Involvement of the linker histone H1Foo in the regulation of oogenesis.

In brief: In oocytes, chromatin structure is loosened during their growth, which seems to be essential for the establishment of competence to accomplish the maturation and further development after fertilization. This paper shows that a linker histone variant, H1foo, is involved in the formation of loosened chromatin structure in growing oocytes. Abstract: During oogenesis, oocytes show a unique mode of division and gene expression patterns. Chromatin structure is thought to be involved in the regulation of these processes. In this study, we investigated the functions of linker histones, which modulate higher-order chromatin structure during oogenesis. Because H1foo is highly expressed in oocytes, we knocked down H1foo using siRNA and observed oocyte growth, maturation, and fertilization. However, H1foo knockdown had no effect on any of these processes. Overexpression of H1b or H1d, which has a high ability to condense chromatin and is expressed at a low level in oocytes, resulting in tightened chromatin and a decreased success rate of oocyte maturation. By contrast, overexpression of H1a, which is expressed at a high level in oocytes and has a low ability to compact chromatin, did not affect growth or maturation. Therefore, H1a, but not other variants, might compensate for the function of H1foo in H1foo-knockdown oocytes. These results implicate H1foo in the formation of loose chromatin structure, which is necessary for oocyte maturation. In addition, the low expression of somatic linker histone variants, for example, H1b and H1d, is important for loosened chromatin and meiotic progression.

Minor zygotic gene activation is essential for mouse preimplantation development.

In mice, transcription initiates at the mid-one-cell stage and transcriptional activity dramatically increases during the two-cell stage, a process called zygotic gene activation (ZGA). Associated with ZGA is a marked change in the pattern of gene expression that occurs after the second round of DNA replication. To distinguish ZGA before and after the second-round DNA replication, the former and latter are called minor and major ZGA, respectively. Although major ZGA are required for development beyond the two-cell stage, the function of minor ZGA is not well understood. Transiently inhibiting minor ZGA with 5, 6-dichloro-1-ß-d-ribofuranosyl-benzimidazole (DRB) resulted in the majority of embryos arresting at the two-cell stage and retention of the H3K4me3 mark that normally decreases. After release from DRB, at which time major ZGA normally occurred, transcription initiated with characteristics of minor ZGA but not major ZGA, although degradation of maternal mRNA normally occurred. Thus, ZGA occurs sequentially starting with minor ZGA that is critical for the maternal-to-zygotic transition.

The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3' processing.

Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3' processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos.

Characterization of gene expression in mouse embryos at the 1-cell stage.

In mice, transcription from the zygotic genome is initiated at the mid-1-cell stage after fertilization. Although a recent high-throughput sequencing (HTS) analysis revealed that this transcription occurs promiscuously throughout almost the entire genome in 1-cell stage embryos, a detailed investigation of this process has yet to be conducted using protein-coding genes. Thus, the present study utilized previous RNA sequencing (RNAseq) data to determine the characteristics and regulatory regions of genes transcribed at the 1-cell stage. While the expression patterns of protein-coding genes of mouse embryos were very different at the 1-cell stage than at other stages and in various tissues, an analysis for the upstream and downstream regions of actively expressed genes did not reveal any elements that were specific to 1-cell stage embryos. Therefore, the unique gene expression pattern observed at the 1-cell stage in mouse embryos appears to be governed by mechanisms independent of a specific promoter element.

Distinct patterns of RNA polymerase II and transcriptional elongation characterize mammalian genome activation.

How transcription is regulated as development commences is fundamental to understand how the transcriptionally silent mature gametes are reprogrammed. The embryonic genome is activated for the first time during zygotic genome activation (ZGA). How RNA polymerase II (Pol II) and productive elongation are regulated during this process remains elusive. Here, we generate genome-wide maps of Serine 5 and Serine 2-phosphorylated Pol II during and after ZGA in mouse embryos. We find that both phosphorylated Pol II forms display similar distributions across genes during ZGA, with typical elongation enrichment of Pol II emerging after ZGA. Serine 2-phosphorylated Pol II occurs at genes prior to their activation, suggesting that Serine 2 phosphorylation may prime gene expression. Functional perturbations demonstrate that CDK9 and SPT5 are major ZGA regulators and that SPT5 prevents precocious activation of some genes. Overall, our work sheds molecular insights into transcriptional regulation at the beginning of mammalian development.

Identification of the small molecule compound which induces hepatic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) are expected to have utility as a cell source in regenerative medicine. Because we previously reported that suppression of the Wnt/ß-catenin signal enhances hepatic differentiation of human MSCs, we synthesized twenty-three derivatives of small molecule compounds originally reported to suppress the Wnt/ß-catenin signal in human colorectal cancer cells. We then screened these compounds for their ability to induce hepatic differentiation of human UE7T-13 MSCs. After screening using WST assay, TCF reporter assay, and albumin mRNA expression, IC-2, a derivative of ICG-001, was identified as a potent inducer of hepatic differentiation of human MSCs. IC-2 potently induced the expression of albumin, complement C3, tryptophan 2,3-dioxygenase (TDO2), EpCAM, C/EBPα, glycogen storage, and urea production. Furthermore, we examined the effects of IC-2 on human bone marrow mononuclear cell fractions sorted according to CD90 and CD271 expression. Consequently, CD90+ CD271+ cells were found to induce the highest production of urea and glycogen, important hepatocyte functions, in response to IC-2 treatment. CD90+ CD271+ cells also highly expressed albumin mRNA. As the CD90+ CD271+ population has been reported to contain a rich fraction of MSCs, IC-2 apparently represents a potent inducer of hepatic differentiation of human MSCs.

Efficient phase conjugation by pico-second four-wave-mixing in solid-dye amplifier.

We demonstrate 2.5 times phase conjugation by four-wave-mixing with the use of pico-second pulses in a rhodamine6G dye polymer amplifier. Phase conjugation pulse shortening by a factor of 2 is also measured.

Global gene silencing is caused by the dissociation of RNA polymerase II from DNA in mouse oocytes.

As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 µm). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP II from the DNA. We also observed dissociation of RPB1 from the DNA in full-grown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.

Expression of c-MYC in nuclear speckles during mouse oocyte growth and preimplantation development.

Myelocytomatosis oncogene (c-myc) is a major transcriptional regulator that controls various biological processes, and its deregulated expression causes carcinogenesis. To investigate the involvement of c-myc in oogenesis and preimplantation development, the expression of c-MYC during these stages was examined by immunocytochemistry. A strong c-MYC signal was detected in the nucleus of growing and fully grown oocytes as well as in preimplantation embryos before the morula stage. The signal intensity decreased slightly at the morula stage, and no signal was detected in blastocysts. Close observation of the nucleus revealed that c-MYC was localized in small granules that appeared to be nuclear speckles controlling pre-mRNA splicing. Although the number of granules decreased during oocyte growth, their size increased. After fertilization, the granules of c-MYC disappeared from the pronuclei, and c-MYC was evenly distributed in the nucleoplasm at the 1-cell stage, but the granules reappeared at the 2-cell stage. These results suggest that c-myc is involved in oocyte growth and preimplantation development and that its role changes during these stages.