Comprehensive analysis of keratin gene clusters in humans and rodents

Here, we present the comparative analysis of the two keratin (K) gene clusters in the genomes of man, mouse and rat. Overall, there is a remarkable but not perfect synteny among the clusters of the three mammalian species. The human type I keratin gene cluster consists of 27 genes and 4 pseudogenes, all in the same orientation. It is interrupted by a domain of multiple genes encoding keratin-associated proteins (KAPs). Cytokeratin, hair and inner root sheath keratin genes are grouped together in small subclusters, indicating that evolution occurred by duplication events. At the end of the rodent type I gene cluster, a novel gene related to K14 and K17 was identified, which is converted to a pseudogene in humans. The human type II cluster consists of 27 genes and 5 pseudogenes, most of which are arranged in the same orientation. Of the 26 type II murine keratin genes now known, the expression of two new genes was identified by RT-PCR. Kb20, the first gene in the cluster, was detected in lung tissue. Kb39, a new ortholog of K1, is expressed in certain stratified epithelia. It represents a candidate gene for those hyperkeratotic skin syndromes in which no K1 mutations were identified so far. Most remarkably, the human K3 gene which causes Meesmann's corneal dystrophy when mutated, lacks a counterpart in the mouse genome. While the human genome has 138 pseudogenes related to K8 and K18, the mouse and rat genomes contain only 4 and 6 such pseudogenes. Our results also provide the basis for a unified keratin nomenclature and for future functional studies.     

Evolution of RH genes in hominoids: characterization of a gorilla RHCE-like gene

The human RH locus is responsible for the expression of the Rh blood group antigens. It consists of two closely linked genes, RHD and RHCE, that exhibit 92% similarity between coding regions. These observations suggest that they are derived from a relatively recent duplication event. Previously a study of nonhuman primate RH-like genes demonstrated that ancestral RH gene duplication occurred in the common ancestor of man, chimpanzees and gorillas. By amplification of intron 3 and intron 4 of gorilla RH-like genes, we have now shown that, like man, gorillas possess two types of RH intron 3 (RHCE intron 3 being 289 bp longer than the RHD intron 3) and two types of intron 4 (RHCE intron 4 being 654 bp longer than the RHD intron 4). Here we report the characterization of a cDNA encoded by a gorilla RH-like gene which possesses introns 3 and 4 of the RHCE type. A comparison of this gorilla RHCE-like coding sequence with previously characterized human and ape cDNA sequences suggests that RH genes experienced complex recombination events after duplication in the common ancestor of humans, chimpanzees and gorillas.     

Copy Number Variation at the APOL1 Locus

Two coding variants in the APOL1 gene (G1 and G2) explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7–30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123) and controls (n = 255) with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03). Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of APOL1 are present.     

Isolation and characterization of cDNA clones for the mouse thymocyte B cell antigen (ThB).

The Thb locus is responsible for the expression of 15-kDa phosphatidyl inositol anchored molecules (ThB) on murine thymocytes and B cells. Thb expression as detected with mAb is polymorphic on B cells with two alleles, Thbh and Thb1 responsible for the high and low expression of ThB on B cells. The regulatory locus for Thb expression had been mapped with the Ly-6 cluster of genes to Chr 15. In our study we used expression cloning in COS cells to isolate cDNA clones that code for ThB after transfection; the cDNA products react with anti- ThB antibodies, but not with Ly-6A.2, -6B.2, -6C.2, or -6D.2 antibodies. One of these clones, pThB-A contains insert of 702 bases which was sequenced. The translated amino acid sequence has 11 cysteine residues, and together with the absence of potential N-linked glycosylation sites is similar to the structure of the Ly-6 molecules. The nucleotide and amino acid sequences of ThB cDNA were compared to those of Ly-6 genes and the Ly-6 related human CD59 and show clear homology. Finally using interspecies crosses, the structural Thb gene has been mapped to Chr 15; thus both structural and regulatory genes map to a similar site. The genetic map location near Ly-6 and the sequence similarity suggest that Thb and Ly-6 may have been derived from the same progenitor by gene duplication.     

The human and murine protocadherin-beta one-exon gene families show high evolutionary conservation, despite the difference in gene number

Extensive cDNA analysis demonstrated that all human and mouse protocadherin-beta genes are one-exon genes. The protein sequences of these genes are highly conserved, especially the three most membrane-proximal extracellular domains. Phylogenetic analysis suggested that this unique gene family evolved by duplication of one single protocadherin-beta gene to 15 copies. The final difference in the number of protocadherin-beta genes in man (#19) and mouse (#22) is probably caused by duplications later in evolution. The complex relationship between human and mouse genes and the lack of pseudogenes in the mouse protocadherin-beta gene cluster suggest a species-specific evolutionary pressure for maintenance of numerous protocadherin-beta genes.     

Block duplications of a zeta-zeta-alpha-theta gene set in the rabbit alpha-like globin gene cluster

In order to understand the coordinate regulation between the alpha-like and beta-like globins during the developmental switches in hemoglobin synthesis, we have studied the rabbit alpha-like globin gene family. A cluster of six linked genes arranged 5'-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3' has been isolated as a set of overlapping clones from a library of rabbit genomic DNA. Blot-hybridization analysis of genomic DNA not only confirms this linkage arrangement but also reveals the presence of additional zeta and theta genes. We propose that this gene cluster was generated by a block duplication of a set of alpha-like genes; the proposed duplication unit is zeta-zeta-alpha-theta. Further duplications of a zeta-zeta-theta set are also proposed to have occurred. As expected for a duplicated locus, the rabbit alpha-like gene cluster contains long blocks of internal homology. The Z homology block is about 7.2 kilobase pairs long and contains the zeta genes; the T homology block is about 4.7 kilobase pairs long and contains a theta gene. Surprisingly, both Z and T homology blocks are flanked by a common junction sequence (J) which contains a region very similar to the 3'-untranslated sequence of an alpha-globin gene. Analysis of the J sequences suggests a recombination mechanism by which the alpha gene could have been deleted from the second set of genes in the cluster (zeta 2-zeta 3-theta 2). The relationships among the genes in characterized alpha-like gene clusters in mammals are summarized. The rabbit gene cluster differs from those of other mammals principally in the loss of a gene orthologous to the human psi alpha 1 and in the block duplication of the zeta-zeta-alpha-theta gene set.     

Duplicated KOX zinc finger gene clusters flank the centromere of human chromosome 10: evidence for a pericentric inversion during primate evolution.

Two related zinc finger (ZNF) gene clusters from the pericentromeric region of human chromosome 10, defined by cDNAs of the KOX series, have been cloned in yeast artificial chromosomes (YACs). The two clusters evolved by duplication of an ancestral gene cluster before the divergence of the human and great ape lineages. Included in cluster A are the ZNF gene sequences ZNF11A, ZNF33A, and ZNF37A, while cluster B comprises the related sequences ZNF11B, ZNF33B and ZNF37B. Genes from both clusters are expressed: cDNAs KOX2, KOX31 and KOX21 derive from ZNF11B, ZNF33A and ZNF37A, respectively. Further YACs have been isolated which link ZNF11A and ZNF33A to another gene, ZNF25, defined by cDNA clone KOX19. Therefore ZNF25 also forms part of cluster A, but has no counterpart in cluster B. Surprisingly, the KOX ZNF gene clusters are located on opposite sides of the centromere: cluster A maps to 10p11.2, while cluster B is in 10q11.2. This suggests the occurrence during primate evolution of a previously undescribed pericentric inversion subsequent to the cluster duplication. The evolution of this subset of KOX ZNF genes has therefore involved three types of genetic event: local gene duplication, gene cluster duplication, and chromosome rearrangement.     

The primate psi beta 1 gene. An ancient beta-globin pseudogene

The human beta-globin gene cluster contains five functional genes plus a single pseudogene termed psi beta 1. Hybridization and comparative sequence analysis show that this pseudogene is not the product of a recent gene duplication, but is ancient and has been maintained in all major primate groups ranging from prosimians to anthropoids, at the same position as in man, between gamma- and delta-globin genes. In the lemur, a prosimian, the central exons of the psi beta 1 and delta-globin genes have undergone an unequal exchange, which has resulted in a contraction of the beta-globin gene cluster and the formation of a Lepore-type psi beta 1-delta globin pseudogene. Comparisons of defects shared by prosimian, New World monkey and human psi beta 1 sequences suggest that the ancestral primate gene was probably a pseudogene with an abnormal initiation codon but few if any additional defects, and that most contemporary pseudogene defects were accumulated relatively recently by slow neutral drift. We suggest that psi beta 1 arose early in primate evolution by silencing of a pre-existing discrete functional gene, and show that psi beta 1-related sequences are also present in other mammalian orders. In view of the antiquity of psi beta 1-related sequences, we propose that this gene be renamed the eta-globin gene.     

Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression

Polycomb group genes were identified as a conserved group of genes whose products are required in multimeric complexes to maintain spatially restricted expression of Hox cluster genes. Unlike in Drosophila, in mammals Polycomb group (PcG) genes are represented as highly related gene pairs, indicative of duplication during metazoan evolution. Mel18 and Bmi1 are mammalian homologs of Drosophila Posterior sex combs. Mice deficient for Mel18 or Bmi1 exhibit similar posterior transformations of the axial skeleton and display severe immune deficiency, suggesting that their gene products act on overlapping pathways/target genes. However unique phenotypes upon loss of either Mel18 or Bmi1 are also observed. We show using embryos doubly deficient for Mel18 and Bmi1 that Mel18 and Bmi1 act in synergy and in a dose-dependent and cell type-specific manner to repress Hox cluster genes and mediate cell survival of embryos during development. In addition, we demonstrate that Mel18 and Bmi1, although essential for maintenance of the appropriate expression domains of Hox cluster genes, are not required for the initial establishment of Hox gene expression. Furthermore, we show an unexpected requirement for Mel18 and Bmi1 gene products to maintain stable expression of Hox cluster genes in regions caudal to the prospective anterior expression boundaries during subsequent development.     

Phylogenetic tests of the hypothesis of block duplication of homologous genes on human chromosomes 6, 9, and 1

There are 10 gene families that have members on both human chromosome 6 (6p21.3, the location of the human major histocompatibility complex [MHC]) and human chromosome 9 (mostly 9q33-34). Six of these families also have members on mouse chromosome 17 (the mouse MHC chromosome) and mouse chromosome 2. In addition, four of these families have members on human chromosome 1 (1q21-25 and 1p13), and two of these have members on mouse chromosome 1. One hypothesis to explain these patterns is that members of the 10 gene families of human chromosomes 6 and 9 were duplicated simultaneously as a result of polyploidization or duplication of a chromosome segment ("block duplication"). A subsequent block duplication has been proposed to account for the presence of representatives of four of these families on human chromosome 1. Phylogenetic analyses of the 9 gene families for which data were available decisively rejected the hypothesis of block duplication as an overall explanation of these patterns. Three to five of the genes on human chromosomes 6 and 9 probably duplicated simultaneously early in vertebrate history, prior to the divergence of jawed and jawless vertebrates, and shortly after that, all four of the genes on chromosomes 1 and 9 probably duplicated as a block. However, the other genes duplicated at different times scattered over at least 1.6 billion years. Since the occurrence of these clusters of related genes cannot be explained by block duplication, one alternative explanation is that they cluster together because of shared functional characteristics relating to expression patterns.      

Oncogene APOL1 promotes proliferation and inhibits apoptosis via activating NOTCH1 signaling pathway in pancreatic cancer

APOL1 encodes a secreted high-density lipoprotein, which has been considered as an aberrantly expressed gene in multiple cancers. Nevertheless, the role of APOL1 in the regulatory mechanisms of pancreatic cancer remains unknown and should be explored. We identified APOL1 was abnormally elevated in human pancreatic cancer tissues compared with that in adjacent tissues and was associated with poor prognosis. The effects of APOL1 in PC cell proliferation, cell cycle, and apoptosis was verified via functional in vitro and in vivo experiments. The results showed that knockdown of APOL1 significantly inhibited the proliferation and promoted apoptosis of pancreatic cancer. In addition, we identified APOL1 could be a regulator of NOTCH1 signaling pathway using bioinformatics tools, qRT-PCR, dual-luciferase reporter assay, and western blotting. In summary, APOL1 could function as an oncogene to promote proliferation and inhibit apoptosis through activating NOTCH1 signaling pathway expression in pancreatic cancer; therefore, it may act as a novel therapeutic target for pancreatic cancer.Subject terms: Oncogenes, Protein-protein interaction networks     

Analysis of the genomic structure of the porcine CD1 gene cluster

CD1 is an MHC class I-like protein that presents lipid antigens to T cell receptors. We determined 470,187 bp of the genomic sequence encompassing the region encoding porcine CD1 genes. We identified 16 genes in this region and newly identified CD1A2, CD1B, CD1C, CD1D, and CD1E. Porcine CD1 genes were located in clusters between KIRREL and olfactory receptor (OR) genes, as observed in humans, although they were divided into two regions by a region encoding OR genes. Comparison of the genomic sequences of CD1 gene loci in pigs with other mammals showed that separation of the CD1 gene cluster by ORs was observed only in pigs. CD1A duplication in the porcine genome was estimated to have occurred after the divergence of the human and porcine. This analysis of the genomic sequence of the porcine CD1 family will contribute to our understanding of the evolution of mammalian CD1 genes.     

DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing.

The genomic sequence of a 66,109 bp long region within the human MHC has been determined by manual and automated DNA sequencing. From cDNA mapping and sequencing data it is known that this region contains a cluster of at least four genes that are believed to be involved in antigen processing. Here, we describe the genomic organization of these genes, which comprise two proteasome-related genes (LMP2 and LMP7), thought to be involved in the proteolytic degradation of cytoplasmic antigens and two ABC transporter genes (TAP1 and TAP2), thought to be involved in pumping of the degraded peptides across the endoplasmic reticulum membrane. Analysis of the sequence homology and the intron/exon structures of the corresponding genes suggests that one gene pair arose by duplication from the other. Comparison of the available sequence data from other organisms shows striking conservation (70 to 84%) of this gene cluster in human, mouse and rat. The presence of several potential interferon stimulated response elements (ISREs) is in agreement with the experimentally observed up-regulation of these genes with gamma-interferon.     

Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish

Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian (Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. Thus neighboring K8 and K18 genes seem a feature shared by all vertebrates. In contrast to the two well defined keratin gene clusters of terrestrial vertebrates, three teleost fish show an excess of type I over type II genes, the lack of a keratin type II gene cluster and a striking dispersal of type I genes, that are probably the result of the teleost-specific whole genome duplication followed by a massive gene loss. This raises the question whether keratin gene clusters extend beyond the ancestral bony vertebrate to cartilage fish and lamprey. We also analyzed the complement of non-keratin IF genes of the chicken. Surprisingly, an additional nuclear lamin gene, previously overlooked by cDNA cloning, is documented on chromosome 10. The two splice variants closely resemble the lamin LIII a + b of amphibia and fish. This lamin gene is lost on the mammalian lineage.