Regulation of antigen-receptor gene assembly in hagfish

Abstract

Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T-cell-like, whereas those expressing VLRB are B-cell-like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single-cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases--in many of which, one allele was functional and the other was defective. In fact, all VLR-assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR-positive lymphocytes.

Figure 1

Assembly of Eptatretus burgeri VLR genes in single lymphocytes. (A) A schematic representation of VLRA and VLRB assembly. Mature VLRA and VLRB genes are generated somatically at each locus, which are distant from each other but on the same chromosome (Kasamatsu et al, 2007). The intervening sequence of the germline VLR is replaced with several LRR gene segments. Gene segments are indicated as follows: signal peptide (grey), amino-terminal LRR plus the first LRR (blue), LRR (green), connecting peptide (red), 5′ half of the carboxy-terminal LRR (orange) and 3′ half of the C-terminal LRR (cream). The number of LRR modules in the middle (green) varies in the assembled VLR genes: one to nine in VLRA and one to seven in VLRB. (B) A schematic representation of single-cell PCR assay of VLRA and VLRB. Primer pairs (arrows) were designed to amplify the germline (shorter) and assembled (longer) VLR gene simultaneously. (C) Agarose gel electrophoresis of single-cell PCR products. Data reflect five representative VLRA or VLRB PCR products (hagfish-a). A germline product along with an assembled product was observed in most lymphocytes. (D) Diallelic VLR assembly. Data reflect eight representative PCR products of the minority of lymphocytes in which two assembled products of either VLRA or VLRB were amplified (hagfish-a). LRR, leucine-rich repeat; VLR, variable lymphocyte receptor; VLRA⁺/B⁺ cells, lymphocytes with assembled VLRA/B.

Figure 2

Analysis of VLR transcription in single lymphocytes. (A) A schematic representation of single-cell RT–PCR assay of VLRA. VLRB was analysed similarly. The blue arrows represent the RT primers, the blue lines represent first-strand cDNA and the black arrows represent PCR primers designed to amplify the cDNAs of the germline (shorter) and assembled (longer) VLR gene. VLRA and VLRB forward PCR primers annealed upstream from introns (∼6 kb for VLRA and ∼0.2 kb for VLRB). (B) Agarose gel electrophoresis of single-cell RT–PCR products. Data reflect a total of nine representative VLRA or VLRB PCR products (hagfish-b, Eptatretus burgeri). VLRA (lanes 1–3, 7–9) or VLRB (lanes 4–6) transcripts were detected from both alleles. In some cases, lymphocytes contained transcripts from two assembled VLRA genes (lanes 7–9). GAPDH transcription was analysed as a control. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT–PCR, reverse transcription–PCR; VLR, variable lymphocyte receptor; VLRA⁺/B⁺, lymphocytes with assembled VLRA/B.

Figure 3

Sequence defects in VLR genes found in lymphocytes with diallelic VLR assembly. (A) A schematic representation of defective VLRA and VLRB genes from lymphocytes with diallelic assembly. All sequence defects were located in LRR (green) modules and are indicated as follows: arrowhead, frame shift; red × , in-frame stop codon. In VLRA #7, an in-frame stop codon (7-1) and a frame shift (7-2) were both found. VLRAs #3 and #6 were identified by single-cell RT–PCR using hagfish-b (Eptatretus burgeri) and hagfish-c, respectively. The rest were identified by single-cell PCR using hagfish-a. The GenBank accession numbers are: VLRA #1–9, AB519982–AB519990; VLRB #1–10, AB520071–AB520080. (B) The nucleotide sequences around the defect sites shown in (A). The sequence defects are indicated as follows: open boxes, possible frame-shift sites; red boxes, in-frame stop codons. For sequences containing frame shifts, short repeats of a specific nucleotide near possible frame-shift sites are underlined, and the number of extra (+) or missing (−) nucleotides is indicated on the right. Consensus amino-acid residues (>90% identity) are indicated above the sequences. LRR, leucine-rich repeat; RT–PCR, reverse transcription–PCR; VLR, variable lymphocyte receptor.