*2.2.9.2 Regulation of transcription and epigenetic modifications*

Epigenetic modifications are necessary for the positive or negative regulation of the activities of different loci. Indeed, besides the presence of sites sensitive to DNase activity, other conditions, controlled by the activating elements and the promoters of the loci concerned, are necessary to initiate gene rearrangements and are correlated with the opening of chromatin to transcription, such as histone acetylation, DNA demethylation, and transcription itself.


**31**

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation*

on the position of the mCpG around the RSS) and induces an inaccessible chromatin configuration. Conversely, CpG demethylation in the heptamer of broken signal ends derived from the 3′ Dβ1 RSS has been shown to allow V(D)J

*2.2.9.3 Establishment of loops-Rosette locus-and contacting segments to recombine*

particular the distal V genes) and thus facilitates rearrangements.

*2.2.9.4 Regulation of loci position at the nucleus*

rearrangements.

**3. Allelic exclusion**

the variable regions.

The establishment of loops is necessary to bring into contact the different segments to recombine giving rise to an image of the so-called rosette locus, which represents one of the prerequisites for V(D)J rearrangements [25]. These loops take place through a number of factors, including transcriptional repressor CCCTCbinding factor (CTCF, also known as 11-zinc finger protein), Yin Yang 1 (YY1), a ubiquitously distributed transcriptional repressor/activator factor of a number of promoters, belonging to the GLI-Kruppel class of zinc finger proteins, and paired box 5 (Pax5), which is important regulators in early development, but not late stages of B-cell differentiation. Such different factors and particularly CTCF, by binding to cohesins, regulate the IG loci reorganization and contraction. This contraction/reorganization allows the juxtaposition of different gene segments (in

The locus nuclear positioning is decisive for the rearrangement. Hence, the IGH locus, anchored *via* the distal VH genes at the nuclear periphery, migrates, in its extended chromatin state, to the center of the nucleus, which facilitates access of the V(D)J recombinase to proximal IGH domain and thus VH-DJH

The allelic exclusion of IG of H and L chain genes allows the production of antibodies from a single chromosome located on 14q32.3 for the H chain [26], and one of the two chromosomes located on 2p12 or 22q11.2 for the L chain [27, 28] (respectively, the Lκ and Lλ chains). This phenomenon constitutes genetic basis of monospecificity of B-cells-a central paradigm in explaining the pathogen-specific production of antibodies (Burnet's clonal selection theory of the adaptive immune system), *i.e.*, each clone of B-cells generates a unique specificity for the appropriate antigen, which is established during the rearrangement of V(D)J gene segments of

During the differentiation of the B-cell, only one fraction of the IG genes, resulting from a first somatic V(D)J recombination on one of the two random chromosomes 14, is functional, *i.e.*, it contains a productive exon V(D)J. By cons, if the rearrangement is abortive (nonproductive), a new recombination is attempted on the other chromosome. The success toward a productive rearrangement of the H chain leads to a temptation to rearrange with the chromosomes encoding the L

The mechanism of allelic exclusion uses pre-BCR–mediated signals. The pre-BCR consists of the H chain resulting from the productive rearrangement of an allele encoding the μ H chain associated with a pseudo-L chain (surrogate L chain). This chain comprises a V polypeptide (called V pre-B) and a type C polypeptide (called λ5 in mice and λ-like in humans) that associate noncovalently. The signals

chains. The absence of rearrangements leads to the sterility of B-cell.

*DOI: http://dx.doi.org/10.5772/intechopen.90637*

cleavage in mouse [24].

#### *Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation DOI: http://dx.doi.org/10.5772/intechopen.90637*

on the position of the mCpG around the RSS) and induces an inaccessible chromatin configuration. Conversely, CpG demethylation in the heptamer of broken signal ends derived from the 3′ Dβ1 RSS has been shown to allow V(D)J cleavage in mouse [24].

## *2.2.9.3 Establishment of loops-Rosette locus-and contacting segments to recombine*

The establishment of loops is necessary to bring into contact the different segments to recombine giving rise to an image of the so-called rosette locus, which represents one of the prerequisites for V(D)J rearrangements [25]. These loops take place through a number of factors, including transcriptional repressor CCCTCbinding factor (CTCF, also known as 11-zinc finger protein), Yin Yang 1 (YY1), a ubiquitously distributed transcriptional repressor/activator factor of a number of promoters, belonging to the GLI-Kruppel class of zinc finger proteins, and paired box 5 (Pax5), which is important regulators in early development, but not late stages of B-cell differentiation. Such different factors and particularly CTCF, by binding to cohesins, regulate the IG loci reorganization and contraction. This contraction/reorganization allows the juxtaposition of different gene segments (in particular the distal V genes) and thus facilitates rearrangements.

#### *2.2.9.4 Regulation of loci position at the nucleus*

The locus nuclear positioning is decisive for the rearrangement. Hence, the IGH locus, anchored *via* the distal VH genes at the nuclear periphery, migrates, in its extended chromatin state, to the center of the nucleus, which facilitates access of the V(D)J recombinase to proximal IGH domain and thus VH-DJH rearrangements.

## **3. Allelic exclusion**

*Normal and Malignant B-cell*

The junction system comprises a number of ubiquitous repair proteins (present in both B-cells and T-cells), which would allow rearrangement of IG genes in B-cell precursors, but rarely in T-cell precursors, and TCR gene rearrangements in T-cell precursors, but rarely in B-cell precursors. It includes in particular the catalytic subunit of a nuclear DNA-dependent serine/threonine protein kinase complex (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related (PIKK) family of protein kinases, composed of a heterodimer of Ku proteins that bind free DNA ends given their strong affinity (Ku70/Ku80 [encoded, respectively, by X-ray cross-complementing gene 5 (XRCC5) and XRCC6 genes in humans, and also called Ku86]) [19], XRCC4, DNA ligase IV and Artemis [3]. TdT is also recruited into the junction system and is involved in the formation of the coding joints, alongside Artemis and DNA-PKcs. Nevertheless, TdT is only rarely recruited into rearrange-

ments that occur during fetal life, so that junctional diversity is limited.

*2.2.9.1 Transcription-sequential/ordered model-of germinal loci and accessibility* 

It should be clearly noted that the VH segments are not silent before the V-DJH recombination steps or before their physical juxtaposition with the Eμ enhancer. It has therefore been shown that they undergo active noncoding germline transcription in B-cell precursors. In addition, many other noncoding transcripts appear as rearrangements occur, in order to allow the opening of chromatin, and thus, the targeting and accessibility by RAG-1/RAG-2 complex, as well as the establishment of the three-dimensional structure of the locus considered. From a kinetic point of view, the first noncoding DH transcripts, also referred to as sterile transcripts (to differentiate them from the coding transcripts, which are initiated at the rearranged VDJ segments), are detected before the D-JH rearrangements and are initiated at JH-proximal DH gene (DQ52), which has both promoter and enhancer activities preferentially active in B-cell precursors [20], generating μ0 transcripts, and at downstream of intronic IGH enhancer Eμ, generating Iμ transcripts. Both μ0 and transcripts Iμ are getting spliced and polyadenylated [ 21]. Once the DJH rearrangement is carried out, new noncoding germinal transcripts appear in VH

Epigenetic modifications are necessary for the positive or negative regulation of the activities of different loci. Indeed, besides the presence of sites sensitive to DNase activity, other conditions, controlled by the activating elements and the promoters of the loci concerned, are necessary to initiate gene rearrangements and are correlated with the opening of chromatin to transcription, such as histone

• *Acetylation of histones*. It occurs prior to V(D)J recombination. It is associated with the VH region after IL-7 stimulation and DJ rearrangement before VH appendage to DJH and is accompanied by increased nuclease sensitivity and

• *DNA methylation/demethylation near RSS*. DNA methylation/demethylation is involved in regulating V(D)J rearrangement. DNA methylation around the RSS inhibits V(D)J cleavage activity of the RAG-1/RAG-2 complex (depending

reorganization of nucleosome structure [22] (for review, see [23]).

*2.2.9 Molecular mechanisms of regulation of V(D)J rearrangement*

*2.2.9.2 Regulation of transcription and epigenetic modifications*

acetylation, DNA demethylation, and transcription itself.

*by RAG-1 and RAG-2 proteins*

regions (for review, see [21]).

**30**

The allelic exclusion of IG of H and L chain genes allows the production of antibodies from a single chromosome located on 14q32.3 for the H chain [26], and one of the two chromosomes located on 2p12 or 22q11.2 for the L chain [27, 28] (respectively, the Lκ and Lλ chains). This phenomenon constitutes genetic basis of monospecificity of B-cells-a central paradigm in explaining the pathogen-specific production of antibodies (Burnet's clonal selection theory of the adaptive immune system), *i.e.*, each clone of B-cells generates a unique specificity for the appropriate antigen, which is established during the rearrangement of V(D)J gene segments of the variable regions.

During the differentiation of the B-cell, only one fraction of the IG genes, resulting from a first somatic V(D)J recombination on one of the two random chromosomes 14, is functional, *i.e.*, it contains a productive exon V(D)J. By cons, if the rearrangement is abortive (nonproductive), a new recombination is attempted on the other chromosome. The success toward a productive rearrangement of the H chain leads to a temptation to rearrange with the chromosomes encoding the L chains. The absence of rearrangements leads to the sterility of B-cell.

The mechanism of allelic exclusion uses pre-BCR–mediated signals. The pre-BCR consists of the H chain resulting from the productive rearrangement of an allele encoding the μ H chain associated with a pseudo-L chain (surrogate L chain). This chain comprises a V polypeptide (called V pre-B) and a type C polypeptide (called λ5 in mice and λ-like in humans) that associate noncovalently. The signals

mediated by this pre-BCR block the accessibility of the RAG recombinases on the second allele of the nonrecombinant μ H chain and redirect them toward the Lκ chain locus to initiate the first recombinations. The formation of a complete BCR combining H and L chain blocks recombinations on other L chain alleles.

Importantly, it has been shown, using genetically engineered mice that carry two functional IGH alleles that are completely recombined and different, that the expression of IG loci does not appear to be monoallelic and that B-cells could have the ability to express H chains by both alleles [29] (for review, see [30]).
