Organization and Expression of Immunoglobulin Genes

1
Organization and Expression of Immunoglobulin
Genes
Patrick C. Swanson, Ph.D. Assistant
Professor Medical Microbiology and
Immunology Creighton University September 23,
2003
LECTURE OBJECTIVES To understand how genetic
diversity in antibodies is achieved To describe
how B cell receptor expression and allelic
exclusion is tied to gene rearrangement To
introduce techniques used to manipulate antibody
genes
2
The paradoxes of antibody structure

• Any model to explain genetics of antibodies must
  account for
• Vast diversity of antibodies
• H and L chain sequence analysis demonstrating
  the presence of
• constant and variable domains (hence, the
  paradox of having a seemingly
• normal gene constant juxtaposed to a gene of
  unknown origin variable
• The existence of H chain isotypes with the same
  antigenic specificity,
• (i.e. same specificity, different H chain
  constant regions
• Early theories
• Germ-line theory cells maintain large
  repertoire of antibody genes
• Somatic variation theory one or a few genes
  undergo mutation or
• recombination in somatic cells (a body cell
  whose genes are not passed
• on to future generations) to generate diverse
  antigenic specificities

3
Dreyer and Bennett provide theoryto resolve
paradox

• The two-gene theory of immunoglobulin genetics
• Two separate genes encode a single Ig heavy or
  light chain
• Each constant region class and subclass is
  encoded in a single gene, whereas
• hundreds or thousands of genes make up the
  pool of potential variable
• region domains
• Somehow, the two types of genes are brought
  together at the DNA level,
• creating a gene that can transcribed and
  translated into a single H or L chain

4
Hozumi and Tonegawa show Ig genes rearrange,
supporting the two-gene theory

• Basic Idea
• Mouse embryo DNA mouse myeloma DNA digested
  with restriction enzyme (RE)
• DNA fragments are fractionated by agarose gel
  electrophoresis and hybridized to
• a radiolabeled nucleic acid probe (original
  method replaced by Southern blotting)

Southern blot loss (or gain) of RE site alters
mobility of hybridizing band
5
A review of gene structure and function
Taken from Benjamini, Sunshine,
Leskowitz Immunology A Short Course
6
Ig Heavy and Light chains are encodedby
multigene families

• Key Points
• Heavy chain variable domain encoded in three
  types of gene segments which
• undergo gene rearrangement to form functional
  exon termed V(D)J recombination
• - Variable (V)
• - Diversity (D)
• - Joining (J)
• Heavy chain constant domains each encoded by
  their own functional exon
• (eight groups of exons, one for each class or
  subclass of H chain)
• Two light chain families (k and l) variable
  domains of both encoded in
• two types of gene segments which undergo V(D)J
  recombination
• - Variable (V)
• - Joining (J)
• k light chain constant domain encoded by a
  single exon
• l light chain constant domain encoded by a
  single exon
• (four total, one for each subclass)

7
Organization of immunoglobulin genes
Mouse Ig loci
(Human chromosome 22 mouse chromosome 16)
Kuby Figure 5.3
(Human chromosome 2 mouse chromosome 6)
Note y pseudogene (nonfunctional)
(Human chromosome 14 mouse chromosome 12)
Assembled to create V exon
C exons intact
8
Combinatorial rearrangementof V, D, and J
segments generates heavy chain variable region
exon
Irreversible! Intervening DNA often lost!
DNA
Irreversible! Intervening DNA often lost!
Functional exon!
Splice site correlated to cell differentiation
status!
RNA
Protein
m heavy chain
Kuby Figure 5-5
VH
CH
9
Light chain gene segmentsalso undergo
rearrangement
Note no D segments!
Kuby Figure 5-4
10
V(D)J recombination is directed byrecombination
signal sequences

• Key Points
• One type of RSS flanks a
• given family of gene
• segments (i.e. all Vk have
• 3 12-RSS)
• Heptamer and nonamer
• sequences conserved
• spacer sequence not well
• conserved

23-RSS
12-RSS
RSSs shown are inverted relative to one another
Kuby Figure 5-4
11
V(D)J recombination follows the 12/23 rule
L
L
V1
Vn
J1
Jn
Ck
V(D)J recombination
L
L
V1
Vn
J1
Jn
Ck

• Recombination only occurs between gene segments
• bearing different length spacers (the 12/23
  rule)
• The 12/23 rule prevents recombination from
  occurring
• among like gene segments

12
Recombination activating genes(RAG-1 and RAG-2)
are required forV(D)J recombination

• Expression
• Restricted to lymphocytes
• Developmentally regulated
• Activity
• Initiates V(D)J recombination
• Partial or complete loss of activity (in
  either RAG-1 or -2)
• causes severe combined immunodeficiency
  (SCID) due
• to arrested lymphocyte development
• - Null T-B-SCID
• - Partial Omenn syndrome
• (No B cells, variable poorly functioning T
  cells)

13
V(D)J recombination involvestwo distinct phases

• Cleavage
• Requires RAG-1, RAG-2, and possibly HMG-1/2
  (high mobility
• group-1 or -2 architectural factors that
  facilitate nucleoprotein
• complex assembly)
• Introduction of DNA double strand breaks
  (DSBs)
• - Signal ends blunt and 5 phosphorylated
  (contains the RSS)
• - Coding ends covalently sealed DNA hairpins
• Joining
• Resolution and repair of DSBs (Non-homologous
  end-joining)
• - Artemis/DNA-PKcs (Processing of coding
  ends)
• - Ku70/Ku80 (End-bridging)
• - XRCC4/DNA ligase IV (Ligation)
• Juxtaposition of signal and coding ends
• - Signal joints generally precise
• - Coding joints may or may not have
  additional nucleotides inserted

14
Cleavage phase involves RAG-mediatedsynapsis and
DNA double-strand breakage

• Three stages to cleavage
• Synapsis coordination of a 12/23
• pair of RSSs by RAG-1/2 into a
• protein-DNA complex (possibly
• with assistance by architectural DNA
• binding proteins HMG-1 or HMG-2)
• Nicking hydrolysis of
• phosphodiester bond at 5 end of
• heptamer, catalyzed by RAG-1/2
• Hairpin formation generation of a
• DNA double strand break (DSB)
• involving a DNA hairpin intermediate,
• catalyzed by RAG-1/2

Synapsis RAG-1/2 HMG-1/2?
Nicking 5 end of heptamer
Hairpin formation DNA-DSB
Kuby Figure 5-7
15
Hairpin formation proceeds viadirect
transesterification(no covalent intermediate)
L
L
V1
Vn
J1
Jn
Ck
12 or 23 base pairs
TG AC
ACAAAAACC TGTTTTTGG
CACAGTG GTGTCAC
Vn
heptamer
nonamer
RAG-1/2
HO
12 or 23 base pairs
1st strand nicking
ACAAAAACC TGTTTTTGG
CACAGTG GTGTCAC
heptamer
nonamer
(5 phosphate 3 hydroxyl)
RAG-1/2
12 or 23 base pairs
direct trans- esterification
TG AC
ACAAAAACC TGTTTTTGG
CACAGTG GTGTCAC
Vn
16
Joining phase involves resolution andrepair of
DNA double-strand breaks

• Three stages to joining
• Resolution of DNA hairpins
• Hairpins opened if asymmetric, can
• lead to formation of palindromic
• (P) element
• Nontemplated (N) nucleotide addition
• Terminal deoxynucleotidyl transferase
• (TdT) adds nucleotides in a template-
• independent fashion, tailing the
• coding end. Expressed early in
• lymphocyte development hence only
• H chain segments affected optional
• DSB repair
• Signal ends ligated (precise) and coding
• ends joined (may be imprecise)
• Involves Ku70/Ku80 and XRCC4/DNA Ligase IV

Hairpin Opening Artemis/DNA-PKcs Source of P
elements
N-nucleotide addition TdT Optional (H chain only)
DSB Repair Ku70/Ku80 XRCC4/DNA Ligase IV
Kuby Figure 5-7
17
Repair of coding ends may be imprecise

• Sources of nucleotide gain or loss at coding
  joints
• Gain
• - P elements
• - N nucleotide addition
• Loss
• - Exonucleolytic trimming of ends (exo)

Note examples shown are only representative of
many different possible outcomes!
Kuby Figure 5-13
(Palindromes)
18
Imprecise joining impacts reading frame of gene

• Key Points
• Coding joints may be productive or
• unproductive the reading frame may be
• altered so that the encoded antibody
• is not functional
• Imprecise joining is a source of
• junctional diversity changing the
• composition or length of the amino
• acid sequence in this region can alter
• the antigenic specificity of the antibody
• Potential for imprecise joining often
• necessitates repeating V(D)J recombination
• (especially for light chain)

(really, just many potential outcomes)
Note P element insertion and N-nucleotide
addition not shown here, but cause similar
effects!
Kuby Figure 5-9
19
Junctional diversity impacts CDR3
FR1
CDR1
FR2
CDR2
FR3
CDR3
FR4
Polypeptide
Light chain gene
P, exo
VL
JL
P, N, exo
VH
JH
D
Heavy chain gene

• Key Point
• V segment contributes CDR1 and CDR2 CDR3
  encoded by joining V, (D) and J

20
Multiple attempts at V(D)J recombination are
often required to generate a productive coding
joint
An example of kappa light chain gene rearrangement
L
L
L
L
Germ-Line
Vk1
Vk2
Vkn
Jk1
Jk2
Jk3
Jk4
Jk5
Vk3
Ck
Attempt 1
L
L
L
Unproductive (hypothetical)
Vk1
Vk2
Jk1
Jk2
Jk3
Jk4
Jk5
Vk3
Ck
Attempt 2
No more options !
L
Vk1
Jk5
Ck
If unproductive, then cell tries to rearrange
other allele or the lambda locus. If all
possible attempts are rearrangement are
unproductive, the cell dies
If productive, then cell lives
21
V(D)J rearrangement is developmentally ordered

• Key Points
• Heavy chain locus rearranged first
• - DH to JH, then VH to DHJH
• Light chain locus rearranged next
• - Starts with either kappa or lambda locus.
• - If rearrangement is unsuccessful at one
  locus,
• rearrangement is attempted at other locus.
• - Results in H2k2 or H2l2, not H2k1l1

22
V(D)J recombination is restricted to certain
stages of B cell development
(DH to JH)
(VH to DHJH)
(yL)
(yL helps select functional heavy chain)
Kuby Figure 5-1
23
Developmentally ordered rearrangements help
ensure one antigenic specificity per cell
Observation The antigen receptor genes (e.g.
heavy chain) are expressed from only a single
chromosome of two homologous chromosomes (termed
allelic exclusion) Basis Rearrangements are
directed toward one genetic locus on
one chromosome at a time. All options for
rearrangements are exhausted before proceeding to
another locus. Importance Keeps a lymphocyte
from expressing receptors of different
antigenic specificity (what would happen if there
were two?)
24
Ordered rearrangement helpsenforce allelic
exclusion
Kuby Figure 5-11
25
Rearranged Ig genes undergo somatic
hypermutation (SHM) after B cell activation

• Key Points
• Occurs in germinal center in secondary
• lymphoid organ, after antigen-specific
• B cell activation
• Requires T cell help
• Targets rearranged V gene
• Visualized by accumulation of silent
• and replacement mutations in rearranged
• V gene that are not present in germ-line
• Rate of SHM is 10-3/bp/cell division
• (105 greater than spontaneous rate of
  10-8/bp/div)
• Mutations occur throughout exon,
• but those improving affinity to antigen

Kuby Figure 5-14
silent
replacement
26

Sources for the generation of diverse antigen
binding specificities in antibodies a brief
review

• Multiple germ-line V, D, and J segments
• (provides baseline for number of possible
  rearrangements)
• Combinatorial rearrangement of V, D, and J
  segments
• Junctional diversity
• - P elements
• - N nucleotide addition
• - Exonuclease trimming
• Somatic hypermutation
• Random pairing of H and L chains

Total B cell diversity gt1010 different
specificities!
27
Factors influencing Ig gene expression

• Heavy chain class switch recombination enables
  expression of
• g, a, and e heavy chains
• - Mediated by unknown switch recombinase that
  recognizes switch regions
• comprised of multiple copies of short
  repeats (GAGCT and TGGGG)
• - Occurs after B cell activation
• - Choice of rearrangement controlled by type
  of cytokines
• present during and after B cell activation
• Differential RNA processing (splicing) enables
  expression
• of m and d heavy chains and dictates the form
  of Ig expressed
• (membrane or secreted)
• - Processing is developmentally regulated
• - Precise mechanisms remain poorly defined

28
Class switch recombination

• Key Steps
• Looping out
• DNA double-strand
• break involving
• activation-induced
• cytidine deaminase
• (AID)
• DNA repair, requiring
• at least Ku70/Ku80

(DNA-DSB and repair)
Excision circle (lost)
Excision circle (lost)
Kuby Figure 5-15
29
Alternative splicing controlsexpression of IgM
and IgD
Note for simplicity, the individual exons
encoding the constant regions are usually shown
as one block (e.g. see previous slide)
(e.g. Pre-, immature and mature B cells)
(mature B cells)
Kuby Figure 5-17
30
Carboxyl-terminus encoded in different exons
Kuby Figure 5-16
31
Alternative splicing controlsform of Ig
expressed
(e.g. plasma cells)
(e.g. mature B cells)
Kuby Figure 5-16
32

Sources of structural diversity in antibodies
that do not influence antigenic specificity a
recap

• Allotypic variation (see previous lecture)
• Class switch recombination
• Alternative RNA processing

33
Synthesis, assembly and expressionof
immunoglobulins
(TGR)

• Key Steps
• Rough endoplasmic reticulum (RER)
• - Separate synthesis of H and L chains
• - Cleavage of leader sequence
• - H and L chain pairing
• - Disulfide bond formation
• Golgi apparatus (CG to TG to TGR)
• - Glycosylation
• Secretory vesicles fuse with membrane
• - Membrane bound Ig anchored
• - Secreted Ig released

(TG)
(CG)
Kuby Figure 5-18
34
Regulation of Ig gene transcription

• Key Points
• Ig gene transcription regulated by three types
  of DNA elements
• - Promoters direct initiation of RNA
  transcription in a specific direction
• bound by ubiquitous and B cell specific
  transcription factors
• - Enhancers activate transcription from a
  promoter in an orientation-independent manner
• when bound by appropriate protein factors
• - Silencers down-regulate transcription in an
  orientation-independent manner
• when bound by appropriate protein factors
• V(D)J recombination juxtaposes promoters and
  enhancers, accelerating transcription

Off
On
Kuby Figure 5-19
35
Antibody genes and antibody engineering

• Basic Idea
• Obtain monoclonal antibody of interest,
  identify rearranged V gene sequence
• Graft V onto human framework (to avoid
  anti-mouse response) for therapeutic use

36
Examples of antibodies in clinical use
37
Alternative outcomes of V(D)J recombination
L
L
V1
Vn
J1
Jn
Ck
Open-shut joint
Transposition (in vitro)
L
L
V1
Vn
J1
Jn
Ck
Hybrid joint
38
Illigitimate V(D)J recombination,
translocations, and cancer
(1)
RAG-1/2
RAG-1/2
(3)
One-ended insertion (transposition)
D
J
ONCOGENE
DSB
(2)
OH
ONCOGENE
D
J
(1)
Cryptic site cleavage
OH
(2)
End donation from spontaneous DSB
D
J
ONCOGENE
D
ONC
and
coding joint
J
ONC
39
Clinical significance of aberrant
V(D)Jrecombination in cancer

• t(1418)(q32q21) translocation involving
  Bcl-2 and IgH locus
• 85 of follicular lymphomas
• 20 of diffuse B cell lymphoma
• T-ALL t(1014) translocation involving TCR and
  HOX11

40
The Yin and Yang of V(D)J recombination
IMMUNO-DEFICIENCY (SCID)
Impaired
Aberrant
The V(D)J recombination process
CANCER
Normal
41
Mechanistic links between V(D)J recombination and
transposition
Non-Replicative Transposition
V(D)J Recombination
RAG-1, RAG-2
Tn5, Tn10
5
'
5
'
5
'
5
'
HO
OH
HO
HO
OH
HO
Signal Joint,
Transposition?
Coding Joint
Yes, in vitro!
OH
HO
Translocation
42
.
The RAGs and the RSSs a disassembled transposon?
Chromosome 11
RAG-2
RAG-1
15 kb (human)
V
J
RAG-1, RAG-2
HO
V
J
HO
?
HO
RAG-1
RAG-2
HO
43
RAG-mediated transposition and the possible
origin of split Ig genes

• Key points
• V(D)J recombination is found within all
  vertebrate organisms, suggesting
• event occurred 400 million years ago
• RAGs are evolutionarily conserved
• RAG-mediated transposition may have split a
  primordial Ig gene,
• an event that was co-opted by vertebrates to
  increase antibody diversity

.
RAG-2
RAG-1
OH
HO
Receptor Gene Exon
array
cluster
"V"
"J"