8. Somatic mutation generates additional diversity

24.7 DNA recombination causes class switching

Key terms defined in this section
Class switching is a change in the expression of the C region of an immunoglobulin heavy chain during lymphocyte differentiation.



Figure 24.17 Immunoglobulin type and function is determined by the heavy chain. J is a joining protein in IgM; all other Ig types exist as tetramers.

The class of immunoglobulin is defined by the type of CH region. Figure 24.17 summarizes the five Ig classes. IgM (the first immunoglobulin to be produced by any B cell) and IgG (the most common immunoglobulin) possess the central ability to activate complement, which leads to destruction of invading cells. IgA is found in secretions (such as saliva), and IgE is associated with the allergic response and defense against parasites.




Figure 24.10 A single gene cluster in man contains all the information for heavy-chain gene assembly.

All lymphocytes start productive life as immature cells engaged in synthesis of IgM. Cells expressing IgM have the germline arrangement of the CH gene segment cluster shown in Figure 24.10. The V-D-J joining reaction triggers expression of the Cµ gene segment. A lymphocyte generally produces only a single class of immunoglobulin at any one time, but the class may change during the cell lineage. A change in expression is called class switching. It is accomplished by a substitution in the type of CH region that is expressed. Switching can be stimulated by environmental effects; for example, the growth factor TGFβ causes switching from Cµ to Ca.


Switching involves only the CH gene segment; the same VH gene segment continues to be expressed. So a given VH gene segment may be expressed successively in combination with more than one CH gene segment. The same light chain continues to be expressed throughout the lineage of the cell. Class switching therefore allows the type of effector response (mediated by the CH region) to change, while maintaining a constant facility to recognize antigen (mediated by the V regions).


Changes in the expression of CH gene segments are made in two ways. The majority occur via further DNA recombination events, involving a system different from that concerned with V-D-J joining (and able to operate only later during B cell development). Another type of change occurs at the level of RNA processing, but generally this is involved with changing the C-terminal sequence of the CH region rather than its class (see next section).




Figure 24.18 Class switching of heavy genes may occur by recombination between switch regions (S), deleting the material between the recombining S sites. Successive switches may occur.

Cells expressing downstream CH gene segments have deletions of Cμ and the other gene segments preceding the expressed CH gene segment. Class switching is accomplished by a recombination to bring a new CH gene segment into juxtaposition with the expressed V-D-J unit. The sequences of switched V-D-J-CH units show that the sites of switching lie upstream of the CH gene segments themselves. The switching sites are called S regions. Figure 24.18 depicts two successive switches.


In the first switch, expression of Cμ is succeeded by expression of Cγ1. The Cγ1 gene segment is brought into the expressed position by recombination between the sites Sμ and Sγ1, deleting the material between. The Sμ site lies between V-D-J and the Cµ gene segment. The Sγ1 site lies upstream of the Cγ1 gene segment.


The linear deletion model imposes a restriction on the heavy-gene locus: once a class switch has been made, it becomes impossible to express any CH gene segment that used to reside between Cµ and the new CH gene segment. In the example of Figure 24.18, cells expressing Cγ1 should be unable to give rise to cells expressing Cγ3, which has been deleted.


However, it should be possible to undertake another switch to any CH gene segment downstream of the expressed gene. The figure shows a second switch to Cα expression, accomplished by recombination between Sα1 and the switch region Sµ,γ1 that was generated by the original switch.


We assume that all of the CH gene segments have S regions upstream of the coding sequences. We do not know whether there are any restrictions on the use of S regions. Sequential switches do occur, but we do not know whether they are optional or an obligatory means to proceed to later CH gene segments. We should like to know whether IgM can switch directly to any other class.


We know that switch sites are not uniquely defined, because different cells expressing the same CH gene segment prove to have recombined at different points. Switch regions vary in length (as defined by the limits of the sites involved in recombination) from 1 V10 kb. They contain groups of short homologous repeats, with repeating units that vary from 20 V80 nucleotides in length. An S region typically is located ~2 kb upstream of a CH gene segment. The switching reaction releases the excised material between the switch sites as a circular DNA molecule. Two of the proteins required for the joining phase of VDJ recombination (and also for general nonhomologous end-joining), Ku and DNA-PKcs, are required, suggesting that there may be similarities with the reaction for nonhomologous end-joining.


A promoter lies immediately upstream of each switch region, and switching may be activated by transcription from the promoter (which itself may be responsive to activators that respond to environmental conditions, such as stimulation by cytokines, thus creating a mechanism to regulate switching. Because the S regions lie within the introns that precede the CH coding regions, switching does not alter the translational reading frame.


Early heavy chain expression can be changed by RNA processing


The period of IgM synthesis that begins lymphocyte development falls into two parts, during which different versions of the µ constant region are synthesized:


As a stem cell differentiates to a pre-B lymphocyte, an accompanying light chain is synthesized, and the IgM molecule (L2µ2) appears at the surface of the cell. This form of IgM contains the µm version of the constant region (m indicates that IgM is located in the membrane). The membrane location may be related to the need to initiate cell proliferation in response to the initial recognition of an antigen.




Figure 24.1 Humoral immunity is conferred by the binding of free antibodies to antigens to form antigen-antibody complexes that are removed from the bloodstream by macrophages or that are attacked directly by the complement proteins.

When the B lymphocyte differentiates further into a plasma cell, the µs version of the constant region is expressed. The IgM actually is secreted as a pentamer IgM5J, in which J is a joining polypeptide (no connection with the J region) that forms disulfide linkages with μ chains. Secretion of the protein is followed by the humoral response depicted in Figure 24.1.




Figure 24.19 The 3 F end controls the use of splicing junctions so that alternative forms of the heavy gene are expressed.

The µm and µs versions of the μ heavy chain differ only at the C-terminal end. The µm chain ends in a hydrophobic sequence that probably secures it in the membrane. This sequence is replaced by a shorter hydrophilic sequence in µs; the substitution allows the μ heavy chain to pass through the membrane. The change of C-terminus is accomplished by an alternative splicing event, which is controlled by the 3′ end of the nuclear RNA, as illustrated in Figure 24.19.


At the membrane-bound stage, the RNA terminates after exon M2, and the constant region is produced by splicing together six exons. The first four exons code for the four domains of the constant region. The last two exons, M1 and M2, code for the 41-residue hydrophobic C-terminal region and its nontranslated trailer. The 5′ splice junction within exon 4 is connected to the 3′ splice junction at the beginning of M1.


At the secreted stage, the nuclear RNA terminates after exon 4. The 5′ splice junction within this exon that had been linked to M1 in the membrane form is ignored. This allows the exon to extend for an additional 20 codons.


A similar transition from membrane to secreted forms is found with other constant regions. The conservation of exon structures suggests that the mechanism is the same.




Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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