Role of UHRF1 in de novo DNA methylation in oocytes and maintenance methylation in preimplantation embryos

The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos

Role of UHRF1 in de novo DNA methylation in oocytes and maintenance methylation in preimplantation embryos.

The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos

Multiple malignant epithelioid mesotheliomas of the liver and greater omentum: a case report and review of the literature

Abstract Background Malignant mesothelioma commonly arises from the pleura, but can also arise from the peritoneum, pericardium, and tunica vaginalis testis. However, malignant mesothelioma of the liver is extremely rare and coexistence with malignant mesothelioma of the greater omentum has not been described in the literature. In this case report, we present a case of multiple malignant mesothelioma of the liver and greater omentum. Case presentation A 36-year-old woman was admitted to our hospital for the evaluation of an elastic hard mass in the right upper abdomen. Abdominal contrast computed tomography showed a cystic mass measuring 13 × 14 × 11 cm in the right liver lobe with enhanced mural nodule. Abnormal accumulation was identified in the liver and lower abdominal area on 18F-fluorodeoxyglucose positron emission tomography. The patient underwent hepatectomy of the posterior segment and partial resection of the omentum. The final pathological diagnosis was low-grade multiple malignant epithelioid mesothelioma based on characteristic immunohistochemical findings. As of 6 months postoperatively, the patient has shown no disease recurrence. Conclusions We present the first case of a 36-year-old woman with multiple malignant mesothelioma of the liver and greater omentum

The role of maternal UHRF1 in CG maintenance methylation in preimplantation embryos.

<p>(A) Violin plots showing the distribution of the 10-kb genomic regions with different CG methylation levels in control [<i>Uhrf1</i>^2lox/+], <i>Uhrf1</i> mat-KO, and <i>Dnmt1</i> mat-KO blastocysts. The horizontal bars indicate the mean CG methylation levels. (B) The genomic distribution of CG methylation across chromosome 14 in control and mat-KO blastocysts. The CG methylation levels of 1-Mb windows are shown. GC contents, RefSeq genes, and CGIs are shown at the bottom. (C) The correlations between the CG methylation levels of the 10-kb genomic windows in the control and mat-KO blastocysts. (D) The CG methylation levels of respective portions of the genic regions, intergenic regions, and various repetitive sequences in control and mat-KO blastocysts. (E) The CG methylation levels of the ICRs in control and mat-KO blastocysts.</p

Dependence of nuclear localization of DNMT1 on UHRF1.

<p>(A,B) Immunostaining of control [<i>Uhrf1</i>^2lox/2lox or <i>Dnmt1</i>^2lox/2lox], <i>Uhrf1</i> KO, and <i>Dnmt1</i> KO FGOs (A) and control [<i>Uhrf1</i>^2lox/+ or <i>Dnmt1</i>^2lox/+], <i>Uhrf1</i> mat-KO, and <i>Dnmt1</i> mat-KO 2-cell embryos (B) was performed with anti-UHRF1 antibodies (green) or anti-DNMT1 antibodies (red). The nuclear/cytoplasmic signal ratios (N/C) were calculated using the mean signal intensities obtained by linear scanning across the cells. Each experiment was done with at least two batches of oocytes/embryos, and the total number (n) of oocytes/embryos is indicated. Each box indicates the 25–75 percentile and the bar in each box indicates the median. Asterisk, <i>P</i> <0.01 (Mann-Whitney <i>U</i> test).</p

UHRF1 facilitates <i>de novo</i> methylation in inactive regions during late oocyte growth.

<p>(A) The classification of the 10-kb genomic regions that were moderately (40–80%) or highly (≥80%) methylated in the control FGOs, based on the extent of methylation change (≥20% or not) in <i>Uhrf1</i> KO FGOs (Groups 1–4). The right panel shows the proportion of moderately and highly methylated 10-kb regions belonging to each group. (B) The distribution of the transcript levels (RPKM) and H3K36me3 enrichment levels [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007042#pgen.1007042.ref020" target="_blank">20</a>] (corrected read count) of the 10-kb regions belonging to each group. The bar in each box indicates the median value. (C) Differences in CG methylation across the transcribed regions between control and <i>Uhrf1</i> KO FGOs. The transcribed regions were classified into four categories based on the expression levels (RPKM) in control FGOs and the results for the respective groups are shown separately. (D) The changes in CG methylation occurring in the three different stages of oocyte growth were determined for the 10-kb regions of the respective groups.</p

The role of UHRF1 in <i>de novo</i> CG methylation during oocyte growth.

<p>(A) Violin plots showing the distribution of the 10-kb genomic regions with different CG methylation levels in NGOs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007042#pgen.1007042.ref005" target="_blank">5</a>], GOs (P12 and P15) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007042#pgen.1007042.ref053" target="_blank">53</a>], and control [<i>Uhrf1</i>^2lox/2lox], <i>Dnmt1</i> KO, and <i>Uhrf1</i> KO FGOs. The horizontal bars indicate the mean CG methylation levels. (B) The genomic distribution of CG methylation across chromosome 14 in control and KO FGOs. The CG methylation levels of the 1-Mb windows are shown. GC contents, RefSeq genes, and CGIs are shown at the bottom. (C) The correlations between the CG methylation levels of the 10-kb genomic windows in control and KO FGOs. (D) The CG methylation levels of respective portions of the genic regions, intergenic regions, and various repetitive sequences in control and respective KO FGOs. (E) The CG methylation levels of the ICRs in control and KO FGOs.</p

Expression and subcellular localization of UHRF1 in oocytes and preimplantation embryos.

<p>(A,B) Immunostaining of C57BL/6J developing oocytes (A), zygote to blastocyst stage embryos (B), <i>Uhrf1</i> KO oocytes (C), and <i>Uhrf1</i> mat-KO embryos (D) performed with an anti-UHRF1 antibody (Th-10a [green]). Each experiment was done with at least two batches of oocytes/embryos, and the total number of oocytes/embryos examined at each stage exceeded 20. The cell nucleus was counterstained with DAPI (blue). NGO, <20 μm; GO, 20–50 μm; FGO, >70 μm. Scale bar, 20 μm.</p