Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

Genes Encoding Teleost Orthologs of Human Haploinsufficient and Monoallelically Expressed Genes Remain in Duplicate More Frequently Than the Whole Genome

Gene dosage is an important issue both in cell and evolutionary biology. Most genes are present in two copies or alleles in diploid eukariotic cells. The most outstanding exception is monoallelic gene expression (MA) that concerns genes localized on the X chromosome or in regions undergoing parental imprinting in eutherians, and many other genes scattered throughout the genome. In diploids, haploinsufficiency (HI) implies that a single functional copy of a gene in a diploid organism is insufficient to ensure a normal biological function. One of the most important mechanisms ensuring functional innovation during evolution is whole genome duplication (WGD). In addition to the two WGDs that have occurred in vertebrate genomes, the teleost genomes underwent an additional WGD, after their divergence from tetrapods. In the present work, we have studied on 57 teleost species whether the orthologs of human MA or HI genes remain more frequently in duplicates or returned more frequently in singleton than the rest of the genome. Our results show that the teleost orthologs of HI human genes remained more frequently in duplicate than the rest of the genome in all of the teleost species studied. No signal was observed for the orthologs of genes mapping to the human X chromosome or subjected to parental imprinting. Surprisingly, the teleost orthologs of the other human MA genes remained in duplicate more frequently than the rest of the genome for most teleost species. These results suggest that the teleost orthologs of MA and HI human genes also undergo selective pressures either related to absolute protein amounts and/or of dosage balance issues. However, these constraints seem to be different for MA genes in teleost in comparison with human genomes.

Meiotic Drive in Chronic Lymphocytic Leukemia compared with other Malignant Blood Disorders

Studies of families with two or more cases of malignant blood disorders (lympoproliferative and/or myeloproliferative) provide a description of the pathway of susceptibility down through the generations towards the proband. The united observations fit into a non-Mendelian operational model consisting of parental genomic imprinting combined with feto-maternal microchimerism. Male affected relatives of a proband are predominant in paternal lines with maternal imprinting, while female affected relatives are predominant in lines with maternal affiliation and paternal imprinting. The findings suggest the influence of a so-called polymorphic equilibrium with segregation distortion related to parental imprinting (fitness optimalization). In the generations before a proband, affected relatives with the same diagnosis may covariate, viz. be present with a higher frequency than expected (relative superiority); or contravariate, that is a lower frequency than expected (mutual minority). Covariation has been observed especially among affected relatives with multiple myeloma, diffuse large B-cell lymphoma, acute myeloid leukemia, Hodgkin’s lymphoma and some few other diagnoses. Contravariation is only seen among affected relatives with chronic lymphocytic leukemia. The dynamic drive of susceptibility in an affected family with birth order effect and/or anticipation is regarded as an additional polymorphic equilibrium with segregation distortion caused by feto-maternal microchimerism.

Genes encoding teleost orthologs of human haplo-insufficient and monoallelic genes remain in duplicate more frequently than the whole genome

Gene dosage is important is an important issue both in cell and evolutionary biology. Most genes are present in two copies in eukaryotic cells. The first outstanding exception is monoallelic gene expression (MA) that concerns genes localized on the X chromosome or in regions undergoing parental imprinting in eutherians, and many other genes scattered throughout the genome. The second exception concerns haploinsufficiency (HI), responsible for the fact that a single functional copy of a gene in a diploid organism is insufficient to ensure a normal biological function. One of the most important mechanisms ensuring functional innovation during evolution is Whole genome duplication (WGD). In addition to the two WGDs that have occurred in vertebrate genomes, the teleost genomes underwent an additional WGD, after their divergence from tetrapod. In the present work, we have studied on 57 teleost species whether the orthologs of human MA or HI genes remain more frequently in duplicates or returned more frequently in singleton than the rest of the genome. Our results show that the teleost orthologs of HI human genes remained more frequently in duplicate than the rest of the genome in all the teleost species studied. No signal was observed for the orthologs of genes localized on the human X chromosome or subjected to parental imprinting. Surprisingly, the teleost orthologs of the other human MA genes remained in duplicate more frequently than the rest of the genome for most teleost species. These results suggest that the teleost orthologs of MA and HI human genes also undergo selective pressures either related to absolute protein amounts and/or of dosage balance issues. However, these constraints seem to be different for MA genes in teleost in comparison with human genomes.

The insulin-like growth factor 2 gene and locus in nonmammalian vertebrates: Organizational simplicity with duplication but limited divergence in fish

The small, secreted peptide, insulin-like growth factor 2 (IGF2), is essential for fetal and prenatal growth in humans and other mammals. Human IGF2 and mouse Igf2 genes are located within a conserved linkage group and are regulated by parental imprinting, with IGF2/Igf2 being expressed from the paternally derived chromosome, and H19 from the maternal chromosome. Here, data retrieved from genomic and gene expression repositories were used to examine the Igf2 gene and locus in 8 terrestrial vertebrates, 11 ray-finned fish, and 1 lobe-finned fish representing >500 million years of evolutionary diversification. The analysis revealed that vertebrate Igf2 genes are simpler than their mammalian counterparts, having fewer exons and lacking multiple gene promoters. Igf2 genes are conserved among these species, especially in protein-coding regions, and IGF2 proteins also are conserved, although less so in fish than in terrestrial vertebrates. The Igf2 locus in terrestrial vertebrates shares additional genes with its mammalian counterparts, including tyrosine hydroxylase (Th), insulin (Ins), mitochondrial ribosomal protein L23 (Mrpl23), and troponin T3, fast skeletal type (Tnnt3), and both Th and Mrpl23 are present in the Igf2 locus in fish. Taken together, these observations support the idea that a recognizable Igf2 was present in the earliest vertebrate ancestors, but that other features developed and diversified in the gene and locus with speciation, especially in mammals. This study also highlights the need for correcting inaccuracies in genome databases to maximize our ability to accurately assess contributions of individual genes and multigene families toward evolution, physiology, and disease.

Similarity and variation in the insulin-like growth factor 2 - H19 locus in primates

Insulin-like growth factor 2 (IGF2), a small, secreted protein, is critical for fetal and prenatal growth in humans and other mammals. The IGF2 gene and its mouse homolog comprise part of a conserved linkage group that is regulated by parental imprinting, with IGF2/ Igf2 being expressed from the paternal chromosome, and the adjacent H19 gene from the maternal chromosome. By using information extracted from public genomic and gene expression databases, I have now analyzed this locus in nine nonhuman primate species representing over 60 million years of evolutionary divergence from a common progenitor. Both IGF2 and H19 genes and the entire locus have been conserved among these primates. Each primate IGF2 gene except for gibbon and marmoset is composed of 10 exons and contains five potential promoters, each with distinctive 5′-untranslated exons. Similarly, except for marmoset and mouse lemur, H19 consists of six exons and has two promoters. DNA sequence conservation is high, not only in orthologous exons and promoters, but also in a putative imprinting control region located 5′ to H19 and in multiple potential distal enhancer elements found 3′ to H19. Collectively, these results support the hypothesis that common regulatory processes shaped the IGF2 - H19 locus before the onset of primate speciation more than 85 million years ago. This study also leads to the conclusion that inaccuracies in data presentation in genetic repositories could limit our ability to develop novel insights about roles of individual genes and multigene loci in mammalian physiology and disease.

The role of DNA methylation: a challenge for the DOHaD paradigm in going beyond the historical debate

A heritage of considerable research into such phenomena as parental imprinting and carcinogenesis is an almost axiomatic association of the DNA methylation epigenetic mark with the silencing of gene expression. However, the increasing technical resolution afforded by burgeoning -omics technologies reveals that a more elaborate interaction may exist between DNA methylation, within sub-regions of gene structure and/or specific dinucleotide sites, and levels of gene activity. Furthermore, seminal observations from the field of DOHaD, which clearly define the alignment of sequential epigenetic modifications and gene activity appear not to support a strictly causal relationship between DNA methylation and gene silencing. The temporal element implicit within DOHaD studies provides a useful framework within which to further explore the role of epigenetic mechanisms and in particular perhaps, to address the question of ‘deterministic intent’ when implicating the epigenetic regulation of gene activity in the manifestation of phenotype.