGACATAAATTTATAACCATAGAAAGATGTATAGGCCAGGCGC GGTGGCTCACGCATGTAATCGCAGCACGTTGGAAGCGATAGGTGGGTGATC

702 753

FIG. 2. Genomic structure of the human Vh3 pseudogene, Humhv3O33. The deduced amino aeid sequence of hv30.33 is shown ahove the nuclcotide sequence. With the exception of Iwo uneertain bases in 2-3, hv3O33 is identical to 2 3 in an overlapped region of 422 bp [6]. The CDR and the conserved heptamer and nonamer are marked.

in humans, it has been demonstrated that ihe

human Vh3 genes during early ontogeny, we

single Vh6 gene and at least one Vh gene of the

screened a genomic library with the cDNA 56P1

Vh5 family are most proximal to the Jh locus [6.

and characterized a new human Vh3 gene, termed

10. 27, 56]. Curiously, no Vh5 sequence was

Humhv3005. It encodes an amino acid sequence

found in fetal liver [55, 56]. Accordingly, the

identical to 56P1, and thus is likely to be the

frequent expression of Hunihv3OO5 and, to a

germline Vh gene for the Vh cDNA 56P1. Fig. 4

lesser extent, VH26, 9-1 and 13-2 in the early

compares the structures of most reported human

human repertoire is unlikely to be accounted for

germline Vh3 genes. The potentially functional

entirely by their locations, which are presently

genes whieh are identical or highly homologous

unknown.

lo the fetal Vh3 cDNA (i.e. Humhv3005, VH26,

To delineate the underlying non-location factors responsible for the biased expression olthese

13-2, 8-IB, and 9-1) are grouped together and shown at the top. The remaining

potentially

Human Earty-Expres.sed Vh3 Genes

261

-1496 -1396

AATTGTTTAAGTGGAATACTGTTGGTGATTAGAATGAACACATTGTTTATACAGAACGAAATTAGTGACTCACAAGTAATTATAGTCAGGAAGAAGGGAG -1397 ACAAATGAAATTGAAAATATAACATTTGAG TTT AACAAAATGTTGAAAAATAGATGTGAATTAA GAAAGATGAGAGG TTGAGTGAGTATGGTGAGAGGGA -129 7

-1296 -1196

AATAATGGGGAGGCAGGAATGAGAGGAAGACAGGGAGGGTTTTAACTGTAATCATCGTTATGTCGATTGTTATTTTGGATACAGAGGTGAGGAGAGGTGA AATTAGGTTTTTTTTGGTTGGGAGACATGGTTTTGCTTTGTGGTGCAGGGTGGAGGTGGGTGGTGAGATGATAGGTGTGTGCAGGGTGAGAGGGTGGGTG

-1197 -1097

-109S -996

TGGAGCAATGCTGCTGGGTGTGGGCTATGTAGGTGGGAGTAGAGGTGTGGAGCAGCATGGTGGGCTTGAGTGGGTATTAAAGGGGAGAGTTCAATTATGG AGTATTTATTATATAGGAATAATGGCTCAATAAGGGTATTAGAAATAAGTGGATAGATAATTTGTTAAAGATTGATGGAAAGATAGATACCAACATGAGA

-997 -897

-B96 -796

AATGTATGACAGTCAAGAAAATAAAAGTGTAGCAAACTTGGTTTTCTTTATATTTGTTAGGTAATCACCACATGTGTACACATGAGACGATGTTGGCATT AGAGAGAAAAGTTCTGCGAAGCTCAGGAGGTGTGAGCCCTCTGTGCTGGGGTTGGTTGAGGGAGAAGTGAGGTGCAGTGGTGAGAAGCACAGGCCCAGAT

-79 7 -697

-696 -596

GCCGAGGGTGAGTGTGAGGAAAAGTCAGGACTGGGGAGATTGTAAAACGGAGGTGTGGTTTTGGTGATAATTTTTGATGTTTAACATGGAAATAATATTG ATAGTATATAGGATGGTTTCTGTGGGTATGTAAAAATAAAAGATGATTGGTGGTAAGTTTAAAAATATGCAGTTTATCTAGATGTATGGTAGCTCA.\TAA

-59 7 -497

-496 -396

AACTGTTTTAAAATAAAAATTAGAAAATTATAAGATTTTTAGGTTTTAAGGTTTAAGTTTATGACAAAAGAAACTGACAATAGGAAAGGAGAATTTGGGA ATGCTTTGAATATGAGAGATGTGGGGGAGGAGATTGTGAGATGGTGTGAGCGCGAGTATGTGCAAAGGGGTGTGAGGGGAGAGGTTAGTATATAGTAGGA *8-mer** . . . . . GATATGGAAATAGAGGGCrCGGTGTGGTGATGAAAACGAGCGGAGCCGTGAGGGTGCAGGTCTGAGAGAGGAGGGGAGGCGTGGGATrTTGAGGTGTTTT

-397 -297

-296

-19

-196 -4 -96

3

M

E

F

G

L

S

W

L

F

L

V

A

I

L

k

G

-4

CATTTGGTGATGAGGAGTGAACAGAGAGAACTCACGATGGAGTTTGGGGTGAGCTGGGTTTTTCTTGTGGCTATTTTAAAAGGTAATTCATGGAGAAATA lyV

E V ** . 1 . GAAAAATTGAGTGTGAATGGATAAGAGTGAGAGAAACAGTGGATACGTGTGGGAGTTTCTGACGAGGCTTTCTTTTTGTTTGGAGGTGTGGAGTGTGAGG Q

L

-197

Q

G

L

-97 2 4 35

5

104

36 105 69 205 305 405

T I

S

R

D

N

S

K

M

T

L

Y

L

Q

M

N

S

L

R

A

E

D

T

A

V

Y

Y

G

A

K

. «7-nier*. ACGATGTCGAGAGAGAATTCGAjf.'^AACACCGTGTATCTCCAAATGAAGAGGGTGAGAGGGGAGGAGAGGGCGGTATATTAGtGTGGCAAAGAGAGAGTGA . **9-mer** . . . . . . . GGGGAAGTGATTGTGAGGGCAGAGACAAAGCTCGCTGGAGGAAGGATGGGGGGGAAATGAGGGGCACGGGGGGGTGAGGACGGGGTGATGAGAGTGATCG GCAGAGGCAGGTCCAGATGGAGGGTGTTTCCTGTCAGGGTGTGGGAGTTGATCTTGTTGTGAGAGTTTCTCTAGTGAACGTGTGTAAGGTCAGAATT

404 501

FIG. 3. Further characterization of the VH26 gene. The deduced amino acid sequence is shown above the nucleotide sequenee. The CDR and the eonscrved nucleotide sequences for promoter, splieing. and rearrangement are marked. The enhaneer-hke sequences arc underlined.

functional Vh3 genes and the Vh3 pseudogenes (whieh are separated by a dashed line) are displayed at the bottom. By visual examination, it is apparent that the fetal-expressed Vh3 genes share a relatively unique stretch of 32 bp, located 16-17 bp downstream of the conserved nonamer, and have a stretch of 38 identical bp, located 74 bp upstream of the translation initiation site. These sequences are underlined in the figure. Of potential interest, the downstream 32 bp contain two partial repeats (i.e. GGGGGaaaTCA and GGGGGcgcTCA, the capital letters representing the matched nueleotides), which are homologous to the enhancer kB motif GGGG(G) A(C)TTTCC [16, 34, 57]. This motif was first identified as a nuclear factor-binding site in the murine kappa light chain enhancer region, and was subsequently shown to be important to the enhancer activity. Recently, this motif was also found in the enhancers of human immunodefi-

ciency virus (GGGACTTTCC) and simian virus 40 (SV4Q) [46, 64], and in the upstream regulatory regions of the class I major histocompatibility complex genes (GGGGATTCCCC) [4]. In addition, it was demonstrated that a single copy of kB motif could act as an activating element, whereas a dinier of kb motif induced a 10-fold increase in the transcription of a reporter gene [50]. However, the precise functions of both upstream and downstream characteristic sequences for the developmentally restricted Vh3 genes is presently unknown. Reeently. rearrangement studies in pre-B eells using construeted substrates showed that the elements to be rearranged were in a DNasesensitive region and were transcriptionally active (reviewed in Ref. 2). In addition, transcription from some unrearranged Vh genes was found to precede VDJ joining in many pre-B cells, but not in mature B cells [2]. Combined, these observa-

C TTC A

C

AO

J005 VH26 Hll AC

TCCC GOGC AG

........—•

C 4

....AC

C

T C

rcACCAT'MAGTTTGGGeTGAGtrilHWrTTTCCTCOTTOCTCTTTTAAGAGOTOATTCATGCAGAAATAO.ACAClCTnjiOTGTGAGTCWieATCACTGAGAWlXAC. . TCCATTTGTGT C T G A A A A T A C T A G AC AC A GA C T T A CC A C AG G A G A T TC G G AG A c C A T T A G 0 C AG A c A T c .G T TG A G T G AG G T CA T TCT A G C AG c A VH105 71-6 15-JB 1-3 303]

A

A CC

c

T

C A G A A A K

C C

G C C

T A

T

. . . .ACAT

TG TG TT C C .TT

G

0

. .T A A T ATG A AAT GG . ..A CAT A G G A T C G T A A C T

•lyv 3005,l VH]E 13-2 S-IB 9-1 Hll 12-2

Q

G AG

A A A

AG AC 0 AG AO

G G A A A AC

CACT

0

GCCArmCTOATAACGO. T G CC . C T T T A CTTTT TC G G ... CAA TC 0 G KCTTA TC 0 G CC G .TGT CC » G CC . C TC 0 G CT . T TC C

VH105 Tl-6 15-JB 2-3,3033 1.9III

C 0 C G A C

TC a

AA IL AA CAAAGA F

3005 VH2t 13-2 e-iB 9-1 Hll 12-2 1.9III

Q

A

I

P G

C AC A CTAC A CCCTCG CTCG GA C CTAC GC GA CTAC

VH10S,'l-6 19-IB J-J

1005 VM26 13-!

c

T S G T G

-4a

CTAC CTAC CTGA

A T D

G

S

H

T C OC T ATACC C T . . TAA ASCA AACTSA OG TAATAG C . CTAO A CA A CT AC G TT

OCTCAC C . . CC SGGACA C G . CC C ACG TACACC C G A

TAC TACTAG AC

CC T

C AC AAG G C G . G TC ACM AO T A G GGTTAC C A CTT C TAC A CA A CT A GG GGGACA C A OTT C TAG A CA A CT A GG GGGACA C A T A A G C A C . O

KNMG AG G AG CA ATTC

T T A 0 A GK C*CO 0 A OA CACS T G T T C A

K G R F T R D N S K N T L y L Q H N S L R CCGTGAAGGCCCCATTCACCATCTCCAGAGACAATTCCAAGAACACGCTCTATCTGCAAATCAACAGCCTGACAGCTGASGACACGOCTCtCTAtTACTCT.

T A VH105 71-6 1S-2B ;-3,)0J] J.9III

T C C T C C T A A G TCACC T A CCG T

OTGAGAOOCACA T

ACACAGTGAGGGGAAgTCATTCTSCGCCCACACACAAACCtCCCTaCAGGAACGCT.CGCCCCAAATCAGCCCCAGGGGGCGCTCAGGAGCCACTGATCAGAGTCAGCCCTGGAGGCAGC A A G C G T CCA

G A A

A TGC

.

T

A

T

C

433

CA

C C G TGCAG

C

T

A ACCA

GCGGT TCCTC

C CTC CTT TO105 71-6 15-JB 3-3 3033 2.9111

A A A

AACT 0 0 0 CC

CC

C A C A CCAC GCAT C A C ACA GGTC CA A M C CTAGG G CCCCA

A A

G

C C.

A A

GT . . . .

0 OG T TC

CACSSCA CT

AA CA

FIG. 4. Comparison of most reported human Vh3 germline genes. The complete nucleolide and amino acid sequences of Humhv3OO5 are given, while sequences of olher genes are given only al the positions where they differ from the hv3OO5 sequence. The potentially functional genes are on the lop. and are separaled from the pseudogenes at the bottom by a dashed line. All sequences were first aligned for maximum homology. The introduced gaps are marked by dots, which also indicate the unsequenced regions. To reduce the complexity of the figure, nucleolide sequences of diffTerent genes are listed together in one line when they are identical over that specific region. The conserved nuclcotide sequences for promoter, splicing, and rearrangement are marked. The potential enhancer-like sequences and Ihe two characteristic stretches for the early-expressed Vh genes are underlined. Genes !3 2, 8- IB, 9-1, 12-2, I.9[II.22-2B, l5-2B,2-3.and2.9IIIwcrereportedby Berman ('/»/. |6|. H i t . VH 105 and 71-6 were reported by three different laboratories [27, 30. 53].

Human Early-Expres.sed Vh3 Genes POSITIOHS FHOH, TO Enhancer OCTA Motif Murine Ig Enhancer OCTA hv30O5 -1836, -1843 hv30O5 -1679, -1.672 hv30O5 -876, -869 hv3005 -519, -526 hv3005 246, 241 hv3005 529, 522 hv3005 562, 555 hv3005 584, 577 VH2 6 -611 -604, 241 VH2 6 248, 2. Human Iq Enhancer OCTA hv3005 -1571, -1578 hv-3005 -54, -47 VH26 -1391, -1398 i. Ig Enhancer E Motif frequency 1. 2.

MhEl HhEl hv3O05 hv3005

3 5 0 , 357 53, 60 -1164, -1157 -1073, -1066

3.

MhE2 hv3005 VH26

4.

HhE2 hv3O05 VH26 VH26

6 . MhE4 6. HhE4 hv3O05 VH26 VH26 VH26 7.

A T T T G C A T A T T T G C A T - - - - - - C -

REFERENCES 1,2,9-11 1,2,9-11

- - - - - a - - - - - a - - g -

-

a c -

-

t c

-

-

-

A -

T q -

T -

T -

G -

C t t

G -

T -

g 4,6

C A G G T G G C 93 79 100 79 100 7 1 100 64 7,8,11 6

3 8 2 , 389 7, 14 - 1 4 6 , -139

C A G C T G G C - - . . . . _ _ t g . _ _ _ _ _ _

7,8,11

87, 94 -14, -7 - 6 4 2 , -649 -14, -7

C A G G T G G _ _ _ _ _ _ t _ _ _ _ _ _ _ t

C „ -

6,8

C C . _ _

T T g _ -

5 3 6 , 529 2 2 9 , 222 -1823, -1830 -1055, -1048 -206, -199 7, 14 80, -S89,

hv300S hv3005 VH26 VH26

-330, -323 417, 424 -701, -694 418, 425

8. HkEl' hv3005 VH26

74, 81 -356, -349 -696, -703

9. HkEl' hv3005 hv3O05 VH26

75, 82 6, -2 416, 409 417, 410

ll.HkE3 hv3aO5 hv30O5

MATCHES

C C -

MkEl hv3005

10.MkE3 VH26 VH26

SEQUENCE

73 -682

A A G A A G c - _ _

A G A G _ . _ _ _ _ _

A A _

G G t _ _ c

T T _

T T _ _ -

G G _

G G _ _ -

G G c

G T _ _ -

C A G A T G G C _ _ _ _ _ _ _ g

_ _ _ _

_ _

_ _ _ _

_ _ _ _

_ _ _ _

_ _ _

t _ c _

184 -330 -189

C A T G T G G T - _ _ - _ _ _ _ _ _ _ t - - - -

177, 155, 160,

184 162 153

C A T A T G G T _ _ _ _ _ _ a a -

A P l / c - j u n Enhancer VH26 -1431, -1425 VH26 ^1318, -1312

D.

SV40 E n h a n c e r GT/C M o t i f hv3005 - 1 7 6 0 , -1767 VH26 -948, -941 VH26 -922, -915

3,11

C -

177, -e23, -196,

C.

3,8,11

a n

CATCTGG C _ _ _ _ _ _ c _ _ _ _ _ _ _ q CACCTGG _ _ _ _ _ _ a _ _ _ _ _ _ , _ _ _ _ _ _ c

6,B

3,8,9,11

T G A C T CA _ _ _ _ _ _ _ _ _ _ _ _ _ _

G c _ a

T G G A A A - - - - - _ _ _ _ t - - - - - -

G -

FIG. 5. Identification of the potential enhancer elements. The sequences and positions of murine enhancer E molifs MhEl, MhE2, MhE4, MkEl, MkEI', and MkE3 are according to Emorinctvc//. [16], Ephriis.si £•;(*/. [17], Churchffa/. [12],Senf(u/. I57],and Lenardof/fl/. [34]. whereas that of the human HhEl. HhE2, HhE4. HkE I', and HkE3 are according to Emorine et at. [16], Church c?fz/. [12]. and Hayday c/ij/. [24]. The references I I6in this figure correspond respectively to references 21. 5, 16.43,49. 24, 17. 12, 57, 20, 34, 60, 7. 61, 25 and 64 of this article.

263

264

P. P. Chen

tions suggested that transcription of the involved elements could be required for rearrangement, and that transcriptional enhancers may play a role in the developmentally regulated expression of Vh genes. This view is supported by the observation that mature B cells exhibited pancreatic deoxyribonuclease- (DNase 1) hypersensitive sites in the Ig enhancer regions within the J-C introns that were absent in the chromatin of other differentiated cells [43. 51]. If transcriptional regulatory elements are involved in the programmed rearrangement and expression of Vh genes, then those Vh genes that are expressed in early ontogeny would be expected to share special promoters and/or enhancers. Because the Ig enhancers are characterized better than Ig promoters, we search for enhancer-like sequences only in the present study. It is remarkable that both hv3005 and VH26 indeed have many short stretches of sequences whieh are identical, or highly homologous, to known enhancer sequences. Fig. 5 shows only the sequences from hv3OO5 and VH26 which (I) share at least seven bases with the known enhaneer sequences, (2) differ from the corresponding enhancers by at most one base, and (3) contain all the known invariable nueleotides of the respective enhancer sequences. The murine Ig enhaneer octamer and Ig enhancer E motifs have been defined by both funetional assays and proteinbinding assays [5, 12.17,18.20,21,34.44,49,57. 60]. ln humans, functional studies have localized the Ig heavy chain enhaneer to a 279-bp Alul fragment which is highly homologous to the murine Ig heavy chain enhancer region [24]. Accordingly, the human heavy chain enhaneer region sequence was aligned with the murine sequenee. and the stretches corresponding to murine octamer, MhEl. MhE2, and MhE4. were tentatively defined as the human oetanier, HhEl, HhE2, and HhE4 [12]. Similarly, although the human kappa light chain enhancer has not been defined by functional analysis, the human kappa intron conserved region was aligned with the murine kappa enhaneer region, and the stretches corresponding to murine MkEI' and MkE3 were tentatively defined as the human HkEl' and HkE3 [12, 16]. API, originally identified as a sequenee-speeifie trans-activator protein, was recently found to be eneoded by the cellular oneogene e-jun [7]. The APl/c-jun protein positively autoregulates its own gene, and interacts with c-fos protein to form a sequenee-speeifie

DNA-binding complex [3, 31]. Additionally, the c-jun gene has been mapped to lp3I-32, a chromosomal region whieh is frequently deleted in neuroblastomas, suggesting that API,/c-jun may be important in regulating eel! differentiation and proliferation [23]. The SV40 enhancer spans about 100 bp and is composed of multiple functional elements, including GT/C, TC, Sph/B, and P motifs [25. 61. 64]. Among them, the most dramatic decrease of enhancer activity (88% reduction) was caused by a single triple base change of TGG to GTT in the GT/C motif (GrGGAAAG) [64]. Furthermore, it was shown that enhancers can be generated by various assortments oi the same or different motifs [25, 64]. The Humhv3OO5 gene contains, in addition to the conserved octamer 'promoter' [15, 19, 22, 40, 44], one completely matched octamer sequence and nine octamer-like sequences (which match 7 out of 8 bases), 13 E motif-like sequences, and one SV40 enhaneer GT/C motif (Eigs 1,4, and 5). In this regard, it should be noted that the genes for two octamer-binding proteins were shown recently to contain the homeodomains (i.e. 'homeobox" domains) [13,29,45,54,58]. Homeoboxes are gene sequences in the homeotic genes of the fruit fly whieh dictate the development of the fruit fly body (reviewed in Ref. 35). Thus, it seems likely that the mammalian homeobox-eontaining genes are also involved in the regulation of tissue and organ development. Similar to Humhv3OO5, VH26 has many sequences which are identieal, or highly homologous, to Ig enhancer E motifs, oetamer, the APl/c-jun binding site, and the SV40 enhancer GT/C motif(Eigs 3 and 5). As can be seen in Fig. 4, two E motif-like sequences (at positions 435-428 and 436-443) are shared by all Vh3 functional genes, while two other E motiflike sequences (at positions —14 to — 1, and 7-14) and 1 oetamer-like sequenee are conserved in both functional and non-functional Vh3 genes. In addition to the last-mentioned three enhaneerlike elements, a similar computer analysis of a 746-bp stretch of the 2.9III Vh3 pseudogene showed up only one additional enhancer-like sequenee. The precise meaning of these potential regulatory elements is still unclear. Funetional analyses of these regions are under way and should reveal their possible role in the early expression of Humhv3OO5 and VH26. In summary, among the developmentally regulated human Vh genes, the single Vh6 gene is the

Human Earty-Expressed Vh3 Genes most Jh-proximal, and this may aeeount for its programmed expression in the fetus. In contrast, the immediate neighbour Vh5 gene(s) was/were not found in the early human repertoire, suggesting other non-positional factors may be involved in the developmental regulation of Vh gene expression. Here, we show that some earlyexpressed Vh3 genes contain many enhaneer-like sequences. It is hypothesized that, in addition to the established positional effects, cis regulatory elements in the flanking regions may influence the early expression of certain Vh3 genes.

ACKNOWLEDGMENTS I greatly appreciate the helpful comments of Dr D. A. Carson during the course of this work. I thank Dr W. Lee for the human genomic library. Dr T. Rabbitts for pVH26-8, and Drs H. W. Schroeder and R. M. Perlmutter for the Vh3 eDNA elone 56P1. I thank S. Sinha for his technical assistance, and the Molecular and Experimental Medicine Word Proeessing Center for their help in preparing this manuscript. Funding for this research is supported In part by grants AR39039, AR33489, and RR00833 from the National Institute of Health. This is publication number 5752MEM from the Research Institute of Scripps Clinie.

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Received 12 June 1989 Accepted in revised form 15 August 1989