Noncoding transcription controls downstream promoters to regulate T-cell receptor alpha recombination

Abstract

The T early alpha (TEA) promoter in the murine Tcra locus generates noncoding transcripts that extend across the 65 kb Jalpha array. Here, we have analyzed the significance of TEA transcription for Tcra locus regulation through the targeted introduction of a transcription terminator downstream of the TEA promoter. We demonstrate that noncoding transcription driven by this single promoter can instruct both positively and negatively the activity of downstream Jalpha promoters, and can similarly instruct alterations in Jalpha chromatin structure and Jalpha recombination. TEA transcription activates promoters associated with relatively proximal Jalpha segments and stimulates histone acetylation, histone methylation and chromatin accessibility in this region. In contrast, at more distal locations, TEA transcription inhibits promoter activity through transcriptional interference and suppresses chromatin accessibility. In combination, these effects target initial Valpha-to-Jalpha recombination to TEA-proximal Jalpha segments and promote the ordered usage of the Jalpha array. The ability of TEA transcription to coordinate the activity of multiple downstream promoters maximizes the biological potential of the Jalpha array and diversifies the Tcra repertoire.

Figure 1

Tcra locus organization and gene targeting. (A) Tcra locus, including the relative positions of V, J and C gene segments and the TEA exon (filled rectangles), promoters (arrows) and Eα (oval). (B) Strategy for the generation of the TEA-T allele. Wild-type 129/Sv allele (129), targeting construct pTEA-T, targeting intermediate allele TEA-T neo^r and mutated allele TEA-T. Term, transcriptional terminator; TK, thymidine kinase (open rectangles); ovals, lox P sites. N, NcoI sites; Pr, probe. (C) Southern blot of NcoI-digested genomic DNA from wild-type (+/+), and targeted (+/TEA-T, TEA-T/TEA-T) mice. Molecular sizes are indicated on the right margin.

Figure 2

Transcription termination in TEA-T mice. In the PCR strategy for analysis of transcription termination (top), arrowheads denote PCR primers. Fivefold serial dilutions (wedges) of cDNA from thymocyte nuclear RNA were PCR amplified and detected by Southern blot and hybridization to radiolabeled oligonucleotide probes to detect unspliced products. Dβ1 transcripts served as loading control. Control PCR reactions: −, no reverse transcriptase; C, water. T (with arrowhead), position of the transcription terminator. Results are representative of two independent experiments.

Figure 3

Jα region germline transcription in TEA-T mice. PCR and Southern blot analysis of threefold serial dilutions of thymocyte cDNA to detect spliced germline Jα transcripts. TEA-Cα, Jα-Cα and total Cα products were detected by Southern blot with a radiolabeled Cα probe. Amplification of Actb controlled for cDNA loading. Control PCR reactions: −, no reverse transcriptase; C, water. Results are representative of two independent experiments.

Figure 4

Jα58, Jα57 and Jα47 promoter activity in TEA-T mice. (A) Agarose gel electrophoresis of 5′ RACE products. M, molecular size markers; −, water. (B) Identification of transcription start sites by sequencing of 5′ RACE products. Start sites are identified by filled circles and the number of circles over a particular nucleotide indicates the number of times that 5′ RACE clone was isolated. Shading identifies the RSS.

Figure 5

Jα region histone modifications in TEA-T mice. Analysis of H3 acetylation (A), H3 K4 di- and trimethylation (B, C) and H3 K36 trimethylation (D) by chromatin immunoprecipitation. −3180 and −1700 indicate nucleotide positions upstream of the TEA transcript start site; TEA is positioned 300 nucleotides downstream of the start site. Oct2, negative control. Actb (exon 4), positive control. Data represent the mean±s.e.m. of triplicate PCR. Results are representative of two independent experiments.

Figure 6

Jα region accessibility in TEA-T mice. (A) Strategy for Southern blot analysis of Jα region DNase I sensitivity using a 5′ Jα probe in SpeI (S) digests and a central Jα probe in BamHI (B) digests. (B) Thymocytes were incubated with DNaseI (5, 10, 15 U) and Southern blots of SpeI (left panels) and BamHI (right panels) digested genomic DNA samples were analyzed by hybridization to the indicated Jα probes (top panels). Blots were then stripped and reanalyzed by hybridization to a control Cd69 probe (bottom panels) to insure equal DNase I digestion and sample loading. Jα segments are indicated on the left. –, no DNaseI. Data are representative of two independent experiments (C) In a third experiment, Southern analysis was quantified for loss of the parental SpeI (left panels) and BamHI (right panels) fragments using a phosphorimager. Blots were sequentially analyzed with Jα and Cd69 probes.

Figure 7

Primary Jα usage in TEA-T mice. Vα8-Cα RT–PCR products of Rorc^−/− (top) and TEA-T Rorc^−/− (bottom) mice were cloned and sequenced for analysis of Jα usage. Data represent the percentage of clones using the indicated Jα segments. Number of clones analyzed: 44 Rorc^−/− and 42 TEA-T Rorc^−/−.

Figure 8

Jα use and genomic rearrangements in TEA-T mice. (A) Southern blot of Vα8-to-Cα RT–PCR products prepared from three mice each of the indicated strains and one mouse lacking the 15 kb TEA-Jα49 genomic region (Δ5′). Jα use was determined by hybridization to radiolabeled Jα or Cα oligonucleotide probes. Analysis of Actb controlled for cDNA loading. (−) control PCR lacking cDNA. Data are representative of two independent experiments. (B) Southern blot of twofold serially diluted genomic DNA samples amplified with Vα8 and different Jα primers. PCR products were detected using radiolabeled Jα oligonucleotide probes. Analysis of Cd14 controlled for DNA loading. (−) control PCR lacking DNA. Data are representative of two independent experiments.