Primate segmental duplication creates novel promoters for the LRRC37 gene family within the 17q21.31 inversion polymorphism region

Abstract

The LRRC37 gene family maps to a complex region of the human genome and has been subjected to multiple rounds of segmental duplication. We investigate the expression and regulation of this gene family in multiple tissues and organisms and show a testis-specific expression of this gene family in mouse but a more ubiquitous pattern of expression among primates. Evolutionary and phylogenetic analyses support a model in which new alternative promoters have been acquired during primate evolution. We identify two promoters, Cl8 and particularly Cl3, both of which are highly active in the cerebellum and fetal brain in human and have been duplicated from a promoter region of two unrelated genes, BPTF and DND1, respectively. Two of these more broadly expressed gene family members, LRRC37A1 and A4, define the boundary of a common human inversion polymorphism mapping to chromosome 17q21.31 (the MAPT locus)-a region associated with risk for frontal temporal dementia, Parkinsonism, and intellectual disability. We propose that the regulation of the LRRC37 family occurred in a stepwise manner, acquiring foreign promoters from BPTF and DND1 via segmental duplication. This unusual evolutionary trajectory altered the regulation of the LRRC37 family, leading to increased expression in the fetal brain and cerebellum.

Figure 1.

Identification of LRRC37 promoter regions in human and macaque. (A) RACE-PCR analysis on the cDNA isolated from the total RNA extracted from human [(Hs) Homo sapiens] and macaque [(Rh) Macaca mulatta] by using testis and cerebellum tissues. Two sets of primer pairs specific for macaque (Rh) and human (Hs) were used for final nested PCR amplification, Rh testis R1 and R2, Hs testis R1 and R2, and Hs cerebellum R1 and R2, respectively. Three PCR products were observed, cloned, and subsequently sequenced. (*) A PCR band for nonspecific amplification. (B) Schematic representation of RACE-PCR transcripts embedded on the structure of the detected LRRC37 gene structure after sequencing analysis. Promoter regions for corresponding transcripts (cerebellum clone 3 [Cl3] and cerebellum clone 8 [Cl8]) and testis-specific promoter (T1) from human (Hs Testis R2) and macaque (Rh Testis R2). (Orange arrow) The transmembrane domain (TMD).

Figure 2.

Synteny map showing expressed duplicated LRRC37 genes and promoter regions. A synteny map for the LRRC37 family between mouse (mm9), macaque (rheMac2), and human (hg19) genome assembly is prepared using the Ensembl genome browser. The locations of the genes and markers are subsequently confirmed using the UCSC genome browser. The map includes only the actively transcribed LRRC37 genes (red) either reported in sequence databases or detected in our expression analysis. The locations of human LRRC37 genes on the human genome (light blue boxes) are calculated using the first exon of reported mRNA (NM_014834) for LRRC37. We have used the beginning of the LRR region to map LRRC37 genes on macaque chromosome 16 (dark green). Lrrc37 in mouse is mapped using mRNA (AK029868) for Lrrc37 in mouse chromosome 11 (cyan), and the location indicates the beginning of AK029868. The position for the promoter regions Cl3 (green), Cl8 (purple), and testis-specific (light blue) indicate the transcription start point on the respective chromosomes of the species. Arrows show the direction of a gene (5′–3′) or the transcriptional activity of the promoter region for the respective gene. Colored dotted lines connecting genes between different chromosomes indicate a possible common evolutionary history for that individual gene based on phylogenetic analysis. The black colored genes are used as syntenic markers.

Figure 3.

Structural polymorphism of LRRC37 genes as H1 and H2 haplotypes. (A) Relative fold expression of the LRRC37 family was detected by real-time PCR using the lymphocyte-derived cell lines. The figure shows the relative fold expression of all LRRC37 genes (All) between GM12236 (H2/H2), GM12156 (H1/H2), and GM18507 (H1/H1) individuals. Expression data were normalized against the housekeeping gene UBE1. (B) PCR analysis is performed to confirm the genotype of H1 and H2 individuals using a diagnostic indel (Evans et al. 2004). (C) A mirorepeats alignment comparing the human chromosome 17 H1 reference sequence to a sequence from an alternate H2 haplotype. The alignment depicts a 1.5-Mb inversion region of the MAPT locus. (Arrow) The transcription start point from Cl3 (green), Cl8 (purple), and testis-specific (light blue) promoters at the 5′ upstream region of LRRC37 genes LRRC37A1, A2, and A4, respectively. (Red lines) The exons for the respective LRRC37 genes. (Green lines) The boundary of the inversion region within the LRRC37 genes. Each line (green or blue) shows 100 bp of homology. The figure is drawn to scale.

Figure 4.

Luciferase activity assay for promoter regions of the LRRC37 family. (Top panel) Luciferase activity of the ancestral copy of the DND1 promoter region from the (Hs) human (H. sapiens) and (Ptr) chimpanzee (Pan troglodytes) DND1 promoters and a duplicated copy of the promoter regions from the human Chr 17q21.31 H1 haplotype (Hs Chr17 H1-R Cl3 p and Hs Chr17 H1-R Cl3 p), H2 haplotype (Hs Chr17 H2-R Cl3 p and Hs Chr17 H2-R Cl3 p), and PTR chimpanzee (Chr 17 H2 Cl3 p) in HEK293FT cells. (Bottom panel) Schematic representation of H1 and H2 haplotypes on the human genome.

Figure 5.

Evolution at the regulatory region of the LRRC37 family. A model for the evolution of promoter regions of LRRC37 is depicted. (A) The LRRC37 family acquired alternative promoter regions, first, from the BPTF promoter region within the macaque lineage; and, second, from the DND1 promoter after the split between New World and Old World monkeys, respectively. An additional promoter, which is amplified from human testis and macaque testis tissues, is detected just upstream of the predicted long coding exon containing the ATG start codon in macaque and human. (B) A schematic representation of the regulation of gene expression within the LRRC37 family. The LRRC37 family evolved from testis-specific expression in mouse to ubiquitous or tissue-specific expression, such as cerebellum and thymus, in human.