Recombination of IgG Genes 2024 - Organization and Expression of Immunoglobulin (Ig) Genes

exam 2 topic

how can genetic model explain Ig structure?

Ig Characteristics

• Diversity of Ig specificities

• presence of Ig Variable region at the amino-terminal end and constant region at the carboxyl-terminal part

• formation of isotypes with the same Ag specificity

which is built first, the heavy chain or the light chain?

• the heavy chain is built first

Genetic Theories

• germ-line theory

• somatic-variation theory

• two-gene theory

• tonewgawa’s theory

germ-line theory

incorrect genetic theory that theres a large repertoire of Ig genes in germ-line cells

somatic-variation

a genetic theory that suggests the genome contains a small number of Ig genes, but mutations/recombinations in the somatic cells will generate a large number of Igs

two-gene theory

incorrect genetic theory that suggests 2 genes encode a single Ig G- or L-chain, one for a V region and the other for a C region

Tonegawa’s theory

correct genetic theory that suggests Ig gene rearrangement

describes how it’s possible to have millions of variable regions

which chain shows VJC

Light chain

which chain expresses VDJC

Heavy chain

Ig chains are encoded by how many separate multigene families

3 separate multigene families

are Ig chains encoded by separate multigene families on the same chromosome or different chromosomes

different

Germ-line DNA

• contains many coding and non-coding regions

• the coding sequences are linked together to form a functional Ig gene

does each multigene germ-line family contain the same or different characteristics?

contains different characteristics

Lambda and Kappa light chain families contain which gene segments

• V,J, and C gene segments

• rearranged VJ segments encode the V region of L-chains

• C gene segments encode C regions of L-chain

H-chain families contain which gene segments?

V,D,J, and C gene segments

• rearranged VDJ segments encode V regions of H-chains

• C segments encode C regions of H-chains

Leader peptide

• V gene segments (of L- and H-chain gene families) are preceded(at 5` end) by and exon that encodes leader peptides (L)

• leads H- or L-chain through the endoplasmic reticulum

• leader peptide is cleaved from the emerged L- and H-chains before assembly of the final form of Ig molecule

functional genes

• encode Ig H- and L-chains

• formed by recombinational events at the DNA level

recombination of genes that encode Ig chains and T-cell receptors

• the ONLY known location-specific DNA rearrangements in vertebrates

variable-region gene rearrangements

• happen in sequenced order during b-cell maturation in the BM

• they are random events that create an enormous variety of V-region specificities

The H-chain V-region genes

• rearrange first , and then the L-chain variable region genes

Which rearrange first? the H-chain V-region genes or the L-chain variable region genes?

• The H-chain V-region genes

How many V-regions are there in a B-cell?

• 1 V-region DNA sequence for its H-chain

• 1 V-region DNA sequence for its L-chain

Process of V-region gene rearrangements

• generates mature, immunocompetent B-cells

• each such B-cell has a BCR with a binding site encoded by a particular sequence of its rearranged V-region genes

recombination of the H-chain C-region genes

• will generate certain Ig classes (isotypes) - that will not affect B-cell specificity for an AG

formation of both kappa and lambda light Ig chains requires…

• rearrangement of the variable-region V and J gene segments

lambda L-chain germ-line DNA (humans)

• and V-lambda gene segment can combine with any of the J-lambda and C-lambda combinations

Kappa L-chain germ-line DNA (humans/mouse)

• any one of the V-Kappa gene segments can combine with any functional J-kappa

Rearranged lambda and kappa chains contain the following regions

• L exon - intron (non-coding sequence) - combined VJ gene segment - second intron - C gene segment

light-chain gene V-J rearrangements steps

1) rearranged lambda and kappa chains

• transcription (RNA polymerase)

2) L chain primary RNA transcript

• polyadenylation, RNA splicing

3) L chain mRNA

• translation

4) Nascent L-chain polypeptide

• (L region pulls Ig into rough endoplasmic reticulum)

• L region cleaved

5) L-chain

generation of the functional H-chain gene requires …

2 rearrangement events within the V region

Heavy-chain V-D-J rearrangement sequence

1) a DH joins to a JH gene segment (DHJH)

2) a DHJH joins to a VH gene segment (VHDHJH)(encodes entire v region)

rearranged H-chain DN contain the following regions

L exon - intron - VDJ - several C gene segments

where is the promoter sequence located (in heavy-chain V-D-J Rearrangements?)

• it is located upstream from the L exon

What does the RNA polymerase transcribe? (In Heavy-chain V-D-J rearrangements?)

• it transcribes the entire rearranged H-chain including introns (both Cμ (mu)and Cδ(delta) gene segments are transcribed)

what do polyadenylation and RNA splicing do (in heavy-chain V-D-J rearrangements)?

• they remove the introns and form the mRNA that includes either the Cμ or the Cdelta segment

The formation of both H-chain mRNAs enables expression of

• both IgM and IgD (with identical specificity for an Ag) on a surface of mature, immunocompetent B-cell

Function of RSS

• function as signals for the recombination process

RSS

Recombination signal sequences

what do RSS do?

• they flank each germ-line V,D, and J gene segment

where is RSS located

• located 3` to each V, 5` to each J, and on both sides to each D gene segment

what does each RSS contain?

• contains a conserved heptamer and a conserved AT-rich nonamer sequence separated by 12- or 23-bp sequence

• these intervening 12- (one-turn RSS) or 23-bp (two-turn RSS) sequences correspond to one or two turns of the DNA helix, respectively

One-turn/two-turn joining rule

• RSS containing one-turn spacer can join only RSS with a two-turn spacer (ensures appropriate order of joining)

Phases of variable-region gene rearrangements

• recognition of RSS by recombinase

• cleavage of single-strand DNA by RAS1/2

• simulate formation of “hair pin” structure and double-strand break at the RSS

• repair and ligation

• coding joint/signal joint

• deletional joining

• inversional joining

where is the cleavage of double-stranded DNA that initiates VDJ rearrangement done?

• it is done exactly at the junction of RSS and coding sequence

is subsequent joining of the coding sequence precise or imprecise?

• imprecise

consequences of the imprecise joining are

• junctional diversity at the VJ and VDJ coding joints is generated by many mechanisms

• nonproductive rearrangement- gene segments may join out of phase so the triple reading frame is not preserved; VJ (VDJ) units will have numerous stop codons that interrupt translation

• if out of phase rearranged H- and L-chains are produced, B-cell dies by apoptosis

• productive rearrangement- gene segments are joined in phase - the VJ (VDJ) unit can be translated entirely resulting with a normal Aby molecule

nonproductive rearrangement

• gene segments may join out of phase so the triple reading frame is not preserved; VJ (VDJ) units will have numerous stop codons that interrupt translation

productive rearrangement

gene segments are joined in phase - the VJ (VDJ) unit can be translated entirely resulting with a normal Aby molecule

B-cells

• diploid cells

• contain both maternal and paternal chromosomes

allelic exclusion

• b-cell expresses the rearranged H-chain genes from ONLY one chromosome, and the rearranged L-chain genes from ONLY one chromosome

what does allelic exclusion ensure?

• it ensures that B-cells never contain more than ONE VHDHJH, and ONE VLJL = crucial for antigenic specificity of B-cells (expression of both alleles would make B-cells multi-specific)

what happens once a productive VJ rearrangement and a productive VDJ rearrangement occur

• the recombination process is turned off - H- and L- chain genes on the homologous chromosome are not expressed

what are the final protein products (in allelic exclusions)?

• H- and L- chain may prevent gene rearrangement of the remaining allele = enabling allelic exclusion (transgenic mice experiments suggest that mechanism)

Properties contributing to the Aby diversity

• multiple germ-line V,D, and J gene segments (occurs in BM)

• multiple combinations of V-J/V-D-J joining (occurs in BM)

• junctional flexibility (occurs in BM)

• P-region nucleotide addition (p-addition) (occurs in BM)

• N-region nucleotide addition (n-addition)(occurs in BM)

• somatic hypermutations (occurs in periphery)

• Combinatorial association of light- and heavy-chains

how much diversity can be achieved by gene rearrangements?

• estimation of human Aby diversity ~ 10^10

are signal sequences (RSS) joined precisely or imprecisely?

• they are joined precisely

are coding sequences joined precisely or imprecisely?

• they are joined imprecisely

Junctional flexibility generates

• many nonproductive rearrangements

• productive rearrangements that encode alternative AA sequences at each coding joining = increase Aby diversity

AA sequence variations created by junctional flexibility in the coding joint are located…

• within the CDR3 region of H- and L- chain (CDR3 region - important for epitope binding!)

cleavage of the hairpin by endonuclease results with a formation of …

• a a single stranded DNA at the end of the coding sequence (very often)

P- addition

-subsequent addition of complementary nucleotides (P-nucleotides) to that strand by repair enzymes that creates a palindromic sequence in the coding joint

variation in the cutting position of hairpin =

• increased variation of the sequence in the coding joint

if cleavage of the hairpin results with a double-stranded end on the coding sequence, does the P-addition occur?

• no, P-addition does not occur

where does the N-addition occur? L- chain or H-chain DNA

• only occurs in H-chain DNA

besides P-nucleotide addition, what else can occur?

• the addition of random N-nucleotides by TdT can occur

How many nucleotides can be added to either DH-JH joints or to VH-DHJJ joints? (in N-addition)

• up to 15 nucleotides

where is the diversity localized (in N-addition)?

• localized in CDR3 region

what does somatic hypermutation do?

• it increases diversity in already rearranged gene segments

where does somatic hypermutation occur?

• it occurs ONLY within germinal centers (periphery) post immunization with Ag

• occurs in MATURE b-cells

Individual nucleotides in VJ/VDJ units are replaced with…

• other nucleotides - influencing the overall affinity to Ag

• affinity maturation of b-cells

whats more frequent, somatic hypermutations or spontaneous mutations

• somatic hypermutations

mechanism for somatic hypermutation

• enzyme AID- mediated

can H-chain DNA be further rearranged?

• yes, H-chain DNA can be further rearranged post Ag exposure of b-cells - VHDHJH unit can be combined with any CH gene (class switching or isotype switching)

what does class switching involves…

• switch region- located upstream of each CH segment (except Cdelta)

switch regions contain…

• multiple “short repeats” - binding site for switch recombinase - performs DNA recombination with resulting class switching

how do cytokines play a major role in class switching?

• cytokines play a major role as switch factors in class switching (determine the isotype to which the B-cell switches)

what does class switching depend on?

• switch regions

• switch recombinase

• cytokines

• AID (activation-induced cytidine deaminase

what is AID

• a key mediator of class switching and somatic hypermutation

• DNA editing enzyme (determines certain cytosines in DNA)

Ig heavy-chain primary transcript can be processed differently

• a single b-cell can produce membrane-bound or secreted form of Ig

• a single b-cell can simultaneously express IgM and IgD

carboxyl-terminal domains of the H-chain

• CH3/CH3 in IgG, IgA, and IgD; or CH4/CH4 in IgM, and IgE

• carboxyl-terminal domains of the H-chain of membrane and secreted Ig form are different

membrane Ig form contains…

• hydrophilic segment outside the cell

• a transmembrane hydrophobic part

• a hydrophilic intracellular segment

secreted Ig form contain hydrophilic AA sequence

differential processing of a primary H-chain transcript allows…

• production of the membrane-bound or secreted type of a H-chain

how many exons does a C mu gene segment contain?

• C mu gene segment contains 4 exons (C mu 1-4) that encode 4 domains of IgM

C mu 4 contains a sequence called…

S (3` end) that encodes the hydrophilic sequence in the CH4 domain of secreted IgM

does M1 encode the transmembrane segment or the cytoplasmic segment?

• transmembrane segment

does M2 encode the transmembrane segment or the cytoplasmic segment?

• cytoplasmic segment

polyadenylation of the side 1 results with …

• a lost of gene segments M1 and M2 - further excision of introns and splicing of exons occur producing mRNA for secreted mu H-chain

polyadenylation of the site 2 results with…

• and excision and lost of S sequence - excision of introns and splicing of exons join mu4 with M1 and M2, producing mRNA for membrane mu H-chain

differential processing of primary H-chain transcript is responsible for …

• the simultaneous expression of membrane IgM and IgD on a single mature B-cell

rearranged H-chain DNA contains both…

• C mu and C delta gene segments that are very close

is there a switch site between C mu and C delta?

• no, there is not a switch site between C mu and C delta

• this allows the entire segment VDJC(mu)C(delta) to be transcribed into a single primary H-chain transcript

how is IgM and IgD simultaneously expressed?

how are the secreted and membrane forms expressed?

are H- and L- chain Mrna translated on the same or separate polyribosomes of RER?

• separate polyribosomes of RER

Synthesis, assembly, and secretion of Igs

1) H- and L-chain mRNAs are translated on separate polyribosomes of RER

2) Nascent Ig chains (H and L) contain 5` L sequence that leads them into the lumen of RER where the L sequence is cleaved

3)The assembly of H- and L-chains into the Ig occurs as the chains pass through RER

4)The assembled Ig molecules enter Golgi apparatus and then secretory vesicles that fuse with the cell membrane

5)Membrane form of Ig is bound to the vesicle membrane, and then inserted to the plasma membrane when vesicle fuses with the plasma membrane

6)Secreted form of Ig is transported in a secretory vesicle as a free molecule, and then it is released from cell post fusion of vesicle and the plasma membrane 

Ig gene transcription is regulated by…

• promoters

• enhancers

• silencers

promotors of Ig gene transcription

• promote initiation of transcription in a specific direction, located upstream of the transcription initiation site

enhancers of Ig gene transcription

activate transcription from the promoter sequence in an independent way of orientation

silencers of Ig gene transcription

down-regulate transcription in an orientation-independent way