Evolution of siglec-11 and siglec-16 genes in hominins

Abstract

We previously reported a human-specific gene conversion of SIGLEC11 by an adjacent paralogous pseudogene (SIGLEC16P), generating a uniquely human form of the Siglec-11 protein, which is expressed in the human brain. Here, we show that Siglec-11 is expressed exclusively in microglia in all human brains studied-a finding of potential relevance to brain evolution, as microglia modulate neuronal survival, and Siglec-11 recruits SHP-1, a tyrosine phosphatase that modulates microglial biology. Following the recent finding of a functional SIGLEC16 allele in human populations, further analysis of the human SIGLEC11 and SIGLEC16/P sequences revealed an unusual series of gene conversion events between two loci. Two tandem and likely simultaneous gene conversions occurred from SIGLEC16P to SIGLEC11 with a potentially deleterious intervening short segment happening to be excluded. One of the conversion events also changed the 5' untranslated sequence, altering predicted transcription factor binding sites. Both of the gene conversions have been dated to ~1-1.2 Ma, after the emergence of the genus Homo, but prior to the emergence of the common ancestor of Denisovans and modern humans about 800,000 years ago, thus suggesting involvement in later stages of hominin brain evolution. In keeping with this, recombinant soluble Siglec-11 binds ligands in the human brain. We also address a second-round more recent gene conversion from SIGLEC11 to SIGLEC16, with the latter showing an allele frequency of ~0.1-0.3 in a worldwide population study. Initial pseudogenization of SIGLEC16 was estimated to occur at least 3 Ma, which thus preceded the gene conversion of SIGLEC11 by SIGLEC16P. As gene conversion usually disrupts the converted gene, the fact that ORFs of hSIGLEC11 and hSIGLEC16 have been maintained after an unusual series of very complex gene conversion events suggests that these events may have been subject to hominin-specific selection forces.

Fig. 1.

Comparison of SIGLEC11 and SIGLEC16/P sequences. (A) Gene structures of SIGLEC11 and SIGLEC16P. Exons are represented by solid and open boxes. (B) Sliding window analysis of the conservation profile of human SIGLEC16P versus SIGLEC11 (window size 20 bp; step size 1 bp). (C) Sliding window analysis of the conservation profile of chimpanzee SIGLEC16 versus SIGLEC11 (window size 20 bp; step size 1 bp).

FIG. 2.

Phylogenetic relationship between the human (h) SIGLEC16P, hSIGLEC16, hSIGLEC11 and chimpanzee (c) SIGLEC11 and cSIGLEC16. Phylogenetic tree of the A/A′ region is constructed using Neighbor-Joining method. The label at the internode represents bootstrap support for 1,000 replications.

FIG. 3.

Phylogenetic analyses of the A/A′ regions of SIGLEC11 and SIGLEC16/P. Phylogenetic relationships between human (h) and chimpanzee (c) SIGLEC11 and SIGLEC16/P for (A) region A/A′1, (B) region A/A′2, and (C) region A/A′1 + A/A′2. (D). Phylogenetic tree of region A/A′1 + A/A′2 of human, chimpanzee, bonobo (b), gorilla (g), and orangutan (o) SIGLEC11 and human SIGLEC16P and chimpanzee SIGLEC16. The label at the internode represents bootstrap support for 1,000 replications. The GenBank accession numbers of bonobo, gorilla, and orangutan SIGLEC11 sequences are AB211392–AB211394.

FIG. 4.

Two possible scenarios for gene conversions from SIGLEC16P to SIGLEC11 during hominin evolution. (A). If the gene conversion occurred in the common ancestor of humans and Denisovans, the inferred phylogeny would then show that Denisovan SIGLEC11 is more closely related to human SIGLEC11 (supported). (B) If the gene conversion occurred only in the human lineage after the human and Denisovan divergence, the inferred phylogeny would then show that Denisovan SIGLEC11 is more closely related to chimpanzee SIGLEC11 (unsupported).

FIG. 5.

Genomic alignments of 5′ noncoding region and exon 1 of SIGLEC11 and SIGLEC16 from multiple hominids. Hsa, Homo sapiens; Ptr, Pan troglodytes; Ppa, Pan paniscus; Ggo, Gorilla gorilla; Ppy, Pongo pygmaeus. Exon 1 is represented by the bars lying on top of the alignment. The open box indicates the ATG start codon. The putative GATA-1-binding sequence is underlined. The “N”s in gorilla and orangutan sequences represent the uncertain sites in sequencing.

FIG. 6.

Detection of Siglec-11 ligands in human brain. (A) Sections of human cerebral cortex or spleen were probed with human Siglec-11-Fc or Siglec-6-Fc (negative control) to detect Siglec binding sites. Brownish-pink staining is positive. Magnification is 400×, scale bar shows 50 μ. (B) Western blot analysis of the biotinylated human brain membrane extract precipitated with human Siglec-11 Fc (Lane 1) or Siglec-6 Fc (Lane 3). Lane 2 is a marker lane. The blot was stripped and reprobed with anti-FLAG antibody to confirm the presence of Siglec-11 Fc and Siglec-6 Fc, both of which have the FLAG tag.

FIG. 7.

SIGLEC16/P haplotype analysis in the human population. (A). Genomic PCR showing the presence of SIGLEC16 or SIGLEC16P alleles. (B). Neighbor-Joining tree of 13 identified haplotypes of human SIGLEC16/P from 27 human individuals. PHASE2.1.1 was used to reconstruct all of the haplotypes. Bootstrap values of more than 50% from 1,000 replications were shown on the tree branches. (C). Sequence alignment of SIGLEC16 and SIGLEC16P haplotypes. SNPs strongly linked to the SIGLEC16 or SIGLEC16P genotype are highlighted in gray. Two SNPs (site 55170744 and 55170890) present in HapMap database were indicated by the arrowheads. The location of each SNP on chromosome 19 is shown above using a sequence of numbers. All of the positions are based on human hg18 assembly. (D). SIGLEC16 genotyping using HapMap SNPs information from 993 individuals. The numbers of individuals are shown in parentheses. The bars represent the frequency of homozygous SIGLEC16P (−/−), black; heterozygous (+/−), light gray; and homozygous SIGLEC16 (+/+), dark gray. The geographic origins of the genotyped individuals are—ASW: African ancestry in Southwest USA; CEU: Utah residents with Northern and Western European ancestry from the CEPH collection; CHB: Han Chinese in Beijing, China; CHD: Chinese in Metropolitan Denver, Colorado; GIH: Gujarati Indians in Houston, Texas; JPT: Japanese in Tokyo, Japan; LWK: Luhya in Webuye, Kenya; MEX: Mexican ancestry in Los Angeles, California; MKK: Maasai in Kinyawa, Kenya; TSI: Tuscan in Italy; YRI: Yoruban in Ibadan, Nigeria.

FIG. 8.

Gene tree of human SIGLEC16 and SIGLEC16P haplotypes. The chimpanzee SIGLEC16 sequence was used to infer the most recent common ancestral sequence. Each dot represents a nucleotide substitution.

FIG. 9.

Proposed scenario of gene conversions between human SIGLEC11 and SIGLEC16 loci. Human SIGLEC16P converted human SIGLEC11 in both of the A/A′1 and A/A′2 regions ∼1.0–1.2 Ma. After these gene conversions, human SIGLEC11 converted human SIGLEC16 in the A/A′1 and 170-bp divergent regions. SIGLEC11 and SIGLEC16 loci are shown in a head-to-head orientation. Arrowheads indicate the 4-bp deletion in human SIGLEC16P.

FIG. 10.

Proposed Scenario for the evolution of SIGLEC11 and SIGLEC16 loci over the last 20 myr. See text for discussion.