DNA double-strand breaks relieve USF-mediated repression of Dß2 germline transcription in developing thymocytes.

Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRß locus (Tcrb) D segments, Dß1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJß2 segment cluster is initially restricted to the J segments and only redirected upstream of Dß2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJß2 recombination is less efficient than that of DJß1. Because inefficient DJß2 assembly offers the potential for V-to-DJß2 recombination to rescue frameshifted V-to-DJß1 joints, we wished to determine how Dß2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dß2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dß2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dß2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dß2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dß2 recombinational accessibility during thymocyte development.

The center of accessibility: Dß control of V(D)J recombination.

Developmental patterning of antigen receptor gene assembly in lymphocyte precursors correlates with decondensation of the chromatin surrounding individual gene segments. Ongoing V(D)J recombination is associated with hyperacetylation of histones H3 and H4 and the expression of sterile germline transcripts across the region of recombinational accessibility. Likewise, histone acetyltransferase and SWI/SNF chromatin remodeling complexes each appear to be required for recombination, and the PHD-finger of RAG-2 preferentially associates with recombination signal sequence (RSS) chromatin that contains H3 trimethylated on lysine 4. However, the regulatory mechanisms that direct chromatin alteration and rearrangement have proven elusive, due in large part to the interdependency of individual stages in gene activation, our limited understanding of functional significance of changes to the histone code, and the difficulty of modeling recombinational accessibility in existing experimental systems. Examining Tcrb assembly in developing thymocytes, we review the central roles of RSS elements and germline promoters as foci for epigenetic reorganization of recombinationally accessible gene segments in light of recent findings and persistent questions.

Promoter activity 5' of Dbeta2 is coordinated by E47, Runx1, and GATA-3.

V(D)J recombination involves the stepwise assembly of B and T cell receptor genes as lymphocytes progress through the early stages of development. While the mechanisms that restrict each step in recombination to its appropriate developmental stage are largely unknown, they share many of the components that regulate transcription. For example, enhancer-dependent modifications in histone acetylation and methylation are essential for both germline transcription and rearrangement of antigen receptor genes. Promoters positioned proximal to individual D and J gene segments in Tcra, Tcrb, Tcrd, IgH, and Igk also contribute to antigen receptor gene assembly, though their effects appear more localized than those of enhancers. Tcrb assembly initiates with D-to-J joining at each of the two D-J-C gene segment clusters in DN1/2 thymocytes. DJ joints are fused with Vbeta elements to complete Tcrb recombination in DN3 cells. We have previously shown that Dbeta2 is flanked by upstream and downstream promoters, with the 5' promoter being held inactive until D-to-J recombination deletes the NFkappaB-dependent 3' promoter. We now report that activity of the 5' promoter reflects a complex interplay among Runx1, GATA-3, and E47 transcription factors. In particular, while multiple E47 and Runx1 binding sites clustered near the Dbeta2 5'RS and overlapping inr elements define the core 5'PDbeta2, they act in concert with an array of upstream GATA-3 sites to overcome the inhibitory effects of a 110bp distal polypurine.polypyrimidine (R.Y) tract. The dependence of 5'PDbeta2 on E47 is consistent with the reported role of E proteins in post-DN1 thymocyte development and V-to-DJbeta recombination.

A streamlined method for rapid and sensitive chromatin immunoprecipitation.

We report a streamlined procedure to efficiently carry samples from chromatin to qPCR-compatible DNA in as little as 4 h. We use this streamlined ChIP to quantify histone H3 modifications at active (cad) and repressed (T early alpha) promoters in a Rag1-deficient pro-T cell line after 1-2 h IP. We further show that the protocol readily quantified histone modifications in chromatin from 10(4) Rag-deficient DN thymocytes. Taken together, these data outline a simple, cost-effective procedure for efficient ChIP analysis.

Differential activation of dual promoters alters Dbeta2 germline transcription during thymocyte development.

Ag receptor genes are assembled through somatic rearrangements of V, D, and J gene segments. This process is directed in part by transcriptional enhancers and promoters positioned within each gene locus. Whereas enhancers coordinate reorganization of large chromatin stretches, promoters are predicted to facilitate the accessibility of proximal downstream gene segments. In TCR beta locus, rearrangement initiates at two D-J cassettes, each of which exhibits transcriptional activity coincident with DJ rearrangement in CD4/CD8 double-negative pro-T cells. Consistent with a model of promoter-facilitated recombination, assembly of the DJbeta1 cassette is dependent on a Dbeta1 promoter (PDbeta1) positioned immediately 5' of the D. Assembly of DJbeta2 proceeds independent from that of DJbeta1, albeit with less efficiency. To gain insight into the mechanisms that selectively alter D usage, we have defined transcriptional regulation at Dbeta2. We find that both DJbeta cassettes generate germline messages in murine CD44+CD25- double-negative 1 cells. However, transcription of unrearranged DJbeta2 initiates at multiple sites 400-550 bp downstream of the Dbeta2. Unexpectedly, loci from which germline promoter activity has been deleted by DJ rearrangement redirect transcription to sites immediately 5' of the new DJbeta2 joint. Our analyses suggest that 3'-PDbeta2 activity is largely controlled by NF-kappaB RelA, whereas 5'-PDbeta2 activity directs germline transcription of DJbeta2 joints from initiator elements 76 bp upstream of the Dbeta2 5' recombination signal sequence. The unique organization and timing of Dbeta2 promoter activity are consistent with a model in which promoter placement selectively regulates the rearrangement potential of Dbeta2 during TCR beta locus assembly.