Regulation of Ig gene transcription (Part 12- Antibody Basics)
Welcome to the 12th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, Part 4, Part 5, Part 6, Part 7, Part 8, Part 9, Part 10, and Part 11
Regulatory sequences in DNA
The immunoglobulin genes are expressed only in B cells, and even in B cells, the genes are expressed at different rates during different developmental stages. As with other eukaryotic genes, there are three major classes of regulatory sequences present in immunoglobulin genes. These classes are Promoters, Enhancers, and Silencers
- First and foremost is the promoter present 200 bp upstream from the transcription initiation site. Each VH and VL gene segment has a promoter located just upstream from the leader sequence (designated as L). In the below Fig, the promoter (designated as P) is shown in the black circle.
The immunoglobulin promoters contain a highly conserved AT-rich sequence called TATA box, which serves as a site for binding of RNA polymerase II to initiate transcription. In addition, it also serves as a site for the binding of various transcription factors that regulate the transcription of immunoglobulin genes. For instance, Ig promoters contain a conserved octamer sequence, which binds to transcription factors oct-1 and oct-2.
2. Apart from promoters, enhancers and silencers are also present on the immunoglobulin genes. These are nucleotide sequences that regulate the transcription of Ig genes. Enhancers have binding sites for the proteins, like transcription factors that activate transcription from the promoter, and silencers have binding sites for the proteins like repressors that downregulate transcription from the promoter.
3. Another essential factor that regulates immunoglobulin transcription is immunoglobulin gene rearrangement.
In germline DNA, RNA polymerase II binds very weakly to the promoters associated with immunoglobulin V gene segments. Also, the variable region enhancers, which have binding sites for transcription factors to activate transcription from the promoters, are present around 200–300kb downstream from the promoters. Since enhancers are quite distant from the promoters, they can hardly influence the transcription of the gene. For this reason, the transcription of VH and VL gene segments is negligible in germline DNA.
At the same time, in the rearranged Ig gene segments, the promoter and enhancer are within a range of 2 kb distance from each other. In this setting, they are close enough for the enhancer to activate the transcription from the promoter region. And as a result, the rate of transcription of rearranged VJ or VDJ unit is 10⁴ times more than that of unarranged VL or VH gene segments.
Allelic exclusion
Allelic exclusion also regulates the expression of immunoglobulin genes. It is a phenomenon that ensures that a mature B cell expresses a single species of antibody with unique specificity for an antigen. But before going into its details, it is important to understand the term allele.
An individual has 23 pairs of chromosomes. And the two chromosomes of a pair are said to be homologous because they are very similar to one another and have the same size and shape. The homologous pair of chromosomes in your genome is formed by pairing of one homologous chromosome that came from your mother and the other homologous chromosome that came from your father. The pairing of homologous chromosomes is done in such a way that the gene encoding for the same trait is always carried on a similar place or position on both of the homologous chromosomes. In other words, homologous chromosomes have the same type of genetic information: that is, each gene resides at the same specific locus on both the homologous chromosomes.
Though one copy of the gene is inherited from the mother and another from the father, it is not necessary that the individual will have the same versions of genes on the two homologous chromosomes of a pair. That’s because he may have inherited two different gene versions from his mother and his father. If this is the situation where two copies of a gene differ from each other, they are known as alleles.
In other words, alleles can be defined as the different versions or forms of the same gene. For instance, the gene coding for eye color can have alleles for brown, blue, green, and other eye colors. Simply put, a gene specifies which trait is required to be expressed, say eye color, and the alleles of the “eye color” gene give directions for making eyes blue, green, brown, and so on. Additionally, a gene may have multiple different alleles, though only two alleles can be present at the gene’s locus in any individual.
Like all somatic cells, B cells are also diploid. It means that each B cell has paired chromosomes: one from each parent. As already discussed, there are three types of immunoglobulin loci on human DNA. These are κ chain locus, ƛ chain locus, and heavy chain locus. Since the B cells are diploid, the immunoglobulin encoding genes are present on both maternal and paternal chromosomes. Or we can say, a B cell contains two heavy chain alleles, two kappa chain alleles, and two lambda light chain alleles. So one heavy chain allele, one kappa chain allele, and one lambda chain allele are present on the maternal chromosome. The second heavy chain allele, kappa chain allele, and the lambda chain allele are present on the paternal chromosome.
Therefore, a B cell has two heavy chain loci and four-light chain loci in its genome. But suppose the immunoglobulin loci present on both the chromosomes are expressed. In that case, B cell might produce two different heavy chains and four different light chains, therefore resulting in B cell receptors or antibodies with 2*4=8 different antigen-binding specificities. But a B cell produces the B cell receptors or antibodies with only one particular antigen-binding specificity. Therefore, even though a B cell is diploid, it expresses rearranged heavy chain genes from only one chromosome. It also expresses light chain genes from only one chromosome, and that chromosome may either be paternal or maternal. The process by which this is accomplished is called allelic exclusion.
The above Fig shows that after rearrangement, the cell can express both the light and heavy chain genes encoded by the maternal chromosome or have heavy chain genes expressed from the maternal chromosome but light chain genes expressed from the paternal chromosome.
Allelic exclusion ensures that a single B cell does not contain more than one VHDHJH unit and one VLDL unit. It is very critical for the antigen specificity of a B cell because expression of both alleles would render B cell multispecific. Because of allelic exclusion, once a productive VHDHJH rearrangement and a productive VLDL rearrangement have occurred, the recombination machinery is turned off, and then the heavy chain and light chain genes from the homologous chromosome are not expressed.
Allelic exclusion also limits the B cells to express either κ light chains or ƛ light chains. But never both of them.
Mechanism by which allelic exclusion occurs in B cells
Once the productive rearrangement of µ heavy chains is obtained from allele 1 in B cell, its encoded protein is expressed, and the presence of this protein signals the maturing B cell to turn off the rearrangement of other µ heavy chain allele and turn on the rearrangement of light chain gene. Whereas if the productive rearrangement of µ heavy chain allele 1 is not obtained, then the expression of µ heavy chain allele 2 is turned on. And in case productive rearrangement is not obtained from any of the two alleles, then the cell undergoes death by apoptosis.
After this, the productive rearrangement of µ heavy chains turns on the rearrangement of κ light chain genes. If a productive κ rearrangement occurs, then κ light chains are produced that pair with µ heavy chains to form a complete antibody molecule. The presence of this antibody molecule then turns off further light chain rearrangement from the other κ light chain allele as well as rearrangement from the ƛ allele.
On the contrary, if κ rearrangement is non-productive for both κ alleles, then rearrangement of the ƛ chain genes begins. If neither ƛ nor κ alleles rearrange productively, the B cell ceases to mature and soon dies by apoptosis.
Therefore, we can conclude that the recombination happens only in one of the alleles and never on both alleles simultaneously. The protein products encoded by rearranged heavy and light chain genes regulate rearrangement of the other allele, thus accounting for allelic exclusion.
Two studies with transgenic mice were done to prove that the protein products encoded by rearranged genes regulate the rearrangement of the remaining alleles. In one study, transgenic mice carrying a rearranged µ heavy chain transgene were prepared. The expressed rearranged µ heavy chain transgene protein then blocked the rearrangement of germline heavy chain genes of mice.
Similarly, the transgenic mice carrying a κ light chain transgene expressed the transgene κ light chain product, and this protein blocked the expression of germline κ light chain genes in mice.
Additionally, transgene κ light chains were found to be associated with the heavy chains to form antibodies.
These studies suggest that the expression of heavy and light chain proteins prevents the gene rearrangement of the remaining alleles and thus accounts for allelic exclusion.
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