The germline organization of Ig and TCR loci described in the preceding section exists in all cell types in the body. The germ line genes cannot be transcribed into mRNAs that encode functional antigen receptor proteins. Functional antigen receptor genes are created only in developing B and T lymphocytes after DNA rearrangement brings randomly chosen V, D, and J gene segments into contiguity.

The process of V(D)J recombination at any Ig or TCR locus involves the joining of one V gene segment, one D segment (only in Ig heavy chain or TCR β or δ chain loci), and one J segment in each lymphocyte to form a single V(D)J exon that will code for the variable region of an antigen receptor protein (Fig.1). In the Ig light chain and TCR α and γ loci, which lack D segments, a single rearrangement event joins a randomly selected V gene segment to a randomly selected J segment. The Ig H and TCR β and δ loci contain D segments, and at these loci two sequential rearrangement events are needed, first joining a D to a J and then a V segment to the fused DJ segment. Each rearrangement event involves a number of steps. First, the chromatin is opened in specific regions of the chromosome to make antigen receptor gene segments accessible to the enzymes that mediate recombination. Next, two selected gene segments are brought next to one another across a considerable chromosomal distance. Then, double-stranded breaks are introduced at the coding ends of these two segments, nucleotides are added or removed at these broken coding ends, and finally the processed ends are ligated to produce antigen receptor genes that can be efficiently transcribed. The C region exons lie downstream of the rearranged V(D)J exon separated by the germline J-C intron. The rearranged gene is transcribed to form a primary (nuclear) RNA transcript. Subsequent RNA splicing brings together the leader exon, the V(D)J exon, and the C region exons, forming an mRNA that can be translated to produce one of the chains of the antigen receptor. The use of different combinations of V, D, and J gene segments and the addition and removal of nucleotides at the junctions contribute to the tremendous diversity of antigen receptors, as we will discuss in more detail later. Also, because the gene segment combinations and the junctions between them are different in each developing B or T lymphocyte, each cell and its clonal progeny produce a distinct antigen receptor.

Fig1. An overview of V(D)J recombination. Ony a small number of V, D, and J gene segments are shown for simplicity. C, Constant; D, diversity; J, joining; V, variable.

Recognition Signals That Drive V(D)J Recombination

Lymphocyte-specific proteins that mediate V(D)J recombination recognize DNA sequences called recombination signal sequences (RSSs) that are located 3′ of each V gene segment, 5′ of each J segment, and flanking each side of every D segment (Fig. 2A). The RSSs consist of a conserved stretch of 7 nucleotides, called the heptamer, usually CACAGTG, located adjacent to the coding sequence, followed by a spacer of either 12 or 23 nonconserved nucleotides, followed by a conserved AT-rich stretch of 9 nucleotides, called the nonamer. Specific residues in the heptamer and nonamer contribute to the binding of the recombinase enzyme that mediates joining, which will be described in the next section. The 12- and 23-nucleotide spacers roughly correspond to one or two turns of a DNA helix, respectively, and they ensure that two distinct RSSs, each adjacent to a different type of coding gene segment, are brought close to one another for recombination, as discussed below.

Fig2. V(D)J recombination. The DNA sequences and mechanisms involved in recombination in the immunoglobulin (Ig) gene loci are depicted. The same sequences and mechanisms apply to recombinations in the TCR loci. (A) Conserved heptamer (7 bp) and nonamer (9 bp) sequences, separated by 12- or 23-bp spacers, are located adjacent to V and J segments (for κ and λ loci) or to V, D, and J segments (in the H chain locus). The V(D)J recombinase recognizes these recombination signal sequences (RSSs) and brings the exons together. (B and C) Recombination of V and J exons may occur by deletion of intervening DNA and ligation of the V and J segments (B) or, if the RSS is 3′ of a J segment, by inversion of the DNA followed by ligation of adjacent gene segments (C). Red arrows indicate the sites where germline sequences are cleaved before their ligation to other Ig or TCR gene segments. D, Diversity; J, joining; V, variable.

During V(D)J recombination, double-stranded breaks are generated between the heptamer of the RSS and the adjacent V, D, or J coding sequence. In Ig light-chain V-to-J recombination, for example, breaks will be made 3′ of a V segment and 5′ of a J segment. The intervening double-stranded DNA, containing signal ends (the ends that contain the heptamer and the rest of the RSS), is removed in the form of a circle, and the V and J coding ends are joined (see Fig. 2B). In some V gene segments, especially in the Ig κ locus, the RSSs are 3′ of a Vκ and 3′ of Jκ and therefore do not face each other. In these cases, the intervening DNA is inverted and the V and J segments are properly aligned; the fused RSSs are not deleted but retained in the chromosome (see Fig. 2C). Most Ig and TCR gene rearrangements occur by deletion; inversion is the basis of up to 50% of rearrangements in the Ig κ locus. In any case, recombination occurs between two segments only if one of the segments is flanked by a 12-nucleotide spacer and the other is flanked by a 23-nucleotide spacer; this is called the 12/23 rule. Thus, the location of flanking RSSs ensures that the appropriate gene segments will recombine. For example, in the Ig heavy-chain locus, the RSSs flanking both V and J segments have 23-nucleotide spacers (two turns of the DNA helix) and therefore cannot join directly; D-to-J recombination occurs first, followed by V-to-DJ recombination, and this is possible because the D segments are flanked on both sides by 12-nucleotide spacers, allowing D-J and then V-DJ joining. The RSSs described here are unique to Ig and TCR genes. Therefore, V(D)J recombination can occur in antigen receptor genes but not in other genes.

One of the consequences of V(D)J recombination is that the promoters located immediately 5′ of V genes are brought close to downstream enhancers that are located in the introns between J and C segments and also 3′ of the C region genes (Fig.3). These enhancers maximize the transcriptional activity of the V gene promoters and are thus important for high-level transcription of rearranged V genes in lymphocytes.

Fig3. Transcriptional regulation of immunoglobulin genes. V(D)J recombination brings quiescent promoter sequences (shown as P, with the green arrow) close to the enhancer (enh). The enhancer promotes transcription of the rearranged V gene (V2, whose active promoter is indicated by a bold green arrow). Many receptor genes have an enhancer in the J-C intron and another 3′ of the C region. Only the 3′ enhancer is depicted here. C, Constant; D, diversity; J, joining; V, variable.

Because Ig and TCR genes are sites of DNA breaks during recombination events in B and T cells, and because these sites become transcriptionally active after recombination, genes from other loci can be abnormally translocated to these loci and, as a result, may be aberrantly transcribed. In tumors of B and T lymphocytes, oncogenes are often translocated to Ig or TCR gene loci. Such chromosomal translocations are frequently accompanied by enhanced transcription of the oncogenes and are a major mechanism leading to the development of lymphoid tumors.

Mechanism of V(D)J Recombination

 Rearrangement of Ig and TCR genes represents a special kind of nonhomologous DNA recombination event that is mediated by the coordinated activities of several enzymes. Some of these enzymes are found only in developing lymphocytes, whereas others are ubiquitous DNA double-stranded break repair (DSBR) enzymes. Although the mechanism of V(D)J recombination is well understood and will be described here, how exactly specific loci are made accessible to the machinery involved in recombination remains to be determined. It is likely that the accessibility of the Ig and TCR loci to the enzymes that mediate recombination is regulated in developing B and T cells by several mechanisms, including epigenetic alterations in chromatin structure and basal transcriptional activity in the gene loci.

The process of V(D)J recombination can be divided into four distinct sequential events (Fig.4):

1. Synapsis: Portions of the chromosome on which the antigen receptor gene is located are made accessible to the recombination machinery. Two steps are involved in achieving accessibility. First, only RSSs that are located in open euchromatin in a specific cell type will be exposed to recombination enzymes. For example, the IgH, Igκ, and Igλ loci will be exposed in a B cell but not in a developing T cell. Second, within this open euchromatin state, gene segments that are actually undergoing recombination acquire additional histone marks, such as the hypermethylation of lysine 4 on histone 3 (H3K4). This modification specifically facilitates recruitment of enzymes, as discussed later. Two selected coding segments and their adjacent RSSs that have acquired these and other histone marks are brought together by a chromosomal looping event and held in position for subsequent cleavage, processing, and joining.

2. Cleavage: Double-stranded breaks are enzymatically generated at the junctions between the RSSs and the coding sequences by a lymphocyte-specific enzyme called the V(D) J recombinase. The V(D)J recombinase is a complex com posed of two molecules each of two different proteins called RAG1 and RAG2, encoded by lymphoid-specific genes called recombination-activating gene 1 and recombination-activating gene 2 (RAG1 and RAG2), respectively. This tetrameric RAG1/RAG2 complex is required for V(D)J recombination, but only RAG1 possesses catalytic activity. The RAG2 protein binds to hypermethylated H3K4 sites in chromatin and associates with and activates RAG1. The RAG1 protein, in a manner similar to a bacterial restriction endonuclease, recognizes the DNA sequence at the junction between a heptamer and a coding segment and cleaves it, but it is enzymatically active only when complexed with the RAG2 protein. RAG1 and RAG2 contribute to holding together gene segments during the process of chromosomal folding or synapsis. RAG1 then makes a nick (on one DNA strand) between the coding end and the heptamer. The released 3′ OH of the coding end then attacks a phosphodiester bond on the other DNA strand, forming a covalent hairpin. The signal end (including the heptamer and the rest of the RSS) does not form a hairpin and is generated as a blunt, double-stranded DNA terminus that undergoes no further processing. This double-stranded break results in a closed hairpin of one coding segment being held in apposition to the closed hairpin of the other coding end and two blunt recombination signal ends being placed next to each other. RAG1 and RAG2, apart from generating the double-stranded breaks, also hold the hairpin ends and the blunt ends together before the modification of the coding ends and the process of ligation begins.

RAG genes are expressed only in developing B and T cells. RAG proteins are produced mainly in the G0 and G1 stages of the cell cycle and are inactivated in proliferating cells. It is thought that limiting DNA cleavage and recombination to the G0 and G1 stages minimizes the risk of generating inappropriate DNA breaks during DNA replication or during mitosis. Mice without functional Rag1 or Rag2 genes (Rag knockout mice) fail to develop B or T lymphocytes, and RAG1 or RAG2 mutations in humans are a cause of severe combined immunodeficiency disease (SCID), in which patients lack B and T lymphocytes.

3. Hairpin opening and end processing: After the formation of double-stranded breaks, hairpins must be opened up at the coding junctions and nucleotides may be added to or removed from the coding ends to create even greater diver sification. ARTEMIS is an endonuclease that opens up the hairpins at the coding ends. In the absence of ARTEMIS, hairpins cannot be opened, and mature T and B cells can not be generated. Mutations in ARTEMIS are a rare cause of SCID, similar to patients with RAG1 or RAG2 mutations. A lymphoid-specific enzyme, called terminal deoxynucleotidyl transferase (TdT), adds nucleotides to broken DNA ends and will be discussed later in the context of junctional diversity.

4. Joining: The broken coding ends as well as the signal ends (the ends that terminate in noncoding RSS sequences) are brought together and ligated by a double-stranded DNA break repair process found in all cells that is called nonhomologous end joining. A number of ubiquitous proteins participate in nonhomologous end joining. KU70 and KU80 are DNA end-binding proteins that bind to the breaks and recruit the catalytic subunit of DNA-dependent protein kinase (DNA-PK), a double-stranded DNA repair enzyme. Mutations affecting DNA-PK result in a failure to produce mature B and T lymphocytes, causing SCID in mice and humans. DNA-PK also phosphorylates and activates ARTEMIS, which, as mentioned before, is involved in end processing. Ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4, the latter being a noncatalytic but essential subunit of the ligase.

Fig4. Sequential events during V(D)J recombination. Synapsis and cleavage of DNA at the heptamer/coding segment boundary are mediated by Rag-1 and Rag-2. The coding end hairpin is opened by the Artemis endonuclease, and broken ends are repaired by the nonhomologous end joining machinery present in all cells. Note that the two strands of DNA are shown in the hairpins but not in other schematic illustrations of genes. D, Diversity; J, joining; PK, protein kinase; RAG, recombination-activating gene; TdT, terminal deoxynucleotidyl transferase; V, variable.

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الآخبار الصحية

المركز الاوروبي : كل ركاب سفينة "هانتا" مخالطون معرضون لخطر كبير

ألمانيا.. رئيس الأطباء يدعو للإسراع بتطبيق "ضريبة السكر"

بين الفائدة والضرر.. جدل حول تأثير الشاي والقهوة على العظام

أمراض خطيرة.. أبحاث تكشف مخاطر الجلوس لفترة طويلة

المزيد

الآخبار التكنلوجية

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مواضيع ذات صلة

Immuno microscopy

Role of Macrophages in Tissue Repair

Ingestion and Killing of Microbes by Activated Phagocytes

Sequence of Events in Inflammation: Vascular Changes and Leukocyte Migration Into Tissues

Double Antibody Sandwich ELISA (DAS ELISA)

Enzyme Immunosorbent Assays

Antibody Structure and Function

Apoptosis in the Immune System

The Major Proinflammatory Cytokines of Innate Immunity: Interleukin-6

The Major Proinflammatory Cytokines of Innate Immunity: Interleukin-1

The Major Proinflammatory Cytokines of Innate Immunity: Tumor Necrosis Factor

The Immune Receptor Family