6 - The Organization and Expression of Lymphocyte Receptor Genes

Question 1

Immunoglobulin

Answer 1

https://www.youtube.com/watch?v=vxWf-66lymg&ab_channel=MEDSimplified

Question 2

Multigene organization of Ig genes

Answer 2

Ig proteins consist of two identical heavy hains and two identical light chains.

The light chains can be kappa light chains or lambda light chains.

The heavy chain, kappa, and lamda chains are encoded on separate chromosomes.

Question 3

K light chain genes

Answer 3

The human and mouse k-chqin loci are arrangen in groups of V and J segments that are located upstream from a single C_k segment.

The precise number of V_k segments in humans varies between individuals.

Some V_k sequences are trc in the opposite direction of the C_k segment, others in the same direction.

Question 4

Lambda light-chain genes

Answer 4

include paired J and C segments.

Recombination of Ig gene segments always occurs in the downstream direction (V to J), Vλ1 variable region is expressed with either Jλ3-Cλ3 or Jλ1-Cλ1, as the Vλ1 is upstream from these.

Jλ2-Cλ2 is upstream from Vλ1, and they are therefore not expressed together. Vλ2 is usually upstream from Jλ2-Cλ2 region, and therefore expressed together (though recombination can fuck up this)

The mouse Ab λ light chain locus has undergone a deletional event, so there are fewer Vλthan V_k gene segments. Consequently, mouse λ light chains are significantly less diverse than their K-chain counterparts.

In mice, 5% of light chains are of the λ-isotype, whereas in humans 40% of light chains carry the λ constant region.

Recombination between Vλ and Jλ gene segments can occur only between Vλ sequences that lie upstream of their Jλ partners.

Question 5

Heavy-chain gene organization

Answer 5

includes VH, D, JH and CH segments
D = diversity region, only in heavy chains and therefore do not need the H.

Three clusters of gene segments (VH, D, JH clusters) encode the variable region of the heavy chain, and are located upstream of a set of eight C-region gene segments.

The precise number of V, D, and J segments avry between individuals

All VH gene segments are transcribed in the same direction as the constant region gene segment.

Human VH locus has approx 45 functional VH segments, 23 functional D segments, 6 functional JH segments.

Question 6

Antibody genes found in mature B cells are the product of DNA recombination

Answer 6

Each B cell uses one Vk or one Vλ coupled with one Jk or one Jλ (respectively), to create a single light-chain variable region.

It also recombines one VH, one D and one JH segment to form a heavy-chain variable region gene.

Question 7

The mechanism of V(D)J-recombination

Answer 7

The DNA encoding a complete V region of an Ab is assembled from V, D, J in heavy chains and V +J in light chains. These are initially separated by thousands of bases in the DNA.

Each developing B cell generates a new pair of VH and VL coding sequences by recombination.

To minimalize the risk of faulty cutting and rejoining of the DNA strand, this recombination is only possible during defined periods of the B and T cell maturation, and only on appropriate regions.
This degree of accuracy is accomplished by the fact that the recombination enzymes recognize DNA seq motifs calles RSS (recombination signal sequences). The RSS also ensure that one of V and J (and D in heavy chains) is included in the recombined genes.

Question 8

V(D)J recombination in lymphocytes is a highly regulated sequential process

Answer 8

step 1 in creating a mature IgR (Ig receptor) is the recombination that brings D and JH together. this happens in the pre-pro-B cell to the early pro-B cell stage. The cell is still in the bone marrow.

step 2: recombination between the VH and D-JH segments happens in the pro-B cell stage.

If this is successful, the Heavy chain is expressed and placed onto cell surface in combination with a nonvariable pair of proteins (VpreB and λ5) (aka surrogate light chain). This results in a pre-B-cell receptor. signaling from this receptor halts heacy-chain recombination, initiates several rounds of proliferation, and calls for the beginning of light-chain recombination.

Light chain recombination may start at the k or λ locus. Expression of an intact membrane IgM BCR shuts off light-chain gene rearrangement.

Question 9

Recombination is directed by recombination signal sequences

Answer 9

RSSs adjacent with each of the variable region gene segments serve to guide the recombination machinery to the correct locations in the genome.

RSSs consist of several heptameric and nonameric sequences separated by either 12 or 23 bp. According to the 12/23 rule, recombination occurs between one region with a 12 bp spacer and a second region with 23 bp spacer.

Question 9

Recombination is directed by recombination signal sequences

Answer 9

RSSs adjacent with each of the variable region gene segments serve to guide the recombination machinery to the correct locations in the genome.

RSSs consist of several heptameric and nonameric sequences separated by either 12 or 23 bp. According to the 12/23 rule, recombination occurs between one region with a 12 bp spacer and a second region with 23 bp spacer.

Question 10

Gene segments are joined by a diverse group of proteins

Answer 10

V(D)J recombination is catalyzed by the lymphocyte-specific recombinase enzymes RAG1 and RAG2 acting in concert with TdT and enzymes from the NHEJ pathway.

TdT = terminal deoxynucleotidyl transferase

the RAG1/2 complex contains two molecules each of RAG1 and RAG2, and is responsible for recognizing and cutting DNA at the precise junction between the IG-encoding regions and the RSS.

Question 11

V(D)J recombination occurs in a series of well-regulated steps (intro)

Answer 11

The DNA between the segments to be joined is deleted/lost as an excision circle.

In cases where the Vk and Jk segments are trc in different directions, the intervening DNA is inverted and the excised sequences are retained on the chromosome upstream of the recombinant gene.

Question 12

Steps 1-10 of V(D)J recombination

Answer 12

1) recognition of the RSS by the RAG1/2 enzyme complex.
The RAG1/2 forms a complex with the RSS next to one of the two segments about to be joined. The binding is usually initiated at the RSS containing the 12bp spacer.
The binding is enhanced by HMMGB1/2 proteins, which may also serve to induce and stabilize the bending of the DNA (thus facillitating its cleavage).

The second RSS is then bound by the RAG1/2 complex, and the two gene segments are brought into close contact. Current studies show that the binding of one type of spacer induces a conformational change in the DBS (DNA binding site) of RAG1/2 that accomodates the binding of the other spacer.

2) One-strand cleavage at the junction of the coding and signal sequences.
The RAG1 protein creates ss nicks, 5’ of the heptameric signal sequence on the coding strand of each V segment (that means the junction between the V segments and the heptamer) and at he heptamer-J junction.

3) Formation of V and J region hairpins and blunt signal ends.
The free 3’ OH at the end of the coding strand of the V segment now attachs the P-group on the noncoding V-strand, forming a new covalent phosphodiester bond across the double helix and yielding a hairpin structure on the V segment side of the break. this is called the coding end.

At the same time, a blunt DNA end is formed at the edge of the heptameric signal sequence. this is the signal end.

this process happens on the J side as well (same time).

Now, RAG1/2 proteins and HMGB1/2 proteins are still associated with the coding and signal ends of both V and J segments.

4) ligation of the signal ends.
DNA ligase IV (NHEJ protein) ligates the blunt ends to form the signal joint.

5) Hairpin cleavage
the hairpins at the ends of the V and J regions are opened by the endonuclease Artemis in one of three ways:
- (1) reopening the identical bond that was formed in step 3, creating a blunt end at the coding joint.
- (2 and 3) the hairpin may be opened asymmetrically (either on top or bottom strand) to yield a 5’ or 3’ overhang, respectively.

Aremis is part of the NHEJ pathway and requires DNA PKcs (NHEJ kinase) to bind to the DNA hairpin ends via its DNA-binding subunits Ku70/80. The most common oberhang is a 2 nt on the 3’ end. the Artemis-DNA-PKcs complex also posesses single- and double stranded DNA endonuclease activity, and can remove several bases on each side of the new joint. Often at the coding joint, rarely signal joint.

6) Overhang extension can lead to addition of palindromic nts.
nt-overhangs can act as substrates for NHEJ DNA repair enzymes, leading to ds-palindromic (P) nts at the coding joint (inverted repeat). This is a direct function of an asymmetric hairpin-opening reaction. P-nt addition can also occur at both the V-D and D-J joints of the heacy chain, but other processes can intervene to add further diversity for light chains.

7) Ligation of light-chain V and J segments
DNA ligase ligates bith the signal joints and the coding joints. at the signal joint is happens without deletions or additions. At the coding joint, some of the enzymes (Artemis can nibble at the ends, DNA pol λ and DNA pol mu are less feithful than the “regular” DNA pol, and the latter can add random nts at the coding joint). Thus, NHEJ repair mechanisms can generate significant nt-diversity at the light-chain coding joint, even in the absence of TdT (acts mainly at heavy-chain joints)

8) Exonuclease trimming
Exonucleases trim back the edges of the V region DNA joints. since the RAG proteins can trim DNA near a 3’ flap, they can cut off some of the lost proteins. Artemis-DNA-PKcs could also be responsible for the V(D)J associated endonuclease function (as in step 5). Extensive trimming is more common in the heavy chain (VD and DJ) joints than the light chain VJ joint.

9) N-nt addition
probably occurs simultaneously with step 8.
nontemplated (N) nts are added by TdT to the coding joints of heavy-chain genes after hairpin cleavage. can add up to 20 nts to each side of the joint. the two ends are helt together by the RAG1/2 complex. TdT-mediated N-nt addition is more common at the coding joints of heavy chain genes than light chain joints

10) Ligation and repair of the heavy-chain gene.
identical to the ligation of the light-chain genes, mediated by DNA ligase IV (in concert with its activator XRCC4)

Unproductive arrangement = when trimming has caused fuck-ups in the reading frame. If this happens at one heavy-chain locus, rearrangement of the other allele is initiated immideatly. If this also fails, apoptosis.

Question 13

5 mechanisms generate Ab diversity in naïve B cells

Answer 13

1) multiple gene segments exist at heavy and light chain loci. These can be combined with one another to provide extensive combinational diversity
2) Heavy-chain/light-chain combinatorial diversity. The same heacy chain can combine with different light chains, and vice versa.
3) P-nt addition results when the DNA hairpin at the coding joint of heavy and light chains is cleaved asymmetrically.
4) exonuclease trimming sometimes occurs at the V-D-J and V-J junctions, causing loss of nts.
5) Nontemplates (N)-nt addition by TdT in heavy chain VD and DJ junctions and from DNA pol mu in both heavy and light chains.

3, 4, 5 result in the formation of the highly variable CDR3 regions of the Ab heavy- and light chains.

Question 14

The regulation of V(D)J ene recombination involves chromaitn alteration

Answer 14

The RAG1/2 complex is tightly regulated, and is inactivated before the cell enters the S phase (as ds breaks in the DNA could interfere with chromatin distribution).

histone code = the nature of histone modofications associated with a gene. Changes in the histone code (methylation, acetylation, ++) of chromatin associated with Ig-DN during B cell development signal the onset of receptiveness of the Ig locus to trc and recombination.

The rosette loop that contains the D, JH and CH regions defines the scope of RAG activity in the earliest B-cell precursors. Once DH-JH recombination has occurred, the loop structure is altered to allow VH-DH recombination.

the manner in which intranuclear localization of the Ag-receptor chromatin is altered in order to make available the relevant genes to the recombinase. Within the nucleus, inactive chromatin is unable to participate in either trc or recombination. inactive chromatin is usually right by the nuclear membrane.
Chromatin in the general nucleoplasm, however, tends to be more active.

Question 15

BCR expression

Answer 15

B cells express Ig heavy- and light-chain genes from only one allele and one light-chain gene family (λ or kappa)

Heavy-chain rearrangement preceeds light-chain rearrangement.

The generation of a functional BCR is energically expensive because of the considerable wastage involved, as a result of significant frequency of nonproductive gene rearrangements.

Heavy chains are produced first, if successful the chain is expressed and placed on the surface along with a surrogate light-chain, signalling for the cell to start recombination of light chain.

Question 16

Receptor editing of potentially autoreactive receptors occurs in light chains

Answer 16

B cells can stille make some changes in its receptor, even when expressed on the surface. if the BCR is autoreactive, it can be switched out (receptor editing). Only has to change either heavy- or light chain, often light chain is chosen. Occurs most frequently in k-chains. receptor editing uses a secondary rearrangement that employs Vk and Jk segment upstrea and downstream, respectively, from the original rearrangement.

Question 17

mRNA splicing regulates the expression of membrane-bound vs secreted Ig

Answer 17

Ig heavy-chain variable region can be expressed in association with more than one heavy-chain constant region.

Both constant regions of an Ab can also be expressed either membrane-bound or soluble. naïve B cells (such as immature B cells and mature B lymphocytes) have not yet encountered their Ag and memory B cells, only make mRNA for the membrane-bound form. In contrast, plasma cells make mRNA encoding soluble Abs.

Immature B cells only express membrane-bound IgM. Upon maturation, they also express IgD (same V-region as IgM for that cell, but have a delta rather than a mu C-region). Cells switch from membrane to secreted IgM and IgD by mRNA splicing. If the Ab is another class, it is called class switch recombination (CSR).

Production of mRNA encoding the membrane-bound form of the mu chain occurs when the cleavage of the primary transcript and addition of the poly(A) tail happens at polyadenylation site 2. RNA splicing then removes the S (secreted) sequence at the 3’ end of the C(mu)4 exon, and the two introns, and joins the reamibder of the C(mu)4 enxon to the M1 and M2 exons (M for membrane). The S sequence includes polyadenylation site 1, and encodes the hydrophilic C-terminal end of the CH4 domain of secreted IgM.

For IgD, polyadenylation sites 3 and 4 encode the secreted and membrane-bound forms, respectively. If polyadenylation happens at site 4, the C(mu) RNA is spliced out and the J segemnt for the V-region is joined to the C(delta) RNA, which is processed to create the mature mRNA encoding the heavy-chain of membrane-bound IgD.

Upon Ag-stimulation, B cells geneate the secreted forms (polyadenation at site 1 for IgM and 3 for IgD).

Membrane bound form: 40 AA, hydrophilic extracellular, hydrophobic transmembrane, hydrophilic intracellular (very short).

Secreted form: 20 AA, hydrophilic

Question 18

What determines which splice sites are used (if the Ig is membrane-bound or soluble?)

Answer 18

not yet fully resolved.

Question 19

TCR

Answer 19

four TCR chains: alpha, beta, gamma, delta.

Two classes of TCR: alphabeta and gammadelta.

As for Ig genes, functional TCR genes are produced by rearrangement of V and J segments (alpha- and gamma-chain families) and V, D, J segments in beta- and delta-families.

Question 20

Recombination of TCR gene segments proceeds at a different rate and occurs at different stages of development in alphbeta vs gamdelta.

Answer 20

T cell development occurs mostly in thymus.

When the lymphoid progenitor cell leaves the bone marrow and enters the thymus, it is reffered to as a double-negative (DN) thymocyte, as it has neither CD4 or CD8 on its cell surface. The DN stage is subdivided into 4 steps (DN1-4) on the basis of the expresion of a number of cell surface markers. at the end of DN4, the T cell acquires forst CD8, then CD4, and becomes DP.

gammadelta receptor T cells complete their differentiation at the DN stage, alpha-beta proceed to the DP stage and undergo positive and negative selection. positive selection = recognize Ags, negative = autoimmunity.

Question 21

The process of TCR gene segment rearrangement is very similar to that of Ig
αβγδ

Answer 21

In DN thymoticyes:
the chromatin regions on which the alpha/delta and beta variable region gene segments reside become accessible to RAG1/2 recombinase. variable region rearrangement happens simultaneously on all three loci.
Similar mechanisms as for Ig.

Animals defect in RAG1/2 or the other enzymes are similarly deficient in the generation of BCRs and TCRs.

Heptamer-nonamer RSSs are found at the 3’ termini for the V seq, 5’ and 3’ for the D seq, and 5’ for the J seq, just like for Ig.

One important difference in the control of receptor gene segment recombination in B and T cells. Ig-gene has 12/23 rule, which means VH and JH recombination is illegal, and all heavy-chain V must therefore include V, D, and J.

In contrast: This applies for both delta and beta, but I just wrote beta

• Dbeta has 12 bp spacer upstream and 23 bp spacer downstream.
• Vbeta has a 23 bp spacer downstream.
• Jbeta has a 12 bp spacer upstream.

This means that according to the 12/23 rule, one V and J could combine without a D segment. It also allows for D-D recombination.

For beta chains, these things do not happen. It is always V, D, J. not really understood what stops the D-D in beta chains.

for delta chains, it is more complicated. Many Vdelta genes are considerably longer than Vbeta genes, and the increased seq length seemed to occur in the parts encoding CRD3 residues that make Ag contact. Firther analysis showed that many Vdelta TCR genes have 2 D region segments, which was responsible for the increased length. D-D-

beta, gamma and detla variable region genes undergo rearrangement at the same time. It seems that a developing T cell allows the RAG recombinase a single cycle of attempted rearragement involving the Vdetlta gene segments. Is fuccessful, and if the cell also ha sa recombined y-chain, the beta-gamme receptor funtionality is tested. If accepted, the cell is fixed as a DN yd T cell and leaves the thymus.
However, if the β-gene rearrangement is successful, the developing thymocyte trc and trn the Vβ gene, expressing the protein on the thymocyte surface in recombination with the pre-Tα protein to form the pre-TCR. this is analogous to the Ig heavy chain.
Signaling from the pre-TCR complex via CD3 turns off recombination of the β, γ and δ loci by inducing down-regulation of RAG1/2 and degradation of RAG2. also results in proliferation that maximizes the use of the β-gene. After several rounds of proliferation, the cell makes the transition to the DP stage and initiates α-chain rearrangement.

α-chain gene rearrangement:
initiated at the most proximal Vα segment. If unsuccessful, they are followed by rearrangements involving distally located α segments. on average 3-5 cycles of Vα to Jα rearrangement. If the resultant αβ TCR is successful during negative selection, rearrangement is terminated, RAG gene expression is ended, and theymocyte devvelopment is complete. the DP loses either CD8 or CD4, and becomes a mature SP (single positive) T cell. If this T cell fails positive selection, it will die.