Sequence divergence and retrotransposon insertion underlie interspecific epigenetic differences in primates.

Changes in the epigenome can affect the phenotype without the presence of changes in the genomic sequence. Given the high identity of the human and chimpanzee genome sequences, a substantial portion of their phenotypic divergence likely arises from epigenomic differences between the two species. In this study, the transcriptome and epigenome were determined for induced pluripotent stem cells (iPSCs) generated from human and chimpanzee individuals. The transcriptome and epigenomes for trimethylated histone H3 at lysine-4 (H3K4me3) and lysine-27 (H3K27me3) showed high levels of similarity between the two species. However, there were some differences in histone modifications. Although such regions, in general, did not show significant enrichment of interspecies nucleotide variations, gains in binding motifs for pluripotency-related transcription factors, especially POU5F1 and SOX2, were frequently found in species-specific H3K4me3 regions. We also revealed that species-specific insertions of retrotransposons, including the LTR5_Hs subfamily in human and a newly identified LTR5_Pt subfamily in chimpanzee, created species-specific H3K4me3 regions associated with increased expression of nearby genes. Human iPSCs have more species-specific H3K27me3 regions, resulting in more abundant bivalent domains. Only a limited number of these species-specific H3K4me3 and H3K27me3 regions overlap with species-biased enhancers in cranial neural crest cells, suggesting that differences in the epigenetic state of developmental enhancers appear late in development. Therefore, iPSCs serve as a suitable starting material for studying evolutionary changes in epigenome dynamics during development.

Conical Ionic Amphiphiles Endowed with Micellization Ability but Lacking Air-Water and Oil-Water Interfacial Activity.

Micellization in water and reduction of the surface tension at water interfaces with air and oil are two archetypical properties of surfactants, caused by self-aggregation and Gibbs monolayer formation at the interfaces, respectively. We present here a new type of amphiphiles that possess a conical shape consisting of a hydrophobic apex and five ionic termini at the base of the cone. The conical shape and the high charge density cooperatively impede monolayer formation at the interfaces, hence preventing foaming and emulsification. On the other hand, the conical shape strongly assists micelle formation in water and hemimicelle formation on a solid surface to promote dissolution of nanoparticles such as magnetic nanoparticles and nanocarbons in water. The well-defined shape and charge locations distinguish the new amphiphiles from known polymer amphiphiles that show similar surface activity.

Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes.

Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.

Three novel ZBTB24 mutations identified in Japanese and Cape Verdean type 2 ICF syndrome patients.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that shows DNA hypomethylation at pericentromeric satellite-2 and -3 repeats in chromosomes 1, 9 and 16. ICF syndrome is classified into two groups: type 1 (ICF1) patients have mutations in the DNMT3B gene and about half of type 2 (ICF2) patients have mutations in the ZBTB24 gene. Besides satellite-2 and -3 repeats, α-satellite repeats are also hypomethylated in ICF2. In this study, we report three novel ZBTB24 mutations in ICF2. A Japanese patient was homozygous for a missense mutation (C383Y), and a Cape Verdean patient was compound heterozygous for a nonsense mutation (K263X) and a frame-shift mutation (C327W fsX54). In addition, the second Japanese patient was homozygous for a previously reported nonsense mutation (R320X). The C383Y mutation abolished a C2H2 motif in one of the eight zinc-finger domains, and the other three mutations caused a complete or large loss of the zinc-finger domains. Our immunofluorescence analysis revealed that mouse Zbtb24 proteins possessing a mutation corresponding to either C383Y or R320X are mislocalized from pericentrometic heterochromatin, suggesting the importance of the zinc-finger domains in proper intranuclear localization of this protein. We further revealed that the proper localization of wild-type Zbtb24 protein does not require DNA methylation.

Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development.

Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. Here, we show that the homeoprotein Nkx2-5 and the 5'-3' exonuclease Xrn2 are involved in the regulation of alternative polyadenylation (APA) during mouse heart development. Nkx2-5 occupied not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affected Xrn2 binding to target loci and resulted in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3'untranslated regions (3' UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 led to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5(+/-)Xrn2(+/-), but neither Nkx2-5(+/-)nor Xrn2(+/-), newborns exhibited a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development. Our findings suggest that tissue-specific transcription factors is involved in the regulation of APA.

Mutations in CDCA7 and HELLS cause immunodeficiency-centromeric instability-facial anomalies syndrome.

The life-threatening Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome is a genetically heterogeneous autosomal recessive disorder. Twenty percent of patients cannot be explained by mutations in the known ICF genes DNA methyltransferase 3B or zinc-finger and BTB domain containing 24. Here we report mutations in the cell division cycle associated 7 and the helicase, lymphoid-specific genes in 10 unexplained ICF cases. Our data highlight the genetic heterogeneity of ICF syndrome; however, they provide evidence that all genes act in common or converging pathways leading to the ICF phenotype.

Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology.

BACKGROUND: Immunodeficiency Centromeric Instability and Facial anomalies (ICF) is a rare autosomal recessive disease characterized by reduction in serum immunoglobulins with severe recurrent infections, facial dysmorphism, and more variable symptoms including mental retardation. ICF is directly related to a genomic methylation defect that mainly affects juxtacentromeric heterochromatin regions of certain chromosomes, leading to chromosomal rearrangements that constitute a hallmark of this syndrome upon cytogenetic testing. Mutations in the de novo DNA methyltransferase DNMT3B, the protein ZBTB24 of unknown function, or loci that remain to be identified, lie at its origin. Despite unifying features, common or distinguishing molecular signatures are still missing for this disease. METHOD: We used the molecular signature that we identified in a mouse model for ICF1 to establish transcriptional biomarkers to facilitate diagnosis and understanding of etiology of the disease. We assayed the expression and methylation status of a set of genes whose expression is normally restricted to germ cells, directly in whole blood samples and epithelial cells of ICF patients. RESULTS: We report that DNA hypomethylation and expression of MAEL and SYCE1 represent robust biomarkers, easily testable directly from uncultured cells to diagnose the most prevalent sub-type of the syndrome. In addition, we identified the first unifying molecular signatures for ICF patients. Of importance, we validated the use of our biomarkers to diagnose a baby born to a family with a sick child. Finally, our analysis revealed unsuspected complex molecular signatures in two ICF patients suggestive of a novel genetic etiology for the disease. CONCLUSIONS: Early diagnosis of ICF syndrome is crucial since early immunoglobulin supplementation can improve the course of disease. However, ICF is probably underdiagnosed, especially in patients that present with incomplete phenotype or born to families with no affected relatives. The specific and robust biomarkers identified in this study could be introduced into routine clinical immunology or neurology departments to facilitate testing of patients with suspected ICF syndrome. In addition, as exemplified by two patients with a combination of molecular defects never described before, our data support the search for new types of mutations at the origin of ICF syndrome.