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Hypothesis: The removal of VH gene downstream sequences is required for its expression in mature B cells
Molecular Immunology, Vol. 26, No. Printed in Great Britain.
8. pp.
121-725,
1989
0161-5890/89$3.00 + 0.00 Q 1989 Maxwell Pergamon Macmillan plc
HYPOTHESIS: THE REMOVAL OF V, GENE DOWNSTREAM SEQUENCES IS REQUIRED FOR EXPRESSION IN MATURE B CELLS
ITS
GIDEON RECHAVI*
Institute of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University,
Israel
(First received 16 January 1989: accepted 24 February 1989) Abstract--In immunoglobulin producing cells only the rearranged V, gene is expressed, while all the other germline V, genes are silent. On the other hand, it was shown that unrearranged V, gene segments are expressed at a high level in the early stages of B lymphocyte differentiation. This expression is independent of the Ig heavy chain enhancer element. It seems, therefore, that unrearranged V, gene expression is depressed in mature B cells by a mechanism that will not operate on the rearranged functional heavy chain gene. We have shown that a highly conserved DNA segment is present upstream to several mouse and human V, genes. DNA sequencing downstream to germline genes revealed a DNA element related to the one described at the 5’ of the V, genes. A model is proposed in which the 5’ and 3’ conserved segments are functional in the regulation of V, gene expression. The model suggests the existence of a protein in mature B cells which recognizes the 5’ and 3’ conserved DNA elements and prevents germline V, gene expression by binding to these segments. V,-D-J, joining results in the deletion of the 3’ flanking sequences of the rearranged V, gene but not of other upstream V, genes. This would result in the release of promoter suppression in the case of the rearranged gene, while all other V, genes would remain transcriptionally silent.
to primary structural changes of the transcription initiation region. Several mechanisms were suggested as an explanation for the transcription activation of Ig heavy chain gene following rearrangment. It was shown that an enhancer element is present in the J&p intron 5’ to the heavy chain switch region DNA rearrangement, which brings a V, gene in the vicinity of the constant region genes, thereby bringing the JH promoter under the influence of the enhancer element (Gillies et a/., 1983; Banerji et al., 1983; Queen and Baltimore, 1983). Other DNA elements were proposed to facilitate Ig gene expression, such as Cp intragenic sequences and sequences located 3’ to the constant region genes (Grosschedl and Baltimore, 1985; Gregor et al., 1986). The generally accepted notion that germline Ig V, gene segments are not expressed and that assembly of a complete heavy chain gene is essential for its activation has recently been challenged. Yancopoulos and Ah (1985) have demonstrated that unrearranged V, gene segments are expressed at a high level in the very early stages of B lymphocyte differentiation. This expression is independent of the enhancer (Yancopoulos and Ah, 1985). In addition, several B cell lines deleted of enhancer element have retained the ability to synthesize and secrete normal amounts of Ig heavy chains (Wabl and Burrows, 1984; Klein et al., 1984, 1985; Eckhardt and Birshtein, 1985; Aguilera et al., 1985; Zaller and Eckhardt, 1984). Moreover, certain human acute and chronic lymphatic leukemias transcribe short polyadenylated VH-RNA. The expressed V, genes are not rearranged
The variable region of the immunoglobulin (Ig) heavy chain is encoded in three gene segments: J,, D and V, (Early et al., 1980; Sakano et al., 1981; Siebenlist et al., 1981). During early B lymphocyte development, one of the D segments recombines with a JH segment. A second rearrangment results in V, segment fusion with the rearranged D-J, segment. The latter creates a complete V,-D-J, gene segment located several thousand basepairs (bp) 5’ to the constant region gene, Cp (Ah et al., 1986; Kemp et al., 1981; Bentley and Rabbit& 1981; Givol et al. 1981; Tonegawa, 1983). Although each V, gene segment has its own promoter, it is usually transcriptionally silent in the germline context (Clarke et al., 1982; Sakano et al., 1980). The Cp has a low constitutive activity (Kemp et al., 1980; Ah et al., 1982). Heavy chain gene expression is turned on in pre-B cells following the rearrangement that positions the V, gene in the vicinity of Cp (Kemp et al., 1980; Alt et al., 1982; Davis et al., 1980). The rearranged V, gene is expressed, while all other germline V, genes are silent in Ig producing cells. No sequence differences were found in the 600 bp long region 5’ to the transcription initiation site between a germline and an active V, gene (Clarke et al., 1982). Therefore, the basis for the expression of the rearranged V, gene is not related
*Address correspondence to the author at: Institute of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer. Israel 52 621. 721
122
GIDEON RECHAVI Downslream
Upstream Inverted Human
HG3
TCATGAATAT
rnO”SB
105
TCATGAATATG
mouse
WA
TCATGAATATG
VI-
mouse
lO8B
TCATGAATATG
wt_
mouse
III
TCATGAATAlZ
L-1
d’rec’
repeat ]_-~__{i~ef~~repea;-)
ml-
- m
CAAATCACCT
repeo’
TCATAATGT
- -I-]
TCTATAAAATG
t--~--{~l - - 4-1
TCTATGAAATG
- - m
- - j?%ii%%-]
CCTGTAAAATT
- - -&)
- -{?i%i%i-]
TCTATAAAATG
- m
Fig. 1. Human and murine conserved Vu flanking sequences. The upstream conserved sequences are located about 130 bp 5’ to the first ATG of the Vu gene. The downstream sequences are about 300 bp 3’ to the terminal codon. Each upstream conserved sequence contains direct and inverted repeats of the downstream sequence. in these cases and it was suggested that the short transcripts are transcribed from a germline Vu gene (Humphries et al., 1988). On the other hand, it has been demonstrated that the heavy chain enhancer is capable of activating transcription of V, promoter located 17.5 kb away and of activating tandem V, promoters (Wang and Calame, 1985). Why, therefore, do V, gene segments in the vicinity of the rearranged Vu remain transcriptionally silent? It is possible that the unrearranged V, genes are silent in Ig producing cells due to an active repression mechanism rather than due to the lack of enhancer effect, per se. It is suggested that such a repression mechanism will be activated only at the V,-D rearrangement stage and will be effective only on unrearranged V, genes, whereas the Vu-D-J, gene will be free of suppression. Since DNA sequences 3’ to the V, are being removed during Vu-D-J, joining, I would like to suggest that this involves the removal of 3’ regulatory sequences leading to the release of V,-D-J, expression from the repression mechanism in mature B cells. STEM
Several years ago, we showed that a highly conserved DNA segment is present approximately 130 bp upstream to the initiator ATG of several V, genes in both mouse and man (Rechavi et al., 1983). An octanucleotide sequence, ATGCAAAT, contained in the conserved region, was found 5’ to many V, genes (Parslow et al., 1984; Calame, 1985; Dreyfus et al., 1987) while an inverted repeat of this octanucleotide sequence, ATTTGCAT, was found 5’ to light chain V, genes. These DNA octamer elements are essential for the efficient function of the immunoglobulin promoter in B cells (Parslow et al., 1984; Calame, 1985; Dreyfus et al., 1987). Recently, nuclear factors were identified that bind to these DNA elements (Neuberger and Cook, 1988). DNA sequencing downstream to germline Vu genes revealed a DNA sequence which is inversely complementary to the one described at the 5’ of the Vu genes (Rechavi et al., 1983). This sequence was found at a distance of approximately 300 bp 3’ to the last codon of the two V, genes that were analyzed. The 5’ and 3’ inverted repeats are flanked by direct repeats, as shown in
CELL
VH gene segments r--‘----
1” gene segments
0 gene segments
Constant region gene*
7
I
..,..w-MH]__
D-C3_C
$4 n n-1: L_..__._-: r__.___________
L.._____________
__.-
cp
C6
cy
_.___________
I
0
promoterVHn+l
,
&in
I I
I I I I
L________--_--__-
I I ,
_----_--
-
---_-
-
_-I
Fig. 2. Immunoglobulin heavy chain genes in stem cells. Vu gene segments, D gene segments, J, gene segments and constant region genes (C-mu, C-delta, C-gamma, etc.) are in the germline configuration. E, enhancer; Vu n + 1, Vun, representative Vu genes. Although the Vu genes are accessible, no nuclear factor is synthesized by the cell and no Vu gene expression is found.
PRF-8
Removal
of V, gene downstream
Rearranged gene r----7
Constant region genes
723
sequences
CELL
V” gene segments
cp
C6
CT
k-4 2-Ex
.“’ ‘.
L___r__-l cl-__-_--__----_---_----_---------___-__,
r-----------
i
I I
nuclear
j factor 8 I I
I
RNA polymerase
b&.@,,A 2;;’
I
, _ i;j:.:j
I I I I I I
....%..$ :.i:s I I promoter VHntl promorer V,n 0 J, I I 0 rearranged gene I L_______________--____-_____--__--_____--__-__-_-_------J
Fig. 3. Immunoglobulin heavy chain genes in pre-B cells. The V,n underwent V&-J, rearrangement, whereas the V,n + 1 remains in the gene line configuration. Nuclear factors are synthesized and both rearranged and non-rearranged V, genes are expressed.
Fig. 1 (Rechavi et al., 1983). We have now identified a similar sequence in the flanking regions of three additional mouse V, genes (Givol et al., 1981; Cohen et al., 1982). In these cases none of the direct or inverted repeats are perfect, but their conservation within a region of highly diverged sequences is striking. A model is proposed in which the 5’ and 3’ conserved segments serve a function in the regulation of V, gene expression (Figs. 2, 3 and 4). This is in analogy to the inverted repeat which constitutes the operator of the E. co/i gal operon (Irani et al., 1983; Kolata, 1983). The model postulates the existence of a repressor protein which recognizes the 5’ and 3’ conserved DNA segments and which prevents V,
B V,
CELL gene
segments
r----------:
0 I
gene expression by binding to these segments. This protein could bc a dimer which brings the two segments in proximity, thereby condensing the chromatin at the site of each V, gene. As a result, the V, promoter would be inaccessibe to RNA polymerase and/or DNA binding factors and therefore the gene would remain silent (Fig. 3). The process of V,-D-J, joining results in the deletion of the 3’ segment of the rearranged V, gene, but not of the other V, genes upstream to it. This would result in the release of the promoter region from suppression in the case of the rearranged gene, while all other VH promoters would remain inactive. Hence, this model can account for the exclusive activation of a single V, gene upon DNA rearrangement (Fig. 4). The demonstration of
Rearranged gene r----7
Constant region genes
_I____________-__--__---_---_---
----
fINA
factor\
promoter
VHn
rear ranged I
-_-_,
nuclear polymerase
D
I !
1~
gene
I VMntl VHn+2 :.________-_ -____ ____ _ _ _______ _____________J
I
Fig. 4. Immunoglobulin heavy chain genes in B cells. The proposed repressor factors are present at this stage and bind to the conserved 5’ and 3’ sequences of all non-rearranged V, gene segments. Only the rearranged gene that had lost its 3’ conserved sequence is accessible to the nuclear factors and to the polymerase. Because of the proximity to the enhancer sequences, the rearranged gene is transcribed at a high level.
724
GIDEON
active germline V, transcription at the pre-B stage of differentiation suggests that transacting factors are produced which are capable of transcription enhancement. It is suggested that, at the subsequent stage, specific production of the putative repressor is responsible for the repression of non-rearranged Vu genes. Immature pre-B cells actively express the germline V, genes and show Vu to D,-Ju joining. In contrast, more mature B cells usually do not show the above mentioned molecular changes (Yancopoulos and Ah, 1985; Blackwell et al., 1986). It seems, therefore, that a permissive V, locus is a prerequisite for both Vu expression and V, to D-J, joining. The proposed model predicts that the putative repressor factor, by condensing and “locking” the germline Vu gene locus, is responsible for the prevention of both Vu expression and recombination. This hypothesis would predict that similar DNA segments are present in other V, subgroups as well as in V, genes. Our model can be experimentally tested by sequencing the flanking regions of more V genes or by the removal of the 3’ segment from a molecularly cloned Vu gene and testing its expression in myeloma cells (Rice and Baltimore, 1982; Oi et al., 1983). Acknowledgements-1 am indebted to Mrs Rivka Amit for expert manuscript preparation and to Mrs Nili Shaked for the preparation of the graphic presentation. I am indebted to Dr David Givol for his help and to Drs Bracha Ramot, Isaac Ben-Bassat and Justus Cohen for stimulating discussion This work is supported in part by the grants given to G. Rechavi by the Maurits Van Beets Fund for Leukemia Research, the Freda and Moise Eskenazy Fund for Cancer Research, the Jacob and Judy Zemel Cancer Research Fund and the Irwin Edelstein Leukemia Research Fund. REFERENCES Aguilera R. J., Hope T. J. and Sakano H. (1985) Characterization of immunoglobulin enhancer deletions in murine plasmacytomas. EMBO J. 4, 3689-3693. Alt F. W., Blackwell T. K., DePinho R. A., Reth M. G. and Yancopoulos G. D. (1986) Regulation of genome rearrangement events during lymphocyte differentiation. Immun. Rev. 89, 5-30. Alt F. W., Rosenberg N., Enea V., Siden E. and Baltimore D. (1982) Multiple immunoglobulin heavy-chain gene transcripts in Abelson murine leukemia virus-transformed lvmphoid cell lines. Molec. Ceil. Biol. 2. 386400. Banerji J., Olson L. and Schaffner W. (1983) A lymphocytespecific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33, 7299740. Bentley D. L. and Rabbitts T. H. (1981) Human V kappa immunoglobulin gene number: implications for the origin of antibody diversity. Cell 24, 613-623. Blackwell T. K.. Moore M. W.. Yancopoulos G. D.. Suh H., Lutzker S., Selsing E. and Al; F. A. (1986) Recombination between immunoglobulin variable region gene segments is enhanced by transcription. Nature 324, 5855589. Calame K. L. (1985) Mechanisms that regulate immunoglobulin gene expression. A. Rec. Immun. 3, 159-195. Clarke C., Berenson J., Goverman J., Bayer P. D., Crews S.. Sin G. and Calame K. L. (1982) An immunoglobulin
RECHAVI promoter region is unaltered by DNA rearrangement and somatic mutation during B-cell development. Nucl. Acids Res. 10, 7131~1149. Cohen J. 9.. Effron K., Rechavi G., Ben-Neriah Y., Zakut R. and Givol D. (1982) Simple DNA sequences in homologous flanking regions near immunoglobulin VH genes: a role in gene interaction. Nucl. Acid.y Res. 10, 335333370. Davis M., Calame K. L., Early P., Livant D., Joho R., Weisman I. and Hood L. (1980) An immunoglobulin heavy-chain gene is formed by at least two recombinational events. Nuture 283. 7333738. Dreyfus M., Doyen N. and Rougeon F. (1987) The conserved decanucleotide from the immunoglobulin heavy chain promoter induces a very high transcriptional activity in B-cells when introduced into an heterologous promoter. EMBO J. 6, 168551690. Early P., Huang M., Davis M., Calame K. L. and Hood L. (1980) An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell 19, 981-992. Eckhardt L. A. and Birshtein B. K. (1985) Independent immunoglobulin class-switch events occurring in a single myeloma cell line. M&c. Cell. Biol. 5, 856868. Gillies S. D., Morrison S. L., Oi V. T. and Tonegawa S. (1983) A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33, 717-728. Givol D., Zakut R., Effron K., Rechavi G., Ram D., and Cohen J. B. (1981) Diversity of germ-line immunoglobulin V, genes. Nature 292, 426430. Gregor P. D., Kobrin B. J., Milcarek C. and Morrison S. L. (1986) Sequences 3’ of immunoglobulin heavy chain genes influence their expression. Immun. Rec. 89, 3147. Grosschedl R. and Baltimore D. (1985) Cell-type specificity of immunoglobulin gene expression is regulated by at least three DNA sequence elements. Cell 41, 8855897. Humphries C. G., Shen A., Kuziel W. A.. Capra J. D., Blattner F. R. and Tucker P. W. (1988) A new immunoglobulin V, family preferentially rearranged in immature B-cell tumors. Nature 331, 4466449. Irani M. H., Orosz L. and Adhya S. (1983) A control element within a structural gene: the gal operon of Escherichia coli. Cell 32, 783-788. Kemp D. J., Harris A.. Cory S. and Adams J. (1980) Expression of the immunoglobulin Cmu gene in mouse T and B lymphoid and myeloid cell lines. Proc. n&n. Acad. Sri. U.S.A. 77, 28762880. Kemp D. J., Taylor B., Bernard O., Gough N., Gerondakis S., Adams J. and Cory S. (1981) Organisation of genes and spacers within the mouse immunoglobulin VH locus. J. m&c. appl. Genet. 1, 2455261. Klein S., Gerster T., Picard D.. Radbruck A. and Schaffner W. (1985) Evidence for transient requirement of the IgH enhancer. Nucl Acids Res. 13, 8901. Klein S., Sablitzky and Radbruck A. (1984) Deletion of the IgH enhancer does not reduce immunoglobulin heavy chain production of a hybridoma IgD class switch variant. EMEO J. 3, 247332476. Kolata G. (1983) Control element found within structural gene. Science 220, 294. Neuberger M. S. and Cook G. P. (1988) The expression of immunoglobulin genes. Immun. Toduy 9, 278-28 1. Oi V. T., Morrison S. L., Herzberg L. A. and Berg P. (1983) Immunoglobulin gene expression in transformed lymphoid cells. Proc. natn. Acad. Sci. U.S.A. 80, 825-829. Parslow T., Blair D., Murphy W. and Granner D. (1984) Structure of the 5’ ends of immunoglobulin genes: a novel conserved sequence. Proc. natn. Acud. Sci. U.S.A. 81, 2650-2654. Queen C. and Baltimore D. (1983) Immunoglobuhn gene transcription is activated by downstream sequence elements Cell 33, 741 748.
Removal
of V, gene downstream
Rechavi G., Ram D., Glazer L., Zakut R. and Givol D. (1983) Evolutionary aspects of immunoglobulin heavy chain variable region (VH) gene subgroups. Proc. mm. Acud. Sri. U.S.A. 80, 855-859. Rice D. and Baltimore D. (1982)Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line. Proc. mtn. Acad. Sci. U.S.A. 77, 7862-7865. Sakano H.. Kurosawa Y., Weigert M. and Tonegawa S. (I 98 I) Identification and nucleotide sequence of a diverse DNA segment (D) of immunoglobulin heavy-chain genes. Nature 290, 562-565. Sakano H.. Maki R., Kurosawa Y., Roeder W. and Tonegawa S. (1980) Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature 286, 676683. Siebenlist U., Ravetch J. V., Korsmeyer S., Waldman T. and Leder P. (1981) Human immunoglobulin D segments
sequences
725
encoded in tandem multigenic families. Nature 294, 631635. Tonegawa S. (1983) Somatic generation of antibody diversity. Nature 302, 575-58 1. Wabl M. and Burrows P. (1984) Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in ris. Proc. mtn. Acad. Sci. U.S.A. 81, 2452-2455. Wang X.-F. and Calame K. L. (1985) The endogenous immunoglobulin heavy chain enhancer can activate tandem VH promoters separated by a large distance. Cell 43, 6599665. Yancopoulus G. D. and Ah F. A. (1985) Developmentally controlled and tissue-specific expression of unrearranged V, gene segments. Cell 40, 271-281. Zaller D. M. and Eckhardt L. A. (1984) Deletion of a B-cell-specific enhancer affects transfected, but not endogenous, immunoglobulin heavy-chain gene expression. Proc. mtn. Acad. Sci. U.S.A. 82, 5088-5092.
8. pp.
121-725,
1989
0161-5890/89$3.00 + 0.00 Q 1989 Maxwell Pergamon Macmillan plc
HYPOTHESIS: THE REMOVAL OF V, GENE DOWNSTREAM SEQUENCES IS REQUIRED FOR EXPRESSION IN MATURE B CELLS
ITS
GIDEON RECHAVI*
Institute of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University,
Israel
(First received 16 January 1989: accepted 24 February 1989) Abstract--In immunoglobulin producing cells only the rearranged V, gene is expressed, while all the other germline V, genes are silent. On the other hand, it was shown that unrearranged V, gene segments are expressed at a high level in the early stages of B lymphocyte differentiation. This expression is independent of the Ig heavy chain enhancer element. It seems, therefore, that unrearranged V, gene expression is depressed in mature B cells by a mechanism that will not operate on the rearranged functional heavy chain gene. We have shown that a highly conserved DNA segment is present upstream to several mouse and human V, genes. DNA sequencing downstream to germline genes revealed a DNA element related to the one described at the 5’ of the V, genes. A model is proposed in which the 5’ and 3’ conserved segments are functional in the regulation of V, gene expression. The model suggests the existence of a protein in mature B cells which recognizes the 5’ and 3’ conserved DNA elements and prevents germline V, gene expression by binding to these segments. V,-D-J, joining results in the deletion of the 3’ flanking sequences of the rearranged V, gene but not of other upstream V, genes. This would result in the release of promoter suppression in the case of the rearranged gene, while all other V, genes would remain transcriptionally silent.
to primary structural changes of the transcription initiation region. Several mechanisms were suggested as an explanation for the transcription activation of Ig heavy chain gene following rearrangment. It was shown that an enhancer element is present in the J&p intron 5’ to the heavy chain switch region DNA rearrangement, which brings a V, gene in the vicinity of the constant region genes, thereby bringing the JH promoter under the influence of the enhancer element (Gillies et a/., 1983; Banerji et al., 1983; Queen and Baltimore, 1983). Other DNA elements were proposed to facilitate Ig gene expression, such as Cp intragenic sequences and sequences located 3’ to the constant region genes (Grosschedl and Baltimore, 1985; Gregor et al., 1986). The generally accepted notion that germline Ig V, gene segments are not expressed and that assembly of a complete heavy chain gene is essential for its activation has recently been challenged. Yancopoulos and Ah (1985) have demonstrated that unrearranged V, gene segments are expressed at a high level in the very early stages of B lymphocyte differentiation. This expression is independent of the enhancer (Yancopoulos and Ah, 1985). In addition, several B cell lines deleted of enhancer element have retained the ability to synthesize and secrete normal amounts of Ig heavy chains (Wabl and Burrows, 1984; Klein et al., 1984, 1985; Eckhardt and Birshtein, 1985; Aguilera et al., 1985; Zaller and Eckhardt, 1984). Moreover, certain human acute and chronic lymphatic leukemias transcribe short polyadenylated VH-RNA. The expressed V, genes are not rearranged
The variable region of the immunoglobulin (Ig) heavy chain is encoded in three gene segments: J,, D and V, (Early et al., 1980; Sakano et al., 1981; Siebenlist et al., 1981). During early B lymphocyte development, one of the D segments recombines with a JH segment. A second rearrangment results in V, segment fusion with the rearranged D-J, segment. The latter creates a complete V,-D-J, gene segment located several thousand basepairs (bp) 5’ to the constant region gene, Cp (Ah et al., 1986; Kemp et al., 1981; Bentley and Rabbit& 1981; Givol et al. 1981; Tonegawa, 1983). Although each V, gene segment has its own promoter, it is usually transcriptionally silent in the germline context (Clarke et al., 1982; Sakano et al., 1980). The Cp has a low constitutive activity (Kemp et al., 1980; Ah et al., 1982). Heavy chain gene expression is turned on in pre-B cells following the rearrangement that positions the V, gene in the vicinity of Cp (Kemp et al., 1980; Alt et al., 1982; Davis et al., 1980). The rearranged V, gene is expressed, while all other germline V, genes are silent in Ig producing cells. No sequence differences were found in the 600 bp long region 5’ to the transcription initiation site between a germline and an active V, gene (Clarke et al., 1982). Therefore, the basis for the expression of the rearranged V, gene is not related
*Address correspondence to the author at: Institute of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer. Israel 52 621. 721
122
GIDEON RECHAVI Downslream
Upstream Inverted Human
HG3
TCATGAATAT
rnO”SB
105
TCATGAATATG
mouse
WA
TCATGAATATG
VI-
mouse
lO8B
TCATGAATATG
wt_
mouse
III
TCATGAATAlZ
L-1
d’rec’
repeat ]_-~__{i~ef~~repea;-)
ml-
- m
CAAATCACCT
repeo’
TCATAATGT
- -I-]
TCTATAAAATG
t--~--{~l - - 4-1
TCTATGAAATG
- - m
- - j?%ii%%-]
CCTGTAAAATT
- - -&)
- -{?i%i%i-]
TCTATAAAATG
- m
Fig. 1. Human and murine conserved Vu flanking sequences. The upstream conserved sequences are located about 130 bp 5’ to the first ATG of the Vu gene. The downstream sequences are about 300 bp 3’ to the terminal codon. Each upstream conserved sequence contains direct and inverted repeats of the downstream sequence. in these cases and it was suggested that the short transcripts are transcribed from a germline Vu gene (Humphries et al., 1988). On the other hand, it has been demonstrated that the heavy chain enhancer is capable of activating transcription of V, promoter located 17.5 kb away and of activating tandem V, promoters (Wang and Calame, 1985). Why, therefore, do V, gene segments in the vicinity of the rearranged Vu remain transcriptionally silent? It is possible that the unrearranged V, genes are silent in Ig producing cells due to an active repression mechanism rather than due to the lack of enhancer effect, per se. It is suggested that such a repression mechanism will be activated only at the V,-D rearrangement stage and will be effective only on unrearranged V, genes, whereas the Vu-D-J, gene will be free of suppression. Since DNA sequences 3’ to the V, are being removed during Vu-D-J, joining, I would like to suggest that this involves the removal of 3’ regulatory sequences leading to the release of V,-D-J, expression from the repression mechanism in mature B cells. STEM
Several years ago, we showed that a highly conserved DNA segment is present approximately 130 bp upstream to the initiator ATG of several V, genes in both mouse and man (Rechavi et al., 1983). An octanucleotide sequence, ATGCAAAT, contained in the conserved region, was found 5’ to many V, genes (Parslow et al., 1984; Calame, 1985; Dreyfus et al., 1987) while an inverted repeat of this octanucleotide sequence, ATTTGCAT, was found 5’ to light chain V, genes. These DNA octamer elements are essential for the efficient function of the immunoglobulin promoter in B cells (Parslow et al., 1984; Calame, 1985; Dreyfus et al., 1987). Recently, nuclear factors were identified that bind to these DNA elements (Neuberger and Cook, 1988). DNA sequencing downstream to germline Vu genes revealed a DNA sequence which is inversely complementary to the one described at the 5’ of the Vu genes (Rechavi et al., 1983). This sequence was found at a distance of approximately 300 bp 3’ to the last codon of the two V, genes that were analyzed. The 5’ and 3’ inverted repeats are flanked by direct repeats, as shown in
CELL
VH gene segments r--‘----
1” gene segments
0 gene segments
Constant region gene*
7
I
..,..w-MH]__
D-C3_C
$4 n n-1: L_..__._-: r__.___________
L.._____________
__.-
cp
C6
cy
_.___________
I
0
promoterVHn+l
,
&in
I I
I I I I
L________--_--__-
I I ,
_----_--
-
---_-
-
_-I
Fig. 2. Immunoglobulin heavy chain genes in stem cells. Vu gene segments, D gene segments, J, gene segments and constant region genes (C-mu, C-delta, C-gamma, etc.) are in the germline configuration. E, enhancer; Vu n + 1, Vun, representative Vu genes. Although the Vu genes are accessible, no nuclear factor is synthesized by the cell and no Vu gene expression is found.
PRF-8
Removal
of V, gene downstream
Rearranged gene r----7
Constant region genes
723
sequences
CELL
V” gene segments
cp
C6
CT
k-4 2-Ex
.“’ ‘.
L___r__-l cl-__-_--__----_---_----_---------___-__,
r-----------
i
I I
nuclear
j factor 8 I I
I
RNA polymerase
b&.@,,A 2;;’
I
, _ i;j:.:j
I I I I I I
....%..$ :.i:s I I promoter VHntl promorer V,n 0 J, I I 0 rearranged gene I L_______________--____-_____--__--_____--__-__-_-_------J
Fig. 3. Immunoglobulin heavy chain genes in pre-B cells. The V,n underwent V&-J, rearrangement, whereas the V,n + 1 remains in the gene line configuration. Nuclear factors are synthesized and both rearranged and non-rearranged V, genes are expressed.
Fig. 1 (Rechavi et al., 1983). We have now identified a similar sequence in the flanking regions of three additional mouse V, genes (Givol et al., 1981; Cohen et al., 1982). In these cases none of the direct or inverted repeats are perfect, but their conservation within a region of highly diverged sequences is striking. A model is proposed in which the 5’ and 3’ conserved segments serve a function in the regulation of V, gene expression (Figs. 2, 3 and 4). This is in analogy to the inverted repeat which constitutes the operator of the E. co/i gal operon (Irani et al., 1983; Kolata, 1983). The model postulates the existence of a repressor protein which recognizes the 5’ and 3’ conserved DNA segments and which prevents V,
B V,
CELL gene
segments
r----------:
0 I
gene expression by binding to these segments. This protein could bc a dimer which brings the two segments in proximity, thereby condensing the chromatin at the site of each V, gene. As a result, the V, promoter would be inaccessibe to RNA polymerase and/or DNA binding factors and therefore the gene would remain silent (Fig. 3). The process of V,-D-J, joining results in the deletion of the 3’ segment of the rearranged V, gene, but not of the other V, genes upstream to it. This would result in the release of the promoter region from suppression in the case of the rearranged gene, while all other VH promoters would remain inactive. Hence, this model can account for the exclusive activation of a single V, gene upon DNA rearrangement (Fig. 4). The demonstration of
Rearranged gene r----7
Constant region genes
_I____________-__--__---_---_---
----
fINA
factor\
promoter
VHn
rear ranged I
-_-_,
nuclear polymerase
D
I !
1~
gene
I VMntl VHn+2 :.________-_ -____ ____ _ _ _______ _____________J
I
Fig. 4. Immunoglobulin heavy chain genes in B cells. The proposed repressor factors are present at this stage and bind to the conserved 5’ and 3’ sequences of all non-rearranged V, gene segments. Only the rearranged gene that had lost its 3’ conserved sequence is accessible to the nuclear factors and to the polymerase. Because of the proximity to the enhancer sequences, the rearranged gene is transcribed at a high level.
724
GIDEON
active germline V, transcription at the pre-B stage of differentiation suggests that transacting factors are produced which are capable of transcription enhancement. It is suggested that, at the subsequent stage, specific production of the putative repressor is responsible for the repression of non-rearranged Vu genes. Immature pre-B cells actively express the germline V, genes and show Vu to D,-Ju joining. In contrast, more mature B cells usually do not show the above mentioned molecular changes (Yancopoulos and Ah, 1985; Blackwell et al., 1986). It seems, therefore, that a permissive V, locus is a prerequisite for both Vu expression and V, to D-J, joining. The proposed model predicts that the putative repressor factor, by condensing and “locking” the germline Vu gene locus, is responsible for the prevention of both Vu expression and recombination. This hypothesis would predict that similar DNA segments are present in other V, subgroups as well as in V, genes. Our model can be experimentally tested by sequencing the flanking regions of more V genes or by the removal of the 3’ segment from a molecularly cloned Vu gene and testing its expression in myeloma cells (Rice and Baltimore, 1982; Oi et al., 1983). Acknowledgements-1 am indebted to Mrs Rivka Amit for expert manuscript preparation and to Mrs Nili Shaked for the preparation of the graphic presentation. I am indebted to Dr David Givol for his help and to Drs Bracha Ramot, Isaac Ben-Bassat and Justus Cohen for stimulating discussion This work is supported in part by the grants given to G. Rechavi by the Maurits Van Beets Fund for Leukemia Research, the Freda and Moise Eskenazy Fund for Cancer Research, the Jacob and Judy Zemel Cancer Research Fund and the Irwin Edelstein Leukemia Research Fund. REFERENCES Aguilera R. J., Hope T. J. and Sakano H. (1985) Characterization of immunoglobulin enhancer deletions in murine plasmacytomas. EMBO J. 4, 3689-3693. Alt F. W., Blackwell T. K., DePinho R. A., Reth M. G. and Yancopoulos G. D. (1986) Regulation of genome rearrangement events during lymphocyte differentiation. Immun. Rev. 89, 5-30. Alt F. W., Rosenberg N., Enea V., Siden E. and Baltimore D. (1982) Multiple immunoglobulin heavy-chain gene transcripts in Abelson murine leukemia virus-transformed lvmphoid cell lines. Molec. Ceil. Biol. 2. 386400. Banerji J., Olson L. and Schaffner W. (1983) A lymphocytespecific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33, 7299740. Bentley D. L. and Rabbitts T. H. (1981) Human V kappa immunoglobulin gene number: implications for the origin of antibody diversity. Cell 24, 613-623. Blackwell T. K.. Moore M. W.. Yancopoulos G. D.. Suh H., Lutzker S., Selsing E. and Al; F. A. (1986) Recombination between immunoglobulin variable region gene segments is enhanced by transcription. Nature 324, 5855589. Calame K. L. (1985) Mechanisms that regulate immunoglobulin gene expression. A. Rec. Immun. 3, 159-195. Clarke C., Berenson J., Goverman J., Bayer P. D., Crews S.. Sin G. and Calame K. L. (1982) An immunoglobulin
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