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-β-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.
After fertilization, the zygotic genome is activated through two phases, minor zygotic activation (ZGA) and major ZGA.
Recently, it was suggested that DUX is expressed during minor ZGA and activates some genes during major ZGA. However, it has not been proven that Dux is expressed during minor ZGA and functions to activate major ZGA genes, because there are several Dux paralogs that may be expressed in zygotes instead of Dux. In this study, we found that more than a dozen Dux paralogs, as well as Dux, are expressed during minor ZGA. Overexpression of some of these genes induced increased expression of major ZGA genes. These results suggest that multiple Dux paralogs are expressed to ensure a sufficient amount of functional Dux and its paralogs which are generated during a short period of minor ZGA with a low transcriptional activity. The mechanism by which multiple Dux paralogs are expressed is discussed.
Sequence-controlled degradable polymers with precisely placed breakable bonds in the main chain were synthesized by controlled alternating cationic copolymerization of vinyl ethers and aldehydes.
Objective• To analyse and then generalize the mechanism by which partial or complete response is achieved among a limited number of patients with metastatic renal cell carcinoma (RCC) treated with interferon or interleukin-2.
Materials and Methods• An expression library of RCC (clear-cell carcinoma) was screened using the sera of patients with metastatic RCC who benefited from partial or complete response to cytokine therapy, the postulation being that those remarkable responders obtained specific cellular immunity against RCC with the antibodies to react with the cancer antigen.• Peripheral blood mononuclear-cells (PBMCs) from healthy volunteers were stimulated with the antigen-derived peptides to induce specific cytotoxic T lymphocytes (CTLs). Specific activities of CTLs were measured by 51Cr-releasing assay.
Results• Among 15 positive clones isolated, two novel genes, galectin 9 and PINCH, were expressed at much higher levels in cancerous lesions than in normal tissues in all the patients with clear-cell carcinoma who were examined.• Both HLA-A*2402-restricted and HLA-A*0201-restricted CTLs were induced by each antigen-derived peptide to exhibit specific and highly cytotoxic activities towards RCC cells.• Specific CTLs were induced abundantly, as shown by flow cytometry analysis of the CTLs labelled with fluorescein isothiocyanate anti-CD107a and APC anti-CD8.• The clonal expansion of the CTLs was shown by the clonality of T-cell receptor Vβ repertoires.
Conclusion• A novel approach based on clinical observations yielded promising tumour antigens as immunotherapy targets of RCC.
Background
Deciphering the 3D structure of the genome is essential for elucidating the regulatory mechanisms of gene expression in detail. Existing methods, such as chromosome conformation capture (3C) and Hi-C have enabled the identification of novel aspects of chromatin structure. Further identification of protein-centric chromatin conformation is enabled by coupling the Hi-C procedure with a conventional chromatin immunoprecipitation assay. However, these methods are time-consuming and require independent methods for validation.
Results
To simultaneously identify protein-centric chromatin conformation and target protein localization, we have developed Cut-C, a method that combines antibody-mediated cleavage by tethered nuclease with chromosome conformation capture to identify chromatin interactions mediated by a protein of interest. Applying Cut-C to H3K4me3, a histone modification enriched at active gene promoters, we have successfully identified chromatin loops mediated by H3K4me3 along with the genome-wide distribution of H3K4me3. Cut-C also identified chromatin loops mediated by CTCF, validating the general applicability of the method.
Conclusions
Cut-C identifies protein-centric chromatin conformations along with the genome-wide distribution of target proteins using simple procedures. The simplified protocol will improve the efficiency of analysing chromatin conformation using precious materials, such as clinical samples.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5989-2) contains supplementary material, which is available to authorized users.
Abstract. Differentiated oocytes acquire totipotency through fertilization. During this transition, genome-wide chromatin remodeling occurs, which leads to change in gene expression. However, the mechanism that underlies this global change in chromatin structure has not been fully elucidated. Histone variants play a key role in defining chromatin structure and are implicated in inheritance of epigenetic information. In this study, we analyzed the nuclear localization and expression of H3.1 to elucidate the role of this histone variant in chromatin remodeling during oogenesis and preimplantation development. Analysis using Flag-tagged H3.1 transgenic mice revealed that Flag-H3.1 was not present in differentiated oocytes or early preimplantation embryos before the morula stage, although Flag-H3.1 mRNA was expressed at all stages examined. In addition, the expression levels of endogenous H3.1 genes were low at the stages where H3.1 was not present in chromatin. These results suggest that H3.1 is not incorporated into chromatin due to the inactivity of the histone chaperone and low mRNA expression level. The significance of the dynamics of H3.1 is evaluated in terms of chromatin remodeling that takes place during development.
The pericentromeric heterochromatin of one-cell embryos forms a unique, ring-like structure around the nucleolar precursor body, which is absent in somatic cells. Here, we found that the histone H3 variants H3.1 and/or H3.2 (H3.1/H3.2) were localized asymmetrically between the male and female perinucleolar regions of the one-cell embryos; moreover, asymmetrical histone localization influenced DNA replication timing. The nuclear deposition of H3.1/3.2 in one-cell embryos was low relative to other preimplantation stages because of reduced H3.1/3.2 mRNA expression and incorporation efficiency. The forced incorporation of H3.1/3.2 into the pronuclei of one-cell embryos triggered a delay in DNA replication, leading to developmental failure. Methylation of lysine residue 27 (H3K27me3) of the deposited H3.1/3.2 in the paternal perinucleolar region caused this delay in DNA replication. These results suggest that reduced H3.1/3.2 in the paternal perinucleolar region is essential for controlled DNA replication and preimplantation development. The nuclear deposition of H3.1/3.2 is presumably maintained at a low level to avoid the detrimental effect of K27me3 methylation on DNA replication in the paternal perinucleolar region.
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