BE301 Eukaryotic Genetics & Expression PDF

11/25/20 Genomic Rearrangements Immunoglobulin Genes: BE301 Structure. Eukaryotic Genetics and...

11/25/20 Genomic Rearrangements Immunoglobulin Genes: BE301 Structure. Eukaryotic Genetics and Expression Segments & Genes Prof. Anne Parle-McDermott Heavy Chain Genes and Diversity. - Somatic Recombination. Topic 6 Genome rearrangements. Immunogloblin Genes - a special Allelic Exclusion- Light chain genes category Increasing Antibody Diversity. - Somatic hypermutation - Class switching 1 2 References The unique organisation and expression of Genes X, Chapter 18 Ig genes Strachan & Read 4, Chapter 4 Section 4.6 ‘Immune System The organisation and expression of the Ig genes are quite cells: function through diversity.’ different from that of most other genes Ig antibodies are produced by B cells These B cells make up part of the adaptive immune defence system The Ig genes in most cells are inactive, but extraordinary shuffling of these genes occurs in B cells in order to activate them. 3 4 Antigen-Antibody complexes are engulfed by macrophages or are attacked by the complement cascade. In mammals, repertoire of B cells is ~ 1012 specificities Immunoglobulins= Antibodies Do we have the coding capacity for 1012 different Ig genes on our chromosomes??? B cell receptor- identical in structure and specificity to those of the antibody that it produces. Antibodies interact with antigens 5 6 1  11/25/20 Each Ig consists of 2 B cells identical light chains and 2 identical heavy B cells – express immunoglobulin molecules chains. THREE human Ig genes Light chains: Lamda or Igs are heterodimers (made up of two kappa. identical heavy chains and two identical light Heavy chains: 9 types. chains) IGH gene specifies the HEAVY chain Both H and L chains IGK (kappa) or have a variable and a constant region. IGL (lambda) gene specifies the LIGHT chain Figure 14.17 Genomes 3 (© Garland Science 2007) 7 8 Ig molecule Ig heavy chains and the light chains have a constant region and a variable region All of the genetic information necessary to make up the heavy chain can be found clustered on chromosome 14; information for kappa light chains – chromosome 2; information for lambda light chains –ch. 22 Antigen recognition triggers clonal expansion Memory cells provide immunity against a subsequent infection 9 10 In vertebrate genomes there are no complete Ig genes. Instead Ig proteins are specified by gene segments. For example, the HEAVY CHAIN part of the Ig molecule is made up of FOUR gene segments: V segment [V for variable region] D segment [D for diversity region] J segment [J for joining region] C segment [C for constant region] One of each of these segments get joined up together to produce an active gene. Table 14.3 Genomes 3 (© Garland Science 2007) 11 12 2  11/25/20 Heavy Chain Genes are Encoded on Chromosome 14 Heavy Chain gene segments In humans, on chromosome 14 are the gene segments for the heavy chain protein. There are 123-129 V segments, 27 D segments, 9 J segments and 11 C segments. In order to make an active heavy chain gene, one of each of these segments must be joined together in the correct order. Diversity via the large number of V genes combining with a small number of C genes Figure 14.18 Genomes 3 (© Garland Science 2007) 13 14 Somatic Recombination: H Chains H Chains are Assembled by 2 Sequential Recombination Events First: one of the D segments recombines with a J H segment. VDJ rearrangement occurs before Second: a V H segment recombines with the already exposure to an recombined D-J H segment. antigen The resulting V H-D-J H DNA sequence is then expressed with the nearest downstream CH gene. Expresses a unique B cell receptor which can be clonally Details of the mechanism of recombination are given in Genes selected. X. Figure 14.19 Genomes 3 (© Garland Science 2007) 15 16 Activation of Gene Expression of Heavy Chain Gene After recombining the various gene segments (V, D, J) next to the C segment(s), the transcriptional promoter located in front of the V segment of the gene is activated as a result of being brought closer to a cis- acting ENHANCER sequence which is located between the J and C segments. Recombination activates a V gene promoter 17 18 3  11/25/20 What about the Ig light chain? Kappa or Lambda Figure 14.19 Genomes 3 (© Garland Science 2007) 19 20 Light Chains are assembled by a single recombination event. Lamda and Kappa genes are encoded on separate Allelic Exclusion chromosomes. Relevant for both Heavy Chains and Light Chains Figure 14.17 Genomes 3 (© Garland Science 2007) 21 22 Allelic Exclusion- Heavy Chains Light Chain Exclusion Remember there are two potential sets of gene segments A light chain synthesised in a single B cell may be a for making heavy chains – as we have two of each kappa chain or a lambda chain, BUT NEVER BOTH. chromosome. As a result there is monoallelic expression at one of the Once an active heavy chain is made (on one of the two functional light chain gene clusters and no chromosomes), any further DNA rearrangements of gene expression at the other. segments on the other chromosome are suppressed. Monoallelic expression of Heavy chain gene segments in a given B cell. 23 24 4  11/25/20 Individual B cells are monospecific Any single B cell makes ONE active heavy chain and ONE active light chain. The decision as to which ‘heavy’ chain and which ’light’ chain combinations are used to create active genes appears to be random. Appears to be some kind of negative feedback regulation. Allelic exclusion limits successful rearrangement to one allele 25 26 Creating Antibody Diversity 1. VDJ (or VJ for light chain) recombination. Increasing 2. Somatic hypermutation (in V region). Antibody Diversity 3. Class switch recombination 27 28 Primary Immune Response vs Secondary Activated B cells (producing a Ig molecule) produce specialised daughter cells called memory cells. Memory cells do not participate in the initial acquired response. Instead they provide a surveillance service after the initial infection has been cleared. As part of the secondary response (to the influx of antigen), the variable regions of the Ig genes in the memory cells undergo rapid mutation in the lymph node. 29 30 5  11/25/20 Hypermutation – additional diversity is produced Somatic hypermutation in the V segment of the immunoglobulin gene Additional Ig diversity results from point mutations being introduced into the V region (mostly) due to error-prone DNA repair This somatic hypermutation leads to a fine tuning of the immune response The mutation rate of these segments is 106 fold higher than the background mutation rate found in the rest of the genome. Figure 16.17 Genomes 3 (© Garland Science 2007) 31 32 Immunoglobulin genes Class Switching FIGURE 24: Somatic mutations occur in VDJ 33 34 Heavy chain class switching Although a B cell produces only ONE type of Ig molecule, the class (or isotype) of the heavy chain can change during development (whether it is IgM or IgD or IgG, etc…). Occurs in peripheral lymphoid organs. 35 36 6  11/25/20 A. Describe the mechanisms that allow B cells to produce Five classes of Immunoglobulins 1012 different types of antibodies? 1. Genomic rearrangements: detail on Heavy Chain- VDJ rearrangement. Despite its name, the constant region is not identical in every immunoglobulin protein. The small variations 2. Somatic hypermutation that occur result in five different classes of immunoglobulins – IgA, IgD, IgE, IgG and IgM – each 3. Class Switching. with its own special role in the immune system. B. How does each B cell produce just 1 type of antibody? Allelic exclusion- explain. 37 38 7