George D. Yancopoulos

Developmentally controlled and tissue-specific expression of unrearranged VH gene segments

It is generally accepted that unrearranged immunoglobulin VH gene segments are not expressed and that assembly of a complete heavy chain gene is required to activate a previously silent VH promoter. We report that unrearranged VH gene segments are indeed expressed at a high level, but only in a developmentally controlled and tissue-specific manner. Unrearranged VH expression is limited to the very early stages of the B-lymphocyte differentiation pathway, and it is most prominent in cells undergoing VH to DJH rearrangement. Germ-line VH expression is independent of the heavy chain enhancer, may be controlled by 5 sequence elements, and is repressible by LPS. In contrast to earlier interpretations, our results demonstrate that the lack of unrearranged VH segment expression in mature, Ig-secreting cells is due to the inactivation of a previously active locus. These findings may provide insight into the mechanisms that control ordered rearrangement and allelic exclusion.

Introduced T cell receptor variable region gene segments recombine in pre-B cells: Evidence that B and T cells use a common recombinase

We have recently proposed that a common recombinase performs all of the many variable region gene assembly events in B and T cells, and that the specificity of these joining events is mediated by regulating the accessibility of the involved gene segments. To test this possibility, we have introduced accessible T cell receptor (TCR) variable region gene segments into a pre-B cell line capable of recombining endogenous and transfected immunoglobulin (Ig) variable region gene segments. Although the corresponding inaccessible endogenous TCR gene segments do not rearrange in this line or in B cells in general, the introduced TCR gene segments join very frequently and, in fact, closely resemble introduced Ig gene segments in their recombination characteristics. These observations suggest a new role for conventional Ig transcriptional enhancers--recombinational enhancement. Our studies provide insight into additional aspects of the joining mechanism such as N region insertion, aberrant joining, and recombination-recognition sequence requirements for joining.

Regulation of Genome Rearrangement Events during Lymphocyte Differentiation

Analyses of A-MuLV transformed cell lines have provided fundamental insights into the molecular mechanisms which control the rearrangement events leading to the expression of specific antigen receptor genes. These studies have clearly indicated that tissue-specific, developmental stage-specific, and allelically excluded assembly of Ig H and L chain and TCR variable region genes are very strictly regulated processes and, furthermore, that this regulation probably is effected at the level of the accessibility of the individual sets of V gene segments to a common recombinase. More preliminary studies have also suggested that accessibility targeting may be involved in the regulation of directed Ig H chain class-switch recombination events. Currently, we do not understand the nature of accessible DNA sequences and we have little understanding of the molecular mechanisms by which Ig (and potentially TCR) chains mediate the regulation of specific recombination events by signaling changes in the accessibility of the various loci. However, an ideal model system for the analysis of these questions is currently available in the form of A-MuLV transformed pre-B cell lines which, in a properly regulated fashion, undergo all of the various recombination events associated with the pre-B stage of B cell differentiation.

The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism

Abelson murine leukemia virus-transformed precursor B lymphocytes from scid (severe combined immunodeficient) mice, like A-MuLV transformants from normal mice, actively rearrange segments of their Ig heavy chain variable region gene locus during growth in culture. Targeting of recombination to appropriate segments appears normal in these lines as evidenced by initial rearrangement of sequences from within the D and JH locus to form aberrant DJH rearrangements and secondary rearrangement of sequences from within the VH locus to the aberrant DJH intermediates. A detailed analysis of the joints in these rearrangements indicates that the VDJ recombinase in scid pre-B cells can correctly recognize heptamernonamer signal sequences and perform precise endonucleolytic scissions at these sequences. We propose that the scid defect involves the inability of scid precursor lymphocytes to join correctly the cleaved ends of the coding strands of variable region gene segments.

Structure and expression of the murine L-myc gene.

We have isolated a 12 kb clone from the murine genome which we show by DNA transfection studies to contain an entire functional L‐myc gene and the transcriptional promoter sequences necessary for its expression. We have also isolated a 3.1 kb cDNA sequence from a murine brain cDNA library which corresponds to most of the L‐myc mRNA. We have identified the L‐myc coding region within the genomic clone by a combination of S1 nuclease analyses. Northern blotting analyses and comparative nucleotide sequence analyses with the cDNA clone. The L‐myc gene appears to be organized similarly to the other well‐characterized myc‐family genes, c‐myc and N‐myc. The predicted amino acid coding sequence of the L‐myc gene indicates that the L‐myc protein is significantly smaller than c‐ and N‐myc, but is highly related. In particular, comparison of the N‐ and c‐myc protein sequences reveals seven relatively conserved regions interspersed among non‐conserved regions; the L‐myc gene retains five of these conserved regions but lacks two others. In addition, a portion of one highly conserved region is encoded within a different region of the L‐myc gene but, due to changes in the size of L‐myc exons relative to those of N‐ and c‐myc, maintains its overall position in the peptide backbone with respect to other conserved regions. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc‐family proteins.

Immunoglobulin genes in transgenic mice

Abstract The introduction of immunoglobulin genese into the germ line of mice has provided an unparalleled system for examining the mechanisms which control the somatic assembly and expression of immunoglobulin genes. These studies have also demonstrated that a mouse can be genetically engineered to produce a desired immune product.

Expression of the Immunoglobulin Heavy-Chain Variable Gene Repertoire

A major challenge in immunology is the elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire. A large body of knowledge concerning murine antibody gene organization and diversity has been accumulated. However, the underlying processes governing usage of these genes remains in question. Knowledge of the mechanisms which regulate immunoglobulin variable (V) gene rearrangement and the relative expression of the various V gene families in developing B–cell populations may be crucial to an understanding of generation of the antibody repertoire in normal as well as disease states. Current investigation of human V H gene organization and diversity will evwntually allow extension of information gained about the murine repertoire to studies of V H gene usage in humans.

Differential Expression of myc -family Genes During Development: Normal and Deregulated N- myc Expression in Transgenic Mice

The myc-family of cellular oncogenes is a dispersed multi-gene family that includes the c-, N- and L-myc genes (For review see Alt et al., 1986). The human and murine c-, N, and L-myc genes encode related but distinct nuclear proteins and have a similar overall organization (Kohl et al., 1986; DePinho et al., 1986, Stanton et al., 1986; DePinho et al., 1987; Legouy et al., 1987; Kay et al., 1988); all have three exons with the first encoding a potentially untranslated leader sequence. All three genes also cooperate similarly with an activated Ha-Ras oncogene to transform primary rat embryo fibroblasts (REFs) (Yancopoulos et al., 1985; DePinho et al., 1987). Despite striking similarities of regions of the myc proteins and the in vitro transforming activities, the three genes are conserved as distinct sequences throughout vertebrate species suggesting unique functional roles. This possibility is supported by findings that the genes are differentially expressed in a stage-and tissue-specific manner during human and murine differentiation (Zimmerman et al., 1986). In addition, deregulated c-myc expression has been causally implicated in the genesis of a wide variety of different tumor types and occurs by a variety of different mechanisms; deregulation of the N- and L-myc genes has been clearly implicated only in a few naturally occurring tumors (eg. human neuroblastomas and small cell lung carcinomas) and only by the mechanism of gene amplification (reviewed by Alt et al., 1986).

Control of Recombination Events During Lymphocyte Differentiation: Heavy Chain Variable Region Gene Assembly and Heavy Chain Class Switching

Our recent studies have focused on the organization of immunoglobulin genes in mice and humans and the mechanism and control of the recombination events that are involved in their assembly and expression. This report describes our progress in this area with particular focus on elucidating factors that influence the generation of the antibody repertoire in normal and diseased states. We present a detailed analysis of the organization of the human VH locus, studies that help to elucidate the nature of the recombination defect in mice with severe combined immunodeficiency, and studies of transgenic mice that focus on the mechanism that regulates tissue-specific variable region gene assembly. In addition, we also characterize mechanisms that control the heavy chain class-switch process. Although the latter process apparently involve a recombination system distinct from that involved in variable region assembly, we find that the two recombination events appear to be controlled by similar mechanisms.

The Effect of the scid Mutation on Mechanism and Control of Immunoglobulin Heavy and Light Chain Gene Rearrangement

Most Abelson murine leukemia virus (A-MuLV)-transformed cell lines derived from scid (severe combined immune deficient) mice actively rearrange their endogenous immunoglobulin (Ig) heavy (H), but not light (L) chain variable region genes. Such cell lines express germline VH segments and other RNA transcripts that are characteristically produced by early precursor (pre)-B lymphocytes, but do not express high levels of transcripts from the germline kappa (k) constant region (C kappa) locus. However, we have derived scid A-MuLV transformants that express germline C kappa transcripts and attempt kappa gene assembly. In one case kappa gene expression and rearrangement occurred in the absence of mu H chain expression, and in another was not induced efficiently by introduction of a mu-expression vector. Although the vast majority of scid H and L chain coding sequence joins are grossly aberrant, scid A-MuLV transformants can form normal coding joints at a very low frequency. In contrast, these cells form generally normal signal sequence joins at an approximately normal efficiency. Thus, these findings mechanistically distinguish coding and signal join formation. Subcloning analyses suggest that scid A-MuLV transformants that do not attempt chromosomal coding sequence joining may have a relative survival advantage, and therefore that these events may often result in unrepaired chromosomal breakage and cell death.