Immunoglobulin heavy-chain constant-region genes

Cell, Vol. 29, 719-721,

July 1982,

Copyright

lmmunoglobulin Constant-Region

0 1982

by MIT

Heavy-Chain Genes

Minireviews

Kenneth B. Marcu Biochemistry Department State University of New York at Stony Brook Stony Brook, New York 11794

human gamma genes are tightly linked (~2-19 kb-y4) (Ellison and Hood, PNAS 79, 1984-l 988, 1982; Krawinkel and Rabbitts, EMBO J., in press). In situ chromosome-hybridization experiments performed with a human y4 probe have successfully mapped the human CH gene locus to band q32 (14q32) of chromosome 14, where translocations commonly found in B-cell lymphomas and leukemias are known to occur (Kirsch et al., Science 276, 301-303, 1982). One unique feature of the human CH gene locus, which sets it apart from the mouse CH genes, is the existence of a duplicated ~--a region (Max et al., Cell 29, 691-699, 1982). One of these two epsilon genes is a pseudogene, with the first two epsilon domains precisely deleted. A second pseudo-E gene has also been identified, but it may not be linked to the C,., gene locus. The expression of C,, genes in terminally differentiated plasma cells has been extensively studied in the murine system and is known to require two independent DNA recombination events. A complete heavychain variable-region gene is created by the somatic recombination of VH, D,, (diversity) and JH (joining) elements 5’ of the mu gene (Early et al., Cell 79,981992, 1980). These events result in the transcription of the mu gene in pre-B lymphocytes. The production of membrane-bound IgM antibody by the immature B lymphocyte awaits the productive rearrangement of a kappa or lambda light-chain gene (Levitt and Cooper, Cell 79, 617-625, 1980; Siden et al., PNAS 78, 1823-l 827, 1981). Individual B lymphocytes then acquire the remarkable ability to switch expression from C, to another CH gene, while maintaining expression of the identical V,., gene (Nossal et al., Cell. Immunol. 2, 41-53, 1971; Pernis et al., CSHS 47, 175-l 83, 1976). CH gene deletions have been observed between the functional V-D-J arrangement and the expressed CH isotype in plasmacytomas. A stochastic mechanism (Coleclough et al., PNAS 77, 1422-1426, 1980) has been postulated for the C,, class switch that may be the result of either intramolecular deletions or sister-chromatid exchanges. Compelling evidence for the role of sister-chromatid exchanges in V.-J, recombination is now available

Antibodies are composed of two identical heavy- and light-chain polypeptides. Each heavy and light chain contains an amino-terminal variable (V) region, responsible for antigen recognition, and a carboxy-proximal constant(C) region, which participates in a variety of immunological processes, including effector-cell recognition and complement fixation. The variable regions are encoded by several hundred germ-line genes, while a limited member of genes encode the constant regions. The mouse CH gene locus resides on the distal arm of chromosome 12 and consists of eight genes (p, 6, 73, yl , y2b, y2a, E and a) that span close to 200 kb of DNA (see figure) (Shimizu et al., Cell 28, 499-506, 1982). The mouse CH gene cluster contains no well conserved pseudo-& genes. &-coding regions are composed of either two (6) three (~3, yl , y2b, (II) or four (cl and E) structural domains, each consisting of -100 amino acids that are separated by small introns. Antibodies of immature B lymphocytes possess hydrophobic carboxy-terminal CH extensions, allowing these molecules to function as transmembrane proteins. Hydrophobic membrane domains are separated from the secreted termini of CH genes by a -2 kb intron (Early et al., Cell 20, 313-319, 1980). Membrane-bound or secreted antibody is thought to be expressed first by regulation of the site of transcriptional termination or poly(A) addition and then by sitespecific RNA splicing (Early et al., op. cit.; Rogers et al., Cell 26, 19-27, 1981). The human CH gene family consists of at least nine functional members (p, 6, yl , 72, y3,y4, c, al and a2). As in the mouse, the human CS gene is located several kilobases 3’ of C,” (Rabbitts et al., NAR 9,4509-4524, 1981). The four human gamma genes display more extensive homology to each other than is observed among the mouse gamma genes, and at least two

ORGANIZATION

OF MOUSE

IMMUNOGLOBULIN

I

I

Kbp 0

= S REGIONS = IO Kbp

=

150 YZb

3 34

I

I

loo r3

-

CHAIN GENES

I

50

55

HEAVY

n

21

115-114;

Y2a

200 c

Q

Cell 720

(Hochtl et al., PNAS 79,1383-l 387,1982; Van Ness et al., PNAS 79, 282-288, 1982). However, only one example of a C,, gene switch has been reported that could be the result of a sister-chromatid exchange (Obata et al., PNAS 78, 2437-2441, 1981). Switchrecombination appears to involve a class of repetitive DNA sequences -2 kb 5’ to each CH gene (S regions; see figure). S regions contain several common, repetitive DNA sequences: GAGCT and GGGGT (Nikaido et al., Nature 292, 845-848, 1981) and YAGGTTG (Marcu et al., Nature, in press). A -3 kb portion of S, mainly consists of tandemly repeated GAGCT sequences. YAGGTTG-like sequences have recently been observed 5’ to the switchTrecombination sites within S, and other SH regions (Marcu et al., op. cit.). An Abelson-virus-transformed pre-B cell line (1881 1 has been observed, unexpectedly, to express the y2b gene (Burrows et al., PNAS 78,584-568, 1981). As anticipated for a pre-B cell, primary subclones only express low levels of mu mRNA and no light-chain mRNA. However, recent studies with long-term tissueculture-adapted subclones of 18-81 (18-81 A-2) have revealed that a switch from mu to y2b occurs without deletion of the mu gene (Alt et al., Nature 296, 325331, 1982). The level of y2b mRNA appears to be inducible with a B-cell mitogen, lipopolysaccharide. However, mu mRNA can no longer be detected in these cells. Deletion events occurring within the J,.,C, intron have been correlated with the loss of mu gene expression and the p + y2b switch. The Jr&Z, intron has been shown to contain repetitive DNA sequences that undergo deletion events (Marcu et al., Cell 22, 187-l 96, 1980). If we assume that the S, y3 and yl genes are present in their germline context, the p + y2b switch in 18-81 A-2 cells may result from the differential RNA splicing of a large (>lOO kb) multi-&-gene transcription unit (Alt et al., op. cit.). The presence of multiple C,, classes on the surface of immature B lymphocytes has been known for some time, but this phenomenon has only recently been analyzed at the molecular level. The close proximity of the mu and delta genes G-4.5 kb-6) initially led to the notion that delta could be expressed by an RNA processing mechanism (Liu et al., Science 209, 1348-l 353, 1980). Studies performed with hybridomas and lymphomas that coexpress mu and delta have suggested that a large p+S transcript is processed by site-specific poly(A) addition coupled with differential RNA splicing to yield mu and delta mRNAs with the identical VH region (Maki et al., Cell 24, 353-365,198l; Moore et al., PNAS 78,1800-l 804, 1981; Knapp et al., PNAS 79, 2996-3000, 1982). In agreement with the postulated p + S transcript, both the mu and delta genes are demethylated in a p+S+ hybridoma but only the mu gene is demethylated in a p+ lymphoma (Rogers and Wall, PNAS 78, 74977501, 1981).

Minor populations of small, splenic B lymphocytes exhibiting p+y, P+E or P+(Y isotypes on their surface are believed to be memory B cells that proliferate and subsequently secrete antibodies during a secondary immune response. A population of presumably >80% B cells isolated from BALB/c mouse pure p+yl+ spleen by immunoadsorption techniques does not display DNA rearrangements 5’ to C,, (Perlmutter and Gilbert, PNAS, in press). Both copies of the mu gene are present in their germline context, and the mu gene copy number is comparable with that of the unique mouse serum albumin gene. A long transcription unit (>90 kb) is proposed to terminate past the yl gene, and then differential RNA splicing subsequently yields ~1and yl mRNAs with identical V-D-J sequences in a p+yl+ B lymphocyte. Similar studies have also recently been performed on P+E+ B lymphocytes, purified on a fluorescence-activated cell sorter, obtained from the spleens of parasite-infected SJA/S mice (a congenic mouse strain with a T-cell defect that apparently blocks IgE secretion), and reveal no evidence for any C&-gene rearrangements or deletions between the Jr, region and the epsilon gene (Yaoita et al., Nature, in press). However, most JH loci are rearranged, presumably by a combination of productive V-D-J and nonproductive D-J recombinations. As in the former study on pL+y+ B cells, these workers have also concluded that a large multi+ transcription unit (-180 kb in this case) terminates beyond the C, gene in p+e+ B cells and yields mu and epsilon mRNAs by a combination of site-specific poly(A) addition and RNA splicing. Both of the above studies have relied on the unsubstantiated fact that these dual-isotype-staining B-lymphocyte populations biosynthetically produce both isotypes. Positive evidence for the existence of such large transcripts is not yet available, and would present a formidable task. The amount of specific CH transcripts in such cells would probably represent less than 0.1% of the immunoglobulin mRNA levels in fully differentiated plasma cells. Dual-isotype-positive B cells may represent a transient stage prior to terminal B-lymphocyte differentiation. We are left with the attractive possibility that B lymphocytes expressing two C,, classes mediate a transient C,, switch at the transcriptional level, and eventually proliferate into an immunoglobulin-secreting plasma cell in which all the C,, genes between V-D-J and the original non-p isotype are deleted. We could explain such a direct switching mechanism conveniently by invoking the activation of a &+-class-specific switch-recombinase late in B-cell differentiation to mediate the final, stable DNA rearrangement. Is this the major pathway responsible for C,, switching in vivo? Do B cells display the potential to switch CH genes via other pathways? Several studies indicate that B cells have the potential to switch CH genes successively (that is, yl + y2b

Cell 721

+ y2a). The immune response to trinitrophenol-Ficoll, a T-cell-independent type II antigen, has been analyzed at the B-cell level (Mongini et al., JEM 755, 884-902, 1982). Following hyperimmunization with trinitrophenol-Ficoll, B-cell clones were isolated by the splenic focus technique in irradiated nude mouse recipients. All B-cell clones expressed mu, and a significant fraction expressed one or more other isotypes. The statistical frequencies of isotypes expressed by in vitro-cultured B-cell clones were observed to follow the C,, gene order (that is, IgM > lgGs > IgG, > IgG*b > IgGn,). T cells were found to increase the amount of lgG2. antibody preferentially. It is difficult to be certain, however, that successive CH switches originate from a single B cell in this system. Moreover, myeloma and hybridoma cell lines have been shown to switch CH genes spontaneously at a low frequency. In the case of MPC-11 (a y2b-producing plasmacytoma), the expressed y2b gene of the parental cell is specifically deleted upon switching to the y2a gene (Eckhardt et al., PNAS 79,3006-3010, 1982). This deletion event encompasses the S,, Syab and C+b regions. The X63 hybridoma has been observed to undergo three forward switches spontaneously (yl + y2b + y2a) and an unexpected backswitch (y2a + yl) at lOO-fold higher frequency (-10e5 versus -10m7 per cell per generation) (Bey-

Cdl, Vol. 29, 721-724,

July 1982.

Copyright

X-Chromosome

Gail R. Martin Department of Anatomy University of California San Francisco, California

reuther et al., ICN-UCLA Symposia 20, 229-244, 1981). Forward switches are correlated with J,, and C, gene rearrangements and deletions (Beyreuther et al., op. cit.). Thus, if we assume that CH deletions are responsible for forward switches, transhomologous chromosome exchange has been invoked to explain the y2a+ yl switch revertant. This latter phenomenon could alsobe explained by tandemly duplicated pairs of C, genes in the X63 hybridoma. It remains to be demonstrated that the phenomena observed in tissueculture cell lines reflect normal B-cell switching events. The molecular requirements and mechanism or mechanisms of regulation of C&gene switching in normal B cells remain important problems. Different steps in class switching could be modulated directly or indirectly by T lymphocytes or their products. T helper factors, which appear to induce or select specific CH isotypes in populations of normal B cells (Isakson et al., JEM 755, 734-748, 1982) conceivably could be used to dissect dynamic steps of CHgene switching. Gene-transfer experiments with multiCH-gene substrates and normal B cells (which express multiple surface isotypes and/or display CH switching potential upon mitogenic stimulation) may also reveal hidden regulatory aspects of this complex biological phenomenon.

63 1982 by MIT

Inactivation

94143

In female mammals there is developmental regulation of X-chromosome activity, resulting in a significant difference between the two X chromosomes in each somatic cell: one is genetically active and the other is not. In the 20 years since the first proposal of a mechanism that supersedes the controls of individual X-linked gene activity, numerous studies have documented the phenomenon and have elucidated the

in Mammals

properties of the inactive X chromosome. It is, for example, heterochromatic (condensed), and in certain cases is clearly visible as the sex chromatin or Barr body lying against the nuclear membrane during interphase. In addition, the inactive X chromosome is known to begin and end its replication later in the S phase of the cell cycle than do the autosomes and the active X chromosome. Its genetic inactivity results in dosage compensation, so that there are similar levels of X-linked gene products in female cells, which have two X chromosomes, and male cells, which have only one. In various species of placental mammals all genes on the affected X chromosome that have been examined are inactive, with the exception of those on