Polygenic control of the wavy coat of the NCT mouse: involvement of an intracisternal A particle insertional mutation of the protease, serine 53 (Prss53) gene, and a modifier gene.

The Nakano cataract mouse (NCT) manifests a wavy coat for their first hair as a genetic trait. In this study, we explored the molecular genetic basis of the wavy coat. We revealed by crossing experiments that the wavy coat is controlled by a major gene on chromosome 7 of NCT, homozygosity of which is a prerequisite for developing the wavy coat, and by a gene on chromosome 9 with a minor effect to reinforce the manifestation of the trait. In humans, a polymorphism of the protease, serine 53 (PRSS53) gene on the homologous chromosome is known to be associated with curly scalp hair. We then investigated the Prss53 gene and discovered that NCT has an insertion of an intracisternal A particle element in the first intron of the gene. Nevertheless, the expression of the Prss53 is not altered in the NCT skin both in transcript and protein levels. Subsequently, we created C57BL/6J-Prss53em1 knockout mice and found that these mice manifest vague wavy coats. A portion of backcross and intercross mice between the C57BL/6J-Prss53em1 and NCT manifested intense or vague wavy coats. These findings demonstrate the polygenic nature of the wavy coat of NCT and Prss53 knockout mice and highlight the similarity of the trait to the curly hair of humans associated with the PRSS53 alteration.

Genomic properties of variably methylated retrotransposons in mouse.

BACKGROUND: Transposable elements (TEs) are enriched in cytosine methylation, preventing their mobility within the genome. We previously identified a genome-wide repertoire of candidate intracisternal A particle (IAP) TEs in mice that exhibit inter-individual variability in this methylation (VM-IAPs) with implications for genome function. RESULTS: Here we validate these metastable epialleles and discover a novel class that exhibit tissue specificity (tsVM-IAPs) in addition to those with uniform methylation in all tissues (constitutive- or cVM-IAPs); both types have the potential to regulate genes in cis. Screening for variable methylation at other TEs shows that this phenomenon is largely limited to IAPs, which are amongst the youngest and most active endogenous retroviruses. We identify sequences enriched within cVM-IAPs, but determine that these are not sufficient to confer epigenetic variability. CTCF is enriched at VM-IAPs with binding inversely correlated with DNA methylation. We uncover dynamic physical interactions between cVM-IAPs with low methylation ranges and other genomic loci, suggesting that VM-IAPs have the potential for long-range regulation. CONCLUSION: Our findings indicate that a recently evolved interplay between genetic sequence, CTCF binding, and DNA methylation at young TEs can result in inter-individual variability in transcriptional outcomes with implications for phenotypic variation.

Endogenous Retroviruses Walk a Fine Line between Priming and Silencing.

Endogenous retroviruses (ERVs) in mammals are closely related to infectious retroviruses and utilize host tRNAs as a primer for reverse transcription and replication, a hallmark of long terminal repeat (LTR) retroelements. Their dependency on tRNA makes these elements vulnerable to targeting by small RNAs derived from the 3'-end of mature tRNAs (3'-tRFs), which are highly expressed during epigenetic reprogramming and potentially protect many tissues in eukaryotes. Here, we review some key functions of ERV reprogramming during mouse and human development and discuss how small RNA-mediated silencing maintains genome stability when ERVs are temporarily released from heterochromatin repression. In particular, we take a closer look at the tRNA primer binding sites (PBS) of two highly active ERV families in mice and their sequence variation that is shaped by the conflict of successful tRNA priming for replication versus evasion of silencing by 3'-tRFs.

Locus-specific hypomethylation of the mouse IAP retrotransposon is associated with transcription factor-binding sites.

BACKGROUND: Intracisternal A particle (IAP) is one of the most transpositionally active retrotransposons in the mouse genome, but its expression varies between cell types. This variation is believed to arise from differences in the epigenetic state (e.g., DNA methylation) of the 5' long terminal repeat (LTR), where transcription starts. However, owing to the high copy number and high sequence similarity between copies, it is difficult to analyze the epigenetic states of individual IAP LTRs in a comprehensive manner. RESULTS: We have developed a method called Target Enrichment after Post-Bisulfite Adaptor Tagging (TEPBAT) to analyze the DNA methylation states of a large number of individual retrotransposon copies at once. Using this method, we determined the DNA methylation levels of >8500 copies of genomic IAP LTRs (almost all copies that we aimed to target by the PCR primers) in the sperm and tail. This revealed that the vast majority of the LTRs were heavily methylated in both sperm and tail; however, hypomethylated copies were more frequently found in the sperm than in the tail. Interestingly, most of these hypomethylated LTRs were solo-type, belonged to specific IAP subfamilies, and carried binding sites for transcription factors (TFs) that are active in male germ cells. CONCLUSIONS: The current study revealed subfamily- and locus-specific hypomethylation of IAP LTRs, and suggests that binding of TFs is involved in the protection from DNA methylation, whereas the IAP internal sequence enhances methylation. Furthermore, the study demonstrated that TEPBAT offers a cost-effective method for a variety of DNA methylome studies that focus on retrotransposon sequences.

Perinatal lead (Pb) exposure results in sex and tissue-dependent adult DNA methylation alterations in murine IAP transposons.

Epidemiological and animal data suggest that adult chronic disease is influenced by early-life exposure-induced changes to the epigenome. Previously, we observed that perinatal lead (Pb) exposure results in persistent murine metabolic- and activity-related effects. Using phylogenetic and DNA methylation analysis, we have also identified novel intracisternal A particle (IAP) retrotransposons exhibiting regions of variable methylation as candidate loci for environmental effects on the epigenome. Here, we now evaluate brain and kidney DNA methylation profiles of four representative IAPs in adult mice exposed to human physiologically relevant levels of Pb two weeks prior to mating through lactation. When IAPs across the genome were evaluated globally, average (sd) methylation levels were 92.84% (3.74) differing by tissue (P < 0.001), but not sex or dose. By contrast, the four individual IAPs displayed tissue-specific Pb and sex effects. Medium Pb-exposed mice had 3.86% less brain methylation at IAP 110 (P < 0.01), while high Pb-exposed mice had 2.83% less brain methylation at IAP 236 (P = 0.01) and 1.77% less at IAP 506 (P = 0.05). Individual IAP DNA methylation differed by sex for IAP 110 in the brain and kidney, IAP 236 in the kidney, and IAP 1259 in the kidney. Using Tomtom, we identified three binding motifs that matched to each of our novel IAPs impacted by Pb, one of which (HMGA2) has been linked to metabolic-related conditions in both mice and humans. Thus, these recently identified IAPs display tissue-specific environmental lability as well as sex-specific differences supporting an epigenetic link between early exposure to Pb and later-in-life health outcomes. Environ. Mol. Mutagen. 58:540-550, 2017. © 2017 Wiley Periodicals, Inc.

Zfp819, a novel KRAB-zinc finger protein, interacts with KAP1 and functions in genomic integrity maintenance of mouse embryonic stem cells.

Pluripotency is maintained by both known and unknown transcriptional regulatory networks. In the present study, we have identified Zfp819, a KRAB-zinc finger protein, as a novel pluripotency-related factor and characterized its role in pluripotent stem cells. We show that Zfp819 is expressed highly in various types of pluripotent stem cells but not in their differentiated counterparts. We identified the presence of non-canonical nuclear localization signals in particular zinc finger motifs and identified them as responsible for the nuclear localization of Zfp819. Analysis of the Zfp819 promoter region revealed the presence of a transcriptionally active chromatin signature. Moreover, we confirmed the binding of pluripotency-related factors, Oct4, Sox2, and Nanog to the distal promoter region of Zfp819, indicating that the expression of this gene is regulated by a pluripotency transcription factor network. We found that the expression of endogenous retroviral elements (ERVs) such as Intracisternal A Particle (IAP) retrotransposons, Long Interspersed Nuclear Elements (LINE1), and Short Interspersed Nuclear Elements (SINE B1) is significantly upregulated in Zfp819-knockdown (Zfp819_KD) cells. In line with the activation of ERVs, we observed the occurrence of spontaneous DNA damage in Zfp819_KD cells. Furthermore, we tested whether Zfp819 can interact with KAP1, a KRAB-associated protein with a transcriptional repression function, and found the interaction between these two proteins in both in vitro and in vivo experiments. The challenging of Zfp819_KD cells with DNA damaging agent revealed that these cells are inefficient in repairing the damaged DNA, as cells showed presence of γH2A.X foci for a prolonged time. Collectively, our study identified Zfp819 as a novel pluripotency-related factor and unveiled its function in genomic integrity maintenance mechanisms of mouse embryonic stem cells.