Jane Skok, PhD

Jane SkokAssociate Professor of Pathology
PhD, 1985 University College London

New York University School of Medicine,
MSB 599, 550 1st Avenue,
New York, NY 10016
Office Tel: (212) 263 0504 Lab Tel: (212) 263 0503
E-mail: jane.skok@med.nyu.edu

Lab website: http://labs.pathology.med.nyu.edu/skok-lab/
Research Theme(s): Nuclear Organization
Keywords: Lymphocyte, B Cell, T Cell, Allelic Exclusion, Locus Contraction, Looping, Immunoglobulin and Tcr Loci, Homologous Pairing, ATM, Pax5, STAT5

Research Summary:

B and T lymphocyte development is driven by V(D)J recombination, a process by which gene segments at the antigen receptor loci are rearranged to create a vast repertoire of antigen receptor loci. In normal circumstances lymphocytes circumvent the dangers associated with the introduction of multiple double strand breaks (DSBs) through deft co-ordination and tight control over 'where and when' breaks are introduced. Research in the Skok lab, employing a combination of sophisticated imaging techniques, molecular biology, and genetics, indicates that nuclear dynamics and pairing of homologous and heterologous chromosomal loci controls accessibility to the RAG1/2 recombinase.  Immunoglobulin (Ig) and T-cell receptor (Tcr) variable region exons are assembled from arrays of V, D, and J coding segments during the development of B and T cells, respectively. Scattered across six different chromosomal locations, the T cell receptor (Tcr) a, b, d, and g loci and immunoglobulin (Ig) heavy (Igh) and light (Igk and Igλ) chain loci each contain large arrays of V, D, and J coding segments flanked by recombination signal sequences (RSS). The lymphoid-specific recombinase, consisting of RAG1 and RAG2 (the protein products of the recombination activating genes 1 and 2), binds to a pair of conserved signal sequences that can be many kilobases apart, cleaves the DNA at the signal sequence borders, and holds the resulting DNA double-strand breaks in a post-cleavage complex. The post-cleavage complex then guides the broken DNA ends into the ubiquitous nonhomologous end joining (NHEJ) machinery for repair, ultimately forming a new antigen receptor gene.

V(D)J rearrangement is regulated by lineage and locus. Despite the fact that both B and T cell loci are similarly organized and undergo recombination through the same RAG proteins and DNA repair factors, full Ig gene recombination is confined to B cells, and Tcr gene recombination is confined to T cells. Rearrangement is also ordered within a given lineage: the Igh is rearranged at the pro-B cell stage of development prior to Igk or Igλ rearrangement in pre-B cells, and in T cells rearrangement of Tcrb occurs in CD4-CD8- double negative (DN) cells prior to Tcra rearrangement in CD4+CD8+ double positive (DP) cells. Furthermore, D-to-J recombination at the Igh and Tcrb loci must take place before V-to-DJ rearrangement can begin. Finally, recombination must also be regulated at the level of the individual allele.

Chromosome interactions play a key role in regulating V(D)J recombination. Precise spatiotemporal control of rearrangement appears to involve a number of different chromosomal interactions. As a result, V(D)J recombination has provided valuable insights into chromosomal interactions in mammalian cells1,2.

  1. Chromosome self-association: looping and contraction: Some of the antigen receptor loci are quite long: the Ig heavy chain (Igh) and Ig kappa (Igk) loci, for example, span about 3Mb in the mouse. The Skok lab discovered that Igh, Igk, Tcrb and Tcra all undergo reversible locus contraction during recombination3-5. Contraction involves an alteration in configuration and the formation of chromatin loops, which enable synapsis and recombination between widely separated gene segments to facilitate rearrangement.
  2. Communication between alleles: homologous pairing. Chromosome pairing is involved in X chromosome inactivation6,7 — a classic instance of monoallelic gene expression. Antigen receptor genes are also largely monoallelically expressed (‘allelically excluded') in B and T lymphocytes. The Skok lab recently discovered that homologous immunoglobulin (Ig) alleles pair up in a stage-specific manner that mirrors the stages of their recombination8. The frequency of homologous Ig pairing is substantially reduced in the absence of the RAG1/RAG2 recombinase, but is rescued in Rag1-/- developing B cells with a transgene expressing an active site mutant form of RAG1 that supports DNA binding but not cleavage. RAG-mediated cleavage on one Ig allele induces the other allele to relocate to repressive pericentromeric heterochromatin (PCH) in a manner that requires the DNA damage response factor, ATM. In the absence of ATM, repositioning at PCH is diminished and the incidence of cleavage on both alleles is significantly increased. ATM appears to be activated by the introduction of a double-strand break on one allele to act in trans on the uncleaved allele, repositioning or maintaining it at PCH to prevent bi-allelic recombination and chromosome breaks or translocations8.
  3. Communication between different loci. We discovered that at the Igh locus, loss of intrachromosomal connections and altered locus accessibility occur as a result of an inter-chromosomal interaction with pericentromerically located Igk alleles. Igk (located on chromosome 6) directs the unrearranged Igh allele (located on chromosome 12) to pericentromeric heterochromatin where the two loci associate, mediated by the 3' enhancer (3'Ek) of the Igk locus. Repositioning of the unrearranged Igh locus at pericentromeric heterochromatin limits access to RAG proteins while decontraction provides a physical barrier to further V gene rearrangement9. Thus, the Igk locus interacts with the Igh locus to co-ordinate the transition from Igh to Igk rearrangement and thus from one stage of B cell development to the next.We are now extending this work to examine chromosome dynamics during class switch recombination, a second genome rearrangement process that B cells undergo to change the effector function of antibodies.In another line of study in our lab, we are visualizing the movement of loci during situations when two different genes must be regulated contemporaneously, such as the Cd4 and Cd8 genes during T lineage commitment.

Selected Publications (2001 to present):