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Generation of Antibody Diversity is Unlike Darwinian Evolution

[Editor's Note: This is part two of a response from microbiologist Don Ewert to arguments from BioLogos's Kathryn Applegate that our immune system shows the creative power Darwinian evolution. Part one can be found here.]

The intricate mechanism for generating antibody diversity from very few germline (existing) genes was discovered over thirty years ago. It involves shuffling gene segments and then fusing them to produce new combining sites for the antibody receptor displayed on individual B cells. How much of this process is pre-programmed and how much is random? Is this an example of the use of a "'blind' system to sustain and preserve life," as Kathryn Applegate suggests? The evidence from decades of research reveals a complex network of highly regulated processes of gene expression that leave very little to chance, but permit the generation of receptor diversity without damaging the function of the immunoglobulin protein or doing damage to other sites in the genome.

The most remarkable aspect of antibody production is the mechanisms that generate the binding site of the antigen receptor. The antigen receptor of B cells are proteins called immunoglobulins. They have an antigen combining site at one end that binds to foreign proteins (variable or V region) and a tail, or constant region (C region), at the other end that controls the interaction with other components of the immune system that are responsible for eliminating the foreign invader. The variable end of the BCR heavy chain is generated by the shuffling and joining of gene segments from separate pools of V (45), D (23), and J (6) segments per cell and the random deletion and insertion of nucleotides at the joining sites. This process is duplicated on the second (light) chain of the immunoglobulin gene. The combined diversity generated by recombination which is limited by the number of gene segments and by nucleotide exchanges, which are unlimited, produces a potential repertoire of about 1011 different receptor specificities. This process occurs during transcription of the DNA and involves a set of coordinated enzymatic reactions. The total number of available receptor specificities is limited by the number of B and T lymphocytes.

The joining of the V, D, and J segments is orchestrated by a set of enzymes, RAG-1 and RAG-2, which cleave the DNA at specific markers called recombination signal sequences (RSS), which flank each V, D, or J gene segment. The RAG-1/2 complex brings the segments together and the segments are joined using the general DNA repair mechanisms of the cell. The shuffling of the V, D, and J gene segments alone has limited ability to generate the necessary diversity of required receptor specificities. The unique feature of this recombinatorial process is the imprecise joining and inclusion of non-templated nucleotides at the junctions of the rearranged V, D, and J gene fragments. During the process, gaps in the joined DNA are filled using repair enzymes (polymerases) that insert nucleotides. These changes generate thousands of new ways of folding the proteins in the V region.

Confining RAG
The RAG proteins belong to a family of proteins called transposases. As the name implies, these proteins are able to cut and paste segments of DNA in the genome, which would otherwise be highly disruptive of normal operations, if not lethal to the organism. Thus, the transposon components of RAG have to be tightly controlled. To be precise, their physical and temporal deployment and function are tightly regulated and highly specific. Remarkably, they function only in jawed vertebrates to rearrange the V(D)J segments and only in the immature B cells (and T cell), while maintaining the integrity of other parts of the genome:

Expression of the Rag proteins is regulated at both the transcriptional and post-transcriptional levels, and co-expression of RAG 1 and RAG 2 is limited to lymphoid committed cells. In order to guarantee that the V(D)J recombinase acts only within the B and T lymphocyte lineage, the reaction is tightly regulated at multiple stages, including expression of the RAG genes themselves as well as controlled access of the recombinatorial machinery to its substrates within chromatin. (Hanasen, J.D. and McBlane, J.F)

B cell development: Irreducibly complex?
The RAG genes do not operate in isolation from the rest of the organism, but are part of a complex gene regulatory network which controls each stage of B cell development. The development of B cells from hematopoietic stem cells is a highly regulated multistep process. Each stage of development can be described on the basis of a constellation of gene expression patterns. Over 100 genes have been identified that are regulated to guide a multipotent hematopeitic stem cell down a pathway that leads to a functional B cell. Some of these are transcription factors which regulate gene expression while others actively suppress genes that are not appropriate for B cell function (Fuxa and Skok). In another recent review Nutt and Lee conclude:

instead of a simple transcriptional hierarchy, efficient B cell commitment and differentiation require the combinatorial activity of multiple transcription factors in a complex gene regulatory network....[T]he transcriptional network controlling B cell specification and commitment is not a simple linear cascade but involves multiple combinatorial inputs and feedback loops. [T]his process involves hierarchical forward steps and feedback loops, with this handful of factors being used in multiple contexts and distinct combinations. It has also demonstrated the surprising requirement for continual reinforcement of the commitment process throughout the life of a B cell.

The irreducible nature of this network has been demonstrated using knockout mice in which individual factors are eliminated (Fuxa and Skok). For example, RAG knockout mice are immuodeficient due to a lack of T or B lymphocytes. Thus failure to rearrange the receptors leads to cell death which has practical implications for survival. Similarly, removal of any of the factors that lead to Ig rearrangement or repress expression of lineage-inappropriate genes will affect B cell survival. Furthermore, this network of regulatory factors is critical for controlling processes that have the potential for causing harm to the genome and cellular function. As noted by Nutt and Kee, B cell transcriptional regulators are targets of mutation or deletion in mouse and human acute lymphoblastic leukemia:

These findings demonstrate that the function of these essential B cell transcription factors needs to be carefully controlled to avoid unwanted outcomes such as malignancy. (Nutt S and Kee B)
Rules of Recombination: The process of V(D)J joining is also highly regulated to ensure the production of functional antibodies and to avoid distal chromosomal damage. The recombination of the V(D)J loci takes place only between gene segments flanked by RSSs with the additional requirement that space lengths of either 12 or 23 bases separate the joining DNA regions. This "12/23" rule prevents non-productive rearrangements by directing recombination of D to J, but not V to V or J to J. Also, recombination occurs sequentially with D to J recombination occurring prior to V to DJ on the heavy chain and heavy chain recombination preceding that of light chains. Each stage is controlled by specific transcription factors:
The transcription factors E2A and EBF control the initial DH-JH rearrangement step by activating expression of the RAG genes and promoting accessibility of the DH-JH region within the IgH locus. PAX, by contrast, induces large-scale contraction of the IgH locus; this is essential for the second stage of VH-DJH recombination. (Fuxa and Skok)

Additionally, recombination is restricted to the G0/G1 phase of the cell cycle in order to avoid chromosomal instability and associated lymphoid cancers.

In conclusion, the mechanism for generation of antibody diversity by V(D)J recombination is, unlike Darwinian evolution, highly regulated and carefully designed to permit expansion of receptor variability but within well defined limits. It is just the kind of system one would design for independent survival of an organism whose encounter with pathogens is not programmed.