Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution

Abstract

Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.

Figure 1. Human and Murine KLF14 Structure and Expression

(A) Human KLF14 and (B) murine Klf14 structure are shown. Genes in 7q32.3 region in the upper panel with maternally and paternally expressed genes are depicted in grey and black, respectively. Striped patterns represent genes with tissue specific imprinting. Arrows indicate transcriptional direction. Lower panels show the gene structure of KLF14/Klf14, including results from RACE, the ORF, CpG island, and primers used in various analyses (represented by thin black bars). The grey block representing AK030435 denotes the fact that evidence of splicing was not identified in our experiments. (C) Expression of Klf14 in murine embryonic and extra-embryonic and (D) brain tissues is shown. Samples lacking reverse transcriptase are indicated by -. Results indicate higher levels of expression in extra-embryonic tissues. (E) Expression of KLF14 in human tissues. Human expression results concur with those of mouse expression, in that there is higher expression in prenatal stages of development.

Figure 2. Imprinting Analysis of Human and Murine KLF14

(A) Imprinted expression of murine Klf14 is shown. Sequence analysis of genomic DNA and RT-PCR products from 15.5-dpc hybrid mice are shown in the left and right panels, respectively. Genomic sequencing results indicate the genotype for JF1 (G) at the polymorphism. RT-PCR sequencing results show the expression of the JF1 allele in all tissues where JF1 is the maternal allele (upper row in right panel) and expression of the BL6 allele in the reciprocal cross (lower row in right panel), indicating maternal expression. (B) Imprinted expression of human KLF14. The first column of panels shows genomic sequencing electropherograms for three fetal samples (rows) heterozygous for a polymorphism in KLF14. The second column presents the genotype for the corresponding maternal samples (maternal DNA was not available for fetus number 62). The third column shows sequencing results of RT-PCR products indicating the monoallelic expression of various tissues, as indicated on the right of the column. Results from fetus number 66, which is informative for parental origin, indicate that KLF14 is maternally expressed. *, sequencing of tongue, stomach, eye, kidney, and intestine cDNA from fetus number 62 showed monoallelic expression. (C) Maternal expression of human KLF14 in somatic cell hybrids. RT-PCR was performed for three independent maternal or paternal monochromosomal hybrid cell lines for human Chromosome 7. Results confirm the maternal expression of KLF14, as seen in (B). The expression of the paternally expressed MEST and mouse A9 cell line, which lacks human Chromosome 7, are also shown.

Figure 3. Epigenetic Modifications of Murine Klf14

(A) The location and distribution of regions analyzed in Mest and Klf14. The CpG islands overlapping Mest exon 1 and Klf14 are depicted by grey bars (row 1). The regions examined in the methylation analysis and ChIP assay are indicated in rows 2 and 3, respectively. The restriction enzymes used in the ChIP assay and the polymorphisms identified in BL6 and JF1 strains are also shown. (B) Analysis of histone modifications by ChIP in fibroblast cells of BL6 × JF1 and JF1 × BL6 hybrids. ChIP was performed using formaldehyde fixed chromatin. Antibodies against histone 3 acetylated at lysines 9 and 14 (H3K9acK14ac), histone 4 acetylated at lysines 5, 8, 12, and 16 (H4ac), and H3K4me2 were used in the ChIP assay. Precipitated DNAs were PCR amplified using primers specific to the CpG islands of Mest and Klf14 and subsequently digested as shown in (A). DNA before immunoprecipitation (input) and the product obtained with no antibody (N.C.) were also included in the analysis. The difference in band intensities between the precipitated products and input DNA reveals that there is preferential precipitation of H3K9acK14ac, H4ac, and H3K4me2 on the paternal allele of the Mest CpG island, but no allelic differences were detected at the Klf14 region. (C) Analysis of histone modifications by native-ChIP in whole embryos of BL6 × JF1 hybrids. Chromatin was immunoprecipitated using antibodies against H3K9ac, H3K4me2, H3K9me3, and H4K20me3. Anti-chicken was used as a nonspecific antibody (mock). Input DNA is denoted by I. Antibody-bound and unbound fractions of the precipitate are denoted by B and U, respectively. Precipitated DNA was PCR amplified using the same primers as in (B). The amplified DNA was analyzed by single strand conformation polymorphism. The results show differences in histone modifications between the two parental alleles in the Mest CpG island, but allelic enrichments were not observed for Klf14. (D) Expression of Klf14 in offspring of Dnmt3a conditional knockout mice. The expression of Mest and Klf14 was examined in two embryos (e1–2) and corresponding extra-embryonic tissues (e1-2ex) from the offspring of female Dnmt3a conditional knockout mice, as well as a wild-type (wt) embryo. Klf14 expression is lost in the knockout mice, suggesting that its expression is dependent upon a maternally methylated region. (E) Model of Klf14 expression in wild-type (wt) and Dnmt3a conditional knockout mice. In wild-type mice (upper panel), the maternally methylated CpG island in Mest (black circle) silences the expression of the gene from the maternal allele (M), while Klf14 is actively transcribed on this allele. The opposite pattern of expression is seen on the paternal strand (P), because of the unmethylated CpG island (white circle). In Dnmt3a −/+ embryos (lower panel), maternal methylation of the Mest CpG island is lost, causing increased expression of Mest and loss of expression of Klf14. (F) Bisulfite sequencing results from 12.5-dpc whole embryos of BL6 × JF1 hybrids. Each block corresponds to a separate region analyzed in the Klf14 CpG island, as shown in (A) (Mest methylation analysis is not shown). Hollow circles and black circles indicate unmethylated and methylated CpG dinucleotides, respectively (N, could not be determined). Each row of circles represents CpGs in an individual PCR product clone. In each block, the top section and bottom sections correspond to clones from the maternal allele (BL6) and paternal (JF1) alleles, respectively, as determined by use of polymorphisms. The three regions analyzed indicate that the Klf14 CpG island is hypomethylated on both alleles.

Figure 4. Retrotransposition of KLF14 and Mammalian Evolution

(A) Genomic distribution of KLF14 and flanking genes is shown. KLF14 is flanked by MKLN1 and TSGA13 in human and mouse. These two genes are present in opossum, yet KLF14 is absent. In chicken, there is a syntenic break in the region, placing genes on different chromosomes. Two microRNAs (miR-29 and miR-29b-2), which lie in the fragile site adjacent to KLF14 (FRA7H), are conserved. (B) Presence of KLF14 and KLF16 in distant mammals is shown. The lower panel shows PCR amplification of KLF16 from mammals of diverse clades, and the upper panel shows the amplification of KLF14. The mammals shown are (L-R) cow (Bos taurus), tree shrew (Tupaia glis), nine-banded armadillo (Dasypus novemcinctus), tamandua (Tamandua tetradactyla), red-necked wallaby (M. rufogriseus), and red-legged short-tailed opossum (M. brevicaudata). It indicates that KLF14 is present in eutherian, but not marsupial mammals, and shows that KLF16 is more ancient than KLF14. 10.1371/journal.pgen.0030065.g004

Figure 5. Haplotype Frequencies of KLF14 ORF in the Human Population

(A) The frequency of eight KLF14 haplotypes in various ethnic populations is shown. The frequency of each haplotype, as defined in Figure S1, identified in ethnic populations is shown (n = number of chromosomes genotyped). (B) KLF14 primate species and human haplotype tree is represented. This tree was created manually by parsimony, with the inferred number of changes shown on each branch (thick lines represent nonsynonymous changes, while thin lines represent synonymous changes). The tree is rooted using two macaque sequences (M. mulatta and M. nemestrina). In orang-utan, gorilla, and chimpanzee, only a single haplotype is represented (where polymorphisms were present, the ancestral allele was used; when descendent alleles were used for polymorphisms, the results did not differ significantly). MRCAs, manually inferred by parsimony, are represented by shaded circles at four nodes in the tree. dN/dS values were calculated for each lineage, including the fixed human sequence (HH-MRCA) and each of the human haplotypes. For the human haplotypes, the dN/dS value is calculated to the human-chimpanzee MRCA, and not to the HH-MRCA. Two methods were utilized: maximum likelihood pairwise comparison (top) and Nei and Gojobori comparison (bottom). Where dS = 0, these methods give values −1.0000. 10.1371/journal.pgen.0030065.g005