Martinez-Barnetche J et al. (2023), Characterization of immunoglobulin loci in the ...

XB-ART-59610

Front Immunol 2023 Jan 01;14:1039274. doi: 10.3389/fimmu.2023.1039274.

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Characterization of immunoglobulin loci in the gigantic genome of Ambystoma mexicanum.

Martinez-Barnetche J , Godoy-Lozano EE , Saint Remy-Hernández S , Pacheco-Olvera DL , Téllez-Sosa J , Valdovinos-Torres H , Pastelin-Palacios R , Mena H , Zambrano L , López-Macías C .

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BACKGROUND: The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates. METHODS: The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison. RESULTS: Canonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality. CONCLUSION: The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.

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Species referenced: Xenopus tropicalis
Genes referenced: igh igl mbp mmp11 rab11b slc2a11

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	FIGURE 1 The heavy chain locus is located in the centromeric portion of chr13q (483 Mbp). (A) Gene density plot of chr13q where the IGH locus is encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole IGH locus (35.967 – 48.91 Mbp). Non-Ig genes (black) in proximal flank OXA1L gene and distal flank ABHD4, IGHC genes (blue), IGHV genes (red), and IGHD and IGHJ (yellow). (C) Zoomed view (36.9 – 38.09 Mbp) of the IGHJ-IGHC gene cluster. The lower panel shows AID hotspots density per 2.5 Kb along the IGHJ-IGHC cluster to map Switch regions. (D) Close-up of the IGHJ cluster and the IGHM gene. The bottom panel shows the spleen RNA-seq coverage histogram of the IGHJ-IGHM region. (E) Schematic representation of IGH locus in A. mexicanum genome (v6) and the corresponding locus in X. tropicalis. Color code as in A X. tropicalis displays the canonical architecture, in which the V, J and C clusters have the same orientation. Note that in A. mexicanum the JH-CH cluster and 3 IGHV segments appear to be inverted, which seems implausible due to the mechanism of V(D)J recombination. Moreover, IGHV segment orientation in axolotl are intercalated, which is atypical. Interestingly, in axolotl, the TRA/TRD locus (Yellow) is not linked to the IGH locus as in X. tropicalis and is coded across the centromere (black circle) in chr13p: 3.3-7.7 Mbp). Not on scale. The IGHD cluster is not shown for simplicity.
	FIGURE 2 The lambda light chain locus is located in the centromeric portion of chr10p (116.7 - 125.8 Mbp). (A) Gene density plot of chr10p where the IGL locus is encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole IGL locus (114.5-128.8 Mbp). Non-Ig genes (black) in proximal flank DGCR2 gene, and distal flank GST. Zoomed view of IGLC genes (blue), IGLV genes (red), and IGLJ (yellow). (C) Zoomed view (116.76-116.88 Mbp) of the IGHC gene cluster. (D) Spleen RNA-seq coverage histogram. Note that there is no expression in Cλ3. (E) Schematic representation of immunoglobulin lambda locus in A. mexicanum genome (chr10p:116-124 Mbp) and X. tropicalis (chr1:144.7-144.9 Mbp). Color code as in A X. tropicalis displays the canonical architecture, in which the V, J, and C clusters are in the same orientation. Note that in A. mexicanum, IGLV genes are in at least two different clusters (IGLV1 and IGLV2), separated by the MMP11, SMARCB11, and OTUB genes, which in Xenopus are located in the distal flank. Also, note that the OTUB gene in axolotl is inverted. Moreover, as in the IGHV locus, IGLV segment orientation in axolotl is intercalated, which is atypical. VJ recombination involving the more distal IGLV segments would delete the OTUB, SMARCB11, MMP11, and the SLC2A11 gene cluster. Not on a scale.
	FIGURE 3 The sigma light chain locus is located in the centromeric portion of chr1p (609.1 - 609.2 Mbp). (A) Gene density plot of chr1p where the IGS and the putative surrogate light chain (SLC) loci are encoded (box). Dark blue colors indicate low gene density. (B) Zoom of the whole putative SLC locus (67.5 - 68.6 Mbp). Non-Ig genes (black) in proximal flank DNAJC18 and distal flank TM4ESF5. Zoomed view of IGLC genes (blue). (C) Zoomed view (68.08 - 68.29 Mbp) of the putative SLC gene. (D) Spleen RNA-seq coverage histogram. (E) Zoom of the whole IGS locus (601 - 616 Mbp). Non-Ig genes (black) in proximal flank RAB11B gene and distal flank PRPF3. (F) Zoomed view of IGSC genes (blue), IGSV genes (red) and IGSJ (yellow) (609.15 - 610 Mbp) (G) Spleen RNA-seq coverage histogram. (H) Schematic representation of Immunoglobulin sigma locus in A. mexicanum genome (v6) (chr1p:609 - 610 Mbp), and X. tropicalis (chr1: 82.1 - 82.22 Mbp). Color code as in A. X. tropicalis displays the canonical architecture, in which the V, J and C clusters are in the same orientation. Note that in A. mexicanum, IGSV gene orientation in axolotl is intercalated, which is atypical. Non-Ig genes in the downstream flank are different in A. mexicanum and at least the PRPF3, RPRD2 and TARS3 genes are located in chr8 106.9 – 107.1 Mbp in X. tropicalis. Also, non Ig genes in the upstream flank correspond to the downstream flank in X. tropicalis. It is possible that this chromosomal configuration is correct, however it is also possible that the IGS locus may be inverted so that the downstream flank is syntenic with X. tropicalis. Not on scale.
	FIGURE 4 Relative immunoglobulin gene transcription (Log10 relative abundance). Publicly available RNA-seq data (SRA: SRP101842) (17) was used to estimate relative gene transcription in 13 adult tissues (spleen, liver, heart, lung, testes, ovary, brain, head, 9 and 15 day post - amputation forelimb blastemas, tail blastema, intact, 1 and 6 days post-injured spinal cord), limb and tail buds. A hierarchical clustering by column (tissue) was performed. (A) Immunoglobulin C region relative transcription. (B) IGHV relative transcription. Each IGHV gene is grouped by IGHV family (rows). (C) IGLV and IGSV relative transcription. Each IGLV segment is grouped according family.
	FIGURE 5 IGHM gene length in Ambystoma mexicanum is evolutionarily constrained. A. mexicanum genome (orange), IGH (purple), IGHC (light blue), IGHV cluster (light green) and individual IGHC gene length size ratios are plotted (from left to right). Comparisons include four placental mammals (human, mouse, dog and a bat), two non-placental mammals (Tasmanian devil and platypus), two bird species (chicken and duck), and one reptile, amphibian and teleost fish. Only the IGHM A. mexicanum: species size ratio was smaller to the respective genome size ratio (Kruskal-Walllis test, Dunnet multiple comparison correction (p = 0.0003).
	FIGURE 6 Distribution of V intron length in tetrapods. (A) The heavy chain and (B) lambda light chain locus in A. mexicanum according to their functionality class (functional, ORF or pseudogene). In A. mexicanum, intron length in functional IGHV genes was shorter than in IGHV pseudogenes. No differences were observed in the lambda locus and (C) D. rerio, (D) H. sapiens, and (E) X. tropicalis. V introns in functional IGHV A. mexicanum were not larger than functional A. mexicanum IGLV genes, or (F) functional IGHV of other tetrapods, however, V introns in (G) non-functional A. mexicanum IGHV genes were larger than other tetrapods. Statistical analysis for multiple comparisons (A, B, F, G) was performed with the robust ANOVA One-Way Trimmed Means Comparisons, with a trimming level of 5% (tr = 0.05), followed by the post hoc Lincon test (30). For two sample comparisons (C-F), Yuen’s robust Tests for Two Independent Groups were performed with a trimming level of 5%. The violin area is scaled for comparability. The median is shown as a black dot.
	FIGURE 7 Intergenic distances by species. Violin plots depicting overall coding to coding gene intergenic distance distribution across the whole genome (A), between IGHV segments (B), and between cytochrome p450 family gene clusters (C) in (D) A. mexicanum (red), (E) X. tropicalis (purple), (F) D. rerio (blue), and (G) H. sapiens (green). Kruskal-Wallis rank sum test followed by Dunn’s multiple comparison tests with Benjamini – Hochberg p adjustment.

References [+] :

André, Structure and diversity of Mexican axolotl lambda light chains. 2000, Pubmed, Xenbase