T and B cell gene rearrangement October 17, Ram Savan

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1 T and B cell gene rearrangement October 17, 2016 Ram Savan savanram@uw.edu 441 Lecture #9 Slide 1 of 28

2 Three lectures on antigen receptors Part 1 (Last Friday): Structural features of the BCR and TCR Janeway Chapter 4 Part 2 (Today): Assembly of antigen receptor genes Janeway Chapter 5; through section 5-10 Part 3 (next Wednesday): Somatic diversification of BCRs: Hypermutation and class switch recombination Janeway Chapter Lecture #9 Slide 2 of 28

3 Generating diversity in the adaptive immune system There are ~ 2 trillion (2 x ) lymphocytes in an average adult human (B + T cells). Roughly equal to the mass of the brain or the liver. In principle, each one of these lymphocytes can have a unique receptor and a unique specificity for antigen. These antigen receptors are assembled in a completely anticipatory fashion, before they ever detect antigen. How in the world is this diversity achieved at the genetic level when we only have ~10 5 genes to work with? 441 Lecture #9 Slide 3 of 28

Ig light chain constant and variable domains Each domain consists of polypeptide strands running in opposite directions, arranged in sheets (antiparallel b sheets).

4 Ig light chain constant and variable domains Each domain consists of polypeptide strands running in opposite directions, arranged in sheets (antiparallel b sheets). The sheets are linked by a disulfide bond and folded (rolled) into b barrels. This domain is the Ig fold, very evolutionarily useful: >750 human genes have them (Heavy chain is similar) 441 Lecture #9 Slide 4 of 28

5 Hypervariable regions form the antigen binding sites Antigen binding site: 3 HV regions from heavy chain 3 HV regions from light chain (Two of these sites per Ab molecule) These regions are complementary to the antigen: complementarity determining regions (CDRs). 441 Lecture #9 Slide 5 of 28

6 Hypervariable regions CDR1 CDR2 CDR3 FR1 FR2 FR3 441 Lecture #9 Slide 6 of 28

7 Nomenclature Genes in multicellular organisms are made of exons and introns: Exons are spliced together into the mature mrna Introns are removed after transcription Open reading frames are the continuous sequences in mature mrnas that encode proteins Codons are units of three bases that encode a single amino acid; codons form the open reading frame Four DNA bases: G, C, A, T. G pairs with C A pairs with T 441 Lecture #9 Slide 7 of 28

Ig genes are rearranged in B cells germline DNA V Restriction enzyme site C B cell DNA V C Isolate and digest DNA Run digested DNA on a gel Transfer separated DNA fragments onto a membrane

8 Ig genes are rearranged in B cells germline DNA V Restriction enzyme site C B cell DNA V C Isolate and digest DNA Run digested DNA on a gel Transfer separated DNA fragments onto a membrane Hybridize radiolabeled V- and C-specific probes to the membrane Develop film B and T cells are the ONLY cells in the body that do this!!! Nobel Prize, 1987: Susumu Tonegawa. 441 Lecture #9 Slide 8 of 28

During B cell development, light chain genes are rearranged, bringing a V and a J together.

9 Ig light chain genes are rearranged in B cells Separate V (variable), J (joining), and C (constant) region gene segments. During B cell development, light chain genes are rearranged, bringing a V and a J together. The intron between the J and C segments is spliced out of the mrna during transcription. The light chain variable domain is encoded by V and J gene segments. 441 Lecture #9 Slide 9 of 28

10 Ig heavy chain gene rearrangements in B cells Separate V, D (diversity), J, and C (constant) region gene segments 1: D-J rearrangement occurs first 2: V DJ rearrangement occurs second 3: mrna splicing joins the VDJ to the constant region exons The heavy chain variable domain consists of V, D, and J gene segments 441 Lecture #9 Slide 10 of 28

11 Germline organization of the Ig loci Each of these loci is on a different chromosome Not drawn to scale: the Heavy Chain locus is over 2 million base pairs long Vs are all together, Ds are together, Js are together. Exception: l locus White boxes ahead of each V are leader sequences encoding signal peptides Constant region introns are left out of this diagram 441 Lecture #9 Slide 11 of 28

Gene segments enable combinatorial diversity There are many copies of V, D, and J segments in germline DNA Any V can pair with any J in light chains (190 k), (165 l) There are two light chain gene

12 Gene segments enable combinatorial diversity There are many copies of V, D, and J segments in germline DNA Any V can pair with any J in light chains (190 k), (165 l) There are two light chain gene loci, Kappa (k) and Lambda (l) Any D can pair with any J in heavy chains (138) Any V can pair with any D-J in heavy chains (6348) Any light chain can pair with any heavy chain (355 x 6348) Start with 156 V, D, J gene segments Total theoretical diversity just by VDJ recombination: 2,253,540! 441 Lecture #9 Slide 12 of 28

13 The control of Ig gene rearrangement Ig gene rearrangement is very carefully controlled Recombination must take place at the precisely right location in the locus VDJ must occur at the correct stage of lymphocyte development (and only then!) Aberrant DNA recombination is responsible for lots of cancers (more on this in another lecture) Today: focus on control at the level of DNA Ig locus first, TCR locus second 441 Lecture #9 Slide 13 of 28

14 RSS control the location and order of Ig gene rearrangement Recognition of recombination signal sequences (RSS) starts rearrangement Each RSS is made of: A ~palindromic heptamer An AT-rich nonamer separated by spacer sequences that are either 12 or 23 bp long Recombination ONLY occurs between RSS elements with 12 and 23 bp spacers This restriction of recombination is called the 12/23 rule 441 Lecture #9 Slide 14 of 28

15 RSS control the location and order of Ig gene rearrangement 23 bp RSS are in purple, 12 bp RSS are in orange In each locus, all Vs, (Ds), and Js have the same type of RSS According to rule, V-J in the heavy chain is forbidden (also V-V, J-J ) 441 Lecture #9 Slide 15 of 28

16 Ig light chain V-J gene rearrangement Initial pairing between V1 and J brings the RSSs together Rearrangement mostly takes place between gene segments found on the same chromosome (not across chromosomes) Some V genes are in the opposite transcriptional orientation; don t worry about these 441 Lecture #9 Slide 16 of 28

Recombination overview Recombination occurs precisely at the ends of the RSS heptamer sequences This occurs by cutting the DNA and sealing it back together into two pieces: The signal joint (includes

17 Recombination overview Recombination occurs precisely at the ends of the RSS heptamer sequences This occurs by cutting the DNA and sealing it back together into two pieces: The signal joint (includes the RSSs) is sealed as a closed circle that is lost forever from the chromosome The coding joint is retained in the chromosome and contains the newly juxtaposed V and J segments What proteins accomplish this??? 441 Lecture #9 Slide 17 of 28

Recombination Activating Genes (RAG-1 and RAG2) RAG-1 and RAG-2 form a DNA endonuclease that recognizes the RSSs Expressed only in lymphocytes, and at specific times during their development RAG-1/2

18 Recombination Activating Genes (RAG-1 and RAG2) RAG-1 and RAG-2 form a DNA endonuclease that recognizes the RSSs Expressed only in lymphocytes, and at specific times during their development RAG-1/2 are likely derived from a DNA transposon that disrupted an ancient antigen receptor gene This occurred in a common ancestor of all jawed vertebrates (all have RAGs) All of this antigen receptor diversity is likely derived from an old virus!!! 441 Lecture #9 Slide 18 of 28

Enzymatic steps in Ig light chain V-J rearrangement Two RAG1/2 complexes align the two RSS Blunt-ended DNA breaks are made between the heptamer and coding sequences The free 3 OH group at the end of

19 Enzymatic steps in Ig light chain V-J rearrangement Two RAG1/2 complexes align the two RSS Blunt-ended DNA breaks are made between the heptamer and coding sequences The free 3 OH group at the end of each coding segment attacks the other strand, forming a closed hairpin Ku70/Ku80 binds to all of these DNA ends and holds them together. (These are general DNA repair molecules expressed in all cells) 441 Lecture #9 Slide 19 of 28

20 Resolving the signal joint (easy) After Ku70/Ku80 binding, no further modification of signal joint DNA occurs DNA ligase / XRCC4 is recruited by the Ku70/Ku80 proteins and the ends are ligated (sealed) The signal joint cannot replicate and is diluted out as cells divide 441 Lecture #9 Slide 20 of 28

Resolving the coding joint: Where all the action happens The DNA ends of the coding joint are covalently closed hairpins Ku70/Ku80 recruits DNA-PK, which brings along an nicking endonuclease called

21 Resolving the coding joint: Where all the action happens The DNA ends of the coding joint are covalently closed hairpins Ku70/Ku80 recruits DNA-PK, which brings along an nicking endonuclease called Artemis Artemis nicks the DNA near each end, opening the hairpins Terminal deoxynucleotidyl transferase (TdT) can add nucleotides randomly to the single-stranded DNA ends DNA ligase / XRCC4 complex joins the ends together The result: an imprecise coding joint that contains new (non-genome-encoded) sequence!! 441 Lecture #9 Slide 21 of 28

Details of the imprecise coding joint resolution heptamers RAG1/2 cleaves the DNA between the heptamers and the coding sequences These blunt ends form covalent hairpins Artemis nicks the DNA near

22 Details of the imprecise coding joint resolution heptamers RAG1/2 cleaves the DNA between the heptamers and the coding sequences These blunt ends form covalent hairpins Artemis nicks the DNA near each end, opening the hairpins These opened ends form the P nucleotides, which are palindromic (read the same backwards and forwards in dsdna) TdT adds random nucleotides (up to 20) to the ends of these ssdna segments this happens mostly in Ig Heavy chain genes The non-templated nucleotides are called N-nucleotides 441 Lecture #9 Slide 22 of 28

Details of the imprecise coding joint resolution The ssdna extensions made by TdT pair up to form regions of microhomology one to two base pairs Unpaired nucleotides are trimmed away by a DNA

23 Details of the imprecise coding joint resolution The ssdna extensions made by TdT pair up to form regions of microhomology one to two base pairs Unpaired nucleotides are trimmed away by a DNA exonuclease (this is a general repair mechanism that occurs during DNA replication) The gaps are filled in by a DNA polymerase (also part of general repair) Ligase seals the strands The newly sealed coding joints are separated by P-N-P nucleotides that greatly increase possible diversity. 441 Lecture #9 Slide 23 of 28

24 The cost of random, imprecise coding joints The V(D)J junctional regions encode the CDR3s of each Ag receptor chain: This enables a massive increase in Ag receptor repertoire diversity BUT: the P/N nucleotide additions are random, and two out of every three of them encode a frameshift that disrupts the reading frame downstream Thus, 2/3 of all VDJ recombination events are non-functional, and millions of developing lymphocytes die every day in our bodies because they don t express a functional antigen receptor. All this diversity must be worth the energy of throwing away all these cells!!! 441 Lecture #9 Slide 24 of 28

25 The T cell receptor loci: similar rules TCRa locus looks similar to IgL locus (V-J-C) TCRb locus looks similar to IgH locus (V-D-J-C) No functional distinctions between Cb1 and Cb2 Constant region introns are left out of this diagram 441 Lecture #9 Slide 25 of 28

26 The T cell receptor loci: similar rules Similar to Ig loci, but more Ds and Js are shown here Remaining Js between the V-J and the C are removed by RNA splicing D-J occurs first, then V DJ second in the TCRb locus 441 Lecture #9 Slide 26 of 28

Mechanics of TCR gene rearrangements are the same (Don t worry about the gd T cells discussed in the book ) The 12/23 rule applies the same way to TCR chain loci Oddity: the Db gene segments have

27 Mechanics of TCR gene rearrangements are the same (Don t worry about the gd T cells discussed in the book ) The 12/23 rule applies the same way to TCR chain loci Oddity: the Db gene segments have both 12 and 23 RSS, and Vb-Jb obey the 12/23 rule In TCRb locus, V-D-J, V-J, V-D-D-J are all possible (but not equally likely) The junctional diversity makes up the CDR3 of the TCRa and TCRb chains CDR3 sits right on top of the peptide!!! 441 Lecture #9 Slide 27 of 28

28 Adding up all the numbers V(D)J recombination + Junctional diversity = Astonishing theoretical diversity of lymphocyte antigen receptors!!! 441 Lecture #9 Slide 28 of 28

29 More diversity on Wednesday Even more diversity in B cell receptors that occurs after they meet antigen 441 Lecture #9 Slide 29 of 28

Chapter 5. Genetic Models. Organization and Expression of Immunoglobulin Genes 3. The two-gene model: Models to Explain Antibody Diversity

Chapter 5. Genetic Models. Organization and Expression of Immunoglobulin Genes 3. The two-gene model: Models to Explain Antibody Diversity Chapter 5 Organization and Expression of Immunoglobulin Genes 3 4 5 6 Genetic Models How to account for: ) Vast diversity of antibody specificities ) Presence of Variable regions at the amino end of Heavy