ReadingThinkingAndWriting

So you’ve been told that all the cells in your body contain the same genetic material? I’ve seen it written as a general principle of Genetics and a quick search around the web will yield many sources indicating that all somatic cells contain the exact same DNA. I did find a few articles in science magazines which “revealed” that this may not be the case, but the general consensus that they do was prevalent.

From all I’ve read, research and texts included, the DNA contained in all somatic cells is the same with one exception; the hematopoietic cells. These cells are the cells of the blood and they all descend from a single progenitor, the Human Hematopoietic Stem cells which can be found in bone marrow. These stem cells give rise to a diverse population of progeny including erythrocytes (red blood cells), T Cells, B Cells, Macrophages, and Natural Killer cells. To be accurate, the stem cells and their immediate descendants contain “germ-line” DNA, which is DNA that was formed during the individual’s fertilization event and is shared with virtually every other somatic cell in the body. However, as the hematopoietic descendants start to specify into the progenitors of T and B cells or “lymphopoietic” progenitors, the process of Somatic Recombination which creates the antigen receptors I’ve been writing about cuts out and discards parts of certain chromosomes thus creating genetically distinct human cells within our own bodies.

In my last post I presented the gene loci on which this process acts and highlighted the first actors in the process; the Recombination Activating Gene 1 & 2 proteins (RAG1 & RAG2). The arrangement of the gene loci is very important in this discussion and the rest of what I’m about to present doesn’t make much sense without this knowledge, so you can click this link to read that post if necessary. The process is performed by the V(D)J Recombinase and the process progresses as the molecules that make it up are assembled, I will introduce them as I describe the process.

It begins with two molecules each containing one RAG1 and one RAG2. These complexes then randomly bind, one each, to two of the hundreds of Recombination Signaling Sequences (RSSs) found surrounding each gene segment in the target antigen locus. It should be noted that the selection is not random across the segment. For example, two V regions will not join; it must be a V and J, for example, in a light chain locus. After binding, the two complexes come together and bind resulting in the formation of a loop of DNA between them which contains all the gene segments that may have been between the two segments brought together.

With the loop formed, the RAG complex will cut the strand exactly at the points where the RSS ends and the coding segment begins (see diagram below). This happens because the RAG complex has an inherent ability to act as an endonuclease. This essentially means that it is a protein that can break a DNA molecule into parts. This is the action that excises a piece of our DNA which is called the signal joint (because the RSS of each segment are joined flush). At this point, because of reasons relating to the chemical properties of the structure of a molecule of DNA, the ends of each strand which are cut fuse to form hairpins.

At this point other proteins join the complex to complete the recombinase and the process. These proteins are actually ubiquitous in human cells and I believe they are present for every mitotic and meiotic event that occurs over an individual’s lifetime. The first two proteins to bind to the growing V(D)J Recombinase are named Ku70:80 and they hold together both the ends of the chromosomal DNA strand, and those of the signal joint. Note, that this is the last I will mention the signal joint as once it’s ends are fused, it is no longer part of our story.

It is here where the process becomes a little unnerving for me. The very precise cut made by the RAG1/RAG2 complex to setup the joining of gene segments is one thing, but what happens next, to me, is another. The following video presents an excellent visualization of the entire process with a voice-over by Dr. Julie Theriot; a brilliant mind. I will, however, go into a little more detail below.

A protein named DNA-PK, or DNA-dependent Protein Kinase, is formed when another protein named DNA-PKcs binds tightly with Ku after the strands are bound. And to this another protein named Artemis joins. Artemis is also an endonuclease without which we would be ending our story with a broken strand of DNA. Artemis cuts the hair pins formed after RAG1/RAG2 excised the signal joint, but the location of its cut appears to be random. It has been found that they occur anywhere along the hair pin and this is where the fun starts.

With the location being random, unequal ends are often left which must be equalized before the DNA can be rejoined. Enter another protein that is found in all our cells and has a great name; Terminal Deoxynucleotidyl Transferase (TdT). TdT randomly adds bases to the cut ends resulting in what is essentially a newly formed random sequence on each. At the same time, two more newcomers bind to each other and then the Recombinase; DNA Ligase IV and XCR44. There are ubiquitous DNA repair proteins which and as soon as they bind, they start trying to re-join the strands; the name Ligase actually comes from the Latin “ligare” meaning “to bind or tie”. With TdT inserting random bases, the repair/ligation complex may need to knock out and replace non-complementary pairings such as a Guanine to a Thiamine.

With the ligation effort winning out in the end and the chromatid now back in one piece, the process of Somatic Recombination is complete. With just a few more steps we have a complete gene which, when transcribed will form one of the two chains in an Immunoglobulin. The process, however, has left the chromatid irrevocably changed; it is unique, different from any other member of its chromosome pair in the body, including other B cells. I’d like to note again that this change is key to the efficacy of our adaptive immune response. Without the enormous diversity generated by this process, neither the B and T cell antigen receptors nor the antibodies secreted by the former would be capable of binding effectively to the diverse and ever changing peptides making up evolving pathogens. However, it also introduces a potential for a number of pathologies, including cancer.

It has been shown in research1 that if the DNA repair mechanisms like DNA Ligase IV\XCR44 and the DNA damage checkpoints are not functioning properly, Somatic Recombination can result in several pathologies. These include aneuploidy, an abnormal number of chromosomes, translocations, where one part of a chromosome is fused with another, and neoplastic transformation, cancer.

In fact, according to both Janeway’s Immuno Biology (p 312) and a paper published in Nature Immunology in 20071, translocations are found in most lymphoma tissues. What is compelling for me about these translocations is that they most often involve what are known as proto-oncogenes which are genes encoding proteins that, when not functioning properly, cause cancer.

What causes DNA checkpoints and the DNA repair proteins to malfunction? I’ve seen the research showing the obvious conclusion; mutations forming in the genes coding for the proteins which carry out the functions. However, I suspect epigenetics may play a role in at least some of the scenarios. What causes these translocations? Given the fact that many lymphomas are age onset, I would conclude that probability must be considered, but the translocations of proto-oncogenes suggest to me that epigenetic factors should be examined. There are genes such as Myc (Myelocytomatosis)that regulate the expression of up to 15% of our entire genome and do so through epigenetic factors such as HAT (Histone Acetyltransferase). It should not be surprising that Myc is an oncogene and it is, in fact, one that is involved in the translocations found in lymphoma tissues.

1. Nature Immunology 8, 801 – 808 (2007)
Published online: 19 July 2007 | doi:10.1038/

It’s been my experience that when bacteria are discussed, it is usually in a negative light. This is despite the fact that there are ten times more bacterial cells in and on our bodies than human cells, and the co-location is actually mutually beneficial. Understandably, viruses get the same treatment, but with their potential sequestering for genetic therapies perhaps there will be a change. I have actually been thinking that viruses may have played a key role in early the evolution of life, but that is another story. This story, the one on somatic recombination, is a very complicated one, so you can review the first two parts, here, and here.  Note that I’m focusing on B cells, but the process is essentially the same in T Cells.

Given negative light in which viruses are seen, it seems ironic then that the mechanism by which a retrovirus integrates its genetic material into the infected cell is very similar to the mechanism by which our B and T cells produce the molecules needed to recognize and defeat bacterial and viral infections ₁. Two proteins, named RAG1 and RAG2, which start off the process in our cells, have been shown to form what is known as a transposase. In retroviruses such as the one which causes AIDS, a transposase is used to splice the viral genetic material into that of the infected cell. RAG1 & 2 combined in a human cell can do the exact same thing₂.

Even the genes which encode these proteins are extraordinary. Mammalian genes usually contain regions called introns which are non-coding and though transcribed, are edited out before the RNA is translated into a protein. However, the RAG genes, like most bacterial genes, contain none. This seems to me, at least, to be a very interesting evolutionary story. How did genes that don’t contain introns and code for proteins which form a transposase end up in our genome?  I’ll have to note this question for another post.

As I mentioned in my last post, the immunoglobulins that Recombination creates have a both a variable (V) regions and a constant (C) regions. They are made up of two identical pairs of protein strands, called chains, with each pair consisting of one light and one heavy (the heavy chains are the longer chains in the image to the right). It is the variable regions we are concerned with here and RAG 1 & 2 proteins join, and then incorporate a few other proteins to form what is known as VDJ recombinase to produce them. The recombinase is the lead actor in somatic recombination.

Before we continue to the process, the genes which code for the light and heavy chains are uncommon as well and therefore worth visiting. Ordinarily, the nucleotide sequence of a given gene is transcribed from the chromosome onto an RNA molecule that is then translated by a ribosome to produce the protein product. The location on a chromosome at which any given gene is located is known as the locus (loci, Pl.)

This is not the case, however, at the loci which encode the heavy and light chains of an Immunoglobulin. An examination of the light chain locus on chromosome 2 will reveal not a single gene but rather a large number of gene segments. Further, there are special sequences of nucleotides to be found surrounding those segments. First, up are the so-called Leader sequences which come into play after translation and mark an immunoglobulin for transport to the plasma membrane. Second are the even more interesting Recombination Signaling Sequences (RSSs) which are the markers to which the RAG 1 and RAG 2 complex binds.

As depicted in the diagram below, there are actually different types of gene segments within the locus which will be combined together in the process. The variable region generated by the VDJ recombinase will include one each of the V, D (heavy chain loci only), and J segments. The C segments code the constant regions and we’ve only found a handful so far. Estimates that I’ve read or heard for the number of V segments, on the other hand, range anywhere from forty to hundreds within a given locus.

The process of somatic recombination forming a Light Chain selects at random a V segment and a J segment and joins them together along with a C segment to form a single gene. The “D” in VDJ refers to the D, or Diversity, segments which exist only in Heavy Chain loci, in fact the proper spelling is V(D)J recombinase.  It is the process of joining the segments that produces the infinite potential for sequence recognition, not the segments themselves.  This process, Somatic Recombination,  is simple in concept, yet complex in execution and within, there is yet another twist which demonstrates the beauty as well as the danger in this amazing biological mechanism. In my next post, the process…

1.Janeway’s Immuno Biology
Seventh Edition
Garland Science

2. Nature. 1998 Aug 20;394(6695):744-51.
Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system.
Agrawal A, Eastman QM, Schatz DG.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.