The strength of selection on ultraconserved elements in the human genome

Ultraconserved elements are stretches of consecutive nucleotides that are perfectly conserved in multiple mammalian genomes. Although these sequences are identical in the reference human, mouse, and rat genomes, we identified numerous polymorphisms within these regions in the human population. To determine whether polymorphisms in ultraconserved elements affect fitness, we genotyped unrelated human DNA samples at loci within these sequences. For all single-nucleotide polymorphisms tested in ultraconserved regions, individuals homozygous for derived alleles (alleles that differ from the rodent reference genomes) were present, viable, and healthy. The distribution of allele frequencies in these samples argues against strong, ongoing selection as the force maintaining the conservation of these sequences. We then used two methods to determine the minimum level of selection required to generate these sequences. Despite the lack of fixed differences in these sequences between humans and rodents, the average level of selection on ultraconserved elements is less than that on essential genes. The strength of selection associated with ultraconserved elements suggests that mutations in these regions may have subtle phenotypic consequences that are not easily detected in the laboratory.     

Tissue- and cell-specific expression of human sn-glycerol-3-phosphate dehydrogenase in transgenic mice.

Comparison of the promoter sequence for the sn-glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) genes in mice and humans showed that there were three promoter domains conserved in evolution (1). To study the functional organization of the GPDH promoter, we generated transgenic mice carrying the complete human gene, GPD1. The level of human and mouse GPDH activity was measured in each tissue and the amount of human-mouse GPDH heterodimer was used as a sensitive indicator of cell-specific expression of GPD1. During postnatal development and in adult tissues of the transgenic mice, human GPDH was expressed at levels that corresponded closely to the expression of the endogenous mouse gene, Gdc-1. Surprisingly, deletion of the evolutionarily conserved fat-specific elements (FSE) in the proximal promoter region failed to reveal any alterations in GPD1 expression that were specific for either white or brown adipose tissue.     

Synergistic effects of germ cell expressed genes on male fertility in mice

Over 200 genes have been shown to be associated with infertility in mouse models. However, knockout mice reveal unexpected functional redundancy of some germ cell expressed genes. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp2), proacrosin (Acr), histone H1.1 (H1.1), histone H1t (H1t) and sperm mitochondria-associated cysteine-rich protein (Smcp) have been generated and analysed. Tnp2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S-phase of the cell cycle. The histone gene Hist1h1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Sperm mitochondria-associated cysteine-rich protein (Smcp) is a major structural element of the mitochondria in the midpiece of the sperm tail. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization four lines of double knockout mice Hist1h1a/Mcsp, Hist1h1t/Mcsp, Tnp2/Mcsp and Acr/Mcsp were established. It was found that even when knockout mice are heterozygous for one allele (-/+) and homozygous for the other allele (-/-), mice were subfertile. Homozygous double knockout mice of all four lines are nearly infertile. However, in the four homozygous double knockout mouse lines, different characteristic abnormalities are prominently manifested: In Hist1h1a-/-/Mcsp-/- the migration of spermatozoa is disturbed in female genital tract, in Hist1h1t-/-/Mcsp-/- spermatozoa show morphological head abnormalities, in Tnp2-/-/Mcsp-/- the motility of sperm is affected, and in Acr-/-/Mcsp-/- the sperm-oocyte interaction is impaired. These findings indicate strongly that male germ cell expressed genes have synergistic effects on male fertility.     

Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region.     

The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche

The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFβ family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvβ3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFβ signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation.     

A haplolethal locus uncovered by deletions in the mouse T complex

Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.     

Functional and comparative genomic analysis of the piebald deletion region of mouse chromosome 14

Several developmentally important genomic regions map within the piebald deletion complex on distal mouse chromosome 14. We have combined computational gene prediction and comparative sequence analysis to characterize an approximately 4.3-Mb segment of the piebald region to identify candidate genes for the phenotypes presented by homozygous deletion mice. As a result we have ordered 13 deletion breakpoints, integrated the sequence with markers from a bacterial artificial chromosome (BAC) physical map, and identified 16 known or predicted genes and >1500 conserved sequence elements (CSEs) across the region. The candidate genes identified include Phr1 (formerly Pam) and Spry2, which are mouse homologs of genes required for development in Drosophila melanogaster. Gene content, order, and position are highly conserved between mouse chromosome 14 and the orthologous region of human chromosome 13. Our studies combining computational gene prediction with genetic and comparative genomic analyses provide insight regarding the functional composition and organization of this defined chromosomal region.     

Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice.     

Trisomic and allelic differences influence phenotypic variability during development of Down syndrome mice

Individuals with full or partial Trisomy 21 (Ts21) present with clinical features collectively referred to as Down syndrome (DS), although DS phenotypes vary in incidence and severity between individuals. Differing genetic and phenotypic content in individuals with DS as well as mouse models of DS facilitate the understanding of the correlation between specific genes and phenotypes associated with Ts21. The Ts1Rhr mouse model is trisomic for 33 genes (the "Down syndrome critical region" or DSCR) hypothesized to be responsible for many clinical DS features, including craniofacial dysmorphology with a small mandible. Experiments with Ts1Rhr mice showed that the DSCR was not sufficient to cause all DS phenotypes by identifying uncharacteristic craniofacial abnormalities not found in individuals with DS or other DS mouse models. We hypothesized that the origins of the larger, dysmorphic mandible observed in adult Ts1Rhr mice develop from larger embryonic craniofacial precursors. Because of phenotypic variability seen in subsequent studies with Ts1Rhr mice, we also hypothesized that genetic background differences would alter Ts1Rhr developmental phenotypes. Using Ts1Rhr offspring from two genetic backgrounds, we found differences in mandibular precursor volume as well as total embryonic volume and postnatal body size of Ts1Rhr and nontrisomic littermates. Additionally, we observed increased relative expression of Dyrk1a and differential expression of Ets2 on the basis of the genetic background in the Ts1Rhr mandibular precursor. Our results suggest that trisomic gene content and allelic differences in trisomic or nontrisomic genes influence variability in gene expression and developmental phenotypes associated with DS.     

Influence of murine maternal diabetes on placental morphology, gene expression, and function

Maternal diabetes causes placental and foetal abnormalities in both rat and humans; however, its effect is less well documented in the mouse. We used a standard approach to induce manifest diabetes in pregnant mice and assessed morphology, function and gene expression in the placentas isolated from these females. We found that diabetic placentas exhibit a consistent abnormal phenotype characterized by increased junctional zone cross sectional area. Lipid profiling of diabetic foetuses and placentas showed that the placental phenotypes do not compromise the lipid transport function of this organ. In a genome-wide survey of mRNA expression by using cDNA micro-arrays, we identified 118 ESTs, corresponding to 59 annotated genes, with differential expression in the diabetic placentas. A significant proportion of these known is involved in metabolism, immunity and defence, and signal transduction. In addition, we found two imprinted genes, Igf2 and Gatm, which exhibited altered expression. The expression of other imprinted genes, Peg1, Gtl2, Peg3, Igf2r and Grb10, was determined by quantitative RT-PCR. For all of these genes, slight changes in gene expression were observed between diabetic placentas and control placentas. Our study thus provides the basis for future work that will address gene action in the diabetic mouse placenta.     

Modeling partial monosomy for human chromosome 21q11.2-q21.1 reveals haploinsufficient genes influencing behavior and fat deposition

Haploinsufficiency of part of human chromosome 21 results in a rare condition known as Monosomy 21. This disease displays a variety of clinical phenotypes, including intellectual disability, craniofacial dysmorphology, skeletal and cardiac abnormalities, and respiratory complications. To search for dosage-sensitive genes involved in this disorder, we used chromosome engineering to generate a mouse model carrying a deletion of the Lipi-Usp25 interval, syntenic with 21q11.2-q21.1 in humans. Haploinsufficiency for the 6 genes in this interval resulted in no gross morphological defects and behavioral analysis performed using an open field test, a test of anxiety, and tests for social interaction were normal in monosomic mice. Monosomic mice did, however, display impaired memory retention compared to control animals. Moreover, when fed a high-fat diet (HFD) monosomic mice exhibited a significant increase in fat mass/fat percentage estimate compared with controls, severe fatty changes in their livers, and thickened subcutaneous fat. Thus, genes within the Lipi-Usp25 interval may participate in memory retention and in the regulation of fat deposition.     

Analysis of CRE-mediated recombination driven by myosin light chain 1/3 regulatory elements in embryonic and adult skeletal muscle: a tool to study fiber specification

An increasing number of genes have been implicated in skeletal muscle fiber diversity. To study the contribution of diverse genetic elements to the regulation of fiber-type composition, we generated a transgenic mouse in which CRE recombinase expression is driven by muscle-specific regulatory sequences of the myosin light chain 1/3 locus (MLC). Using ROSA26 conditional reporter mice, we detected expression of the MLC-Cre transgene starting from embryonic day 12.5 (E12.5). By E15, recombination was detected in all muscle-derived structures. Immunohistochemical analysis revealed CRE activity was restricted to fast-twitch (type II) and excluded from slow-twitch (type I) fibers of skeletal muscle. The MLC-Cre transgenic mouse can be used in conjunction with conditional alleles to study both developmental patterning and maintenance of fast fiber-type phenotypes.