Project description:The integrity of chromatin, which provides a dynamic template for all DNA related processes in eukaryotes, is maintained through replication dependent and independent assembly pathways. To address the role of replication independent histone deposition, we deleted the histone H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is highly dynamic, and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and, ultimately, a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. In contrast to the only minor transcriptional phenotype observed in mouse pluripotent cells, we unequivocally show the importance of histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in an in vivo context. RNA-Seq on 4 Hiraf/f and 4 Hiraf/f, Gdf9-Cre+ single MII oocytes
Project description:The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of replication-independent histone deposition, we deleted the histone H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is highly dynamic, and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and, ultimately, a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. In contrast to the only minor transcriptional phenotype observed in mouse pluripotent cells, we unequivocally show the importance of histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in an in vivo context. RNA-Seq on 4 Hiraf/f and 4 Hiraf/f, Zp3-Cre single MII oocytes.
Project description:To investigate the physiological function of TCF12 in oocyte maturation and preimplantational development, we crossed Tcf12fl/fl mice with Gdf9-Cre mice to specifically delete Tcf12 from the primordial follicle stage. We then performed gene expression profiling analysis using data obtained from RNA-seq on Tcf12fl/fl and Tcf12fl/fl;Gdf9-Cre growing GV oocytes (gGO), fully grown GV oocytes (fGO), embryos at 2-cell and 4-cell stage.
Project description:The goal of this study is to identify the differentially expressed genes in Zp3-Cre mediated Mtor oocyte-specific knockout (CKO) Metaphase II (MII) oocytes by comparing the transcriptomes of Wild-Type (WT) and CKO Mouse MII oocytes via RNA-Seq Analysis. Methods: mRNA profiles of 3 sets of super-ovulated MII oocytes collected from 24-day old WT- and Mtor CKO female mice were generated by deep sequencing using an Illumina Hiseq2500 platform with 41bp single read. All RNA-seq reads passed filter were trimmed to remove low-quality bases and adaptor sequences. Reads then were aligned to the mm10 reference genome using tophat2 (v2.0.8b). Reads aligned to genes are counted by cufflinks (v2.1.1). The FPKMs are normalized usingcuffnorm. Differentially expressed genes are calculated using cuffdiff.
Project description:HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. HIRA is essential for mouse development, as the standard knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigated the effect of Hira KO on hematopoiesis using Vav-Cre Loxp system. We show that Hira KO dramatically reduces bone marrow LSK cells, resulting in anemia, thrombopenia and severe, combined immunodeficiency. To investigate the molecular mechanisms, RNA-seq and ATAC-Seq were performed using LSK cells isolated from WT and Hira conditional KO mice.
Project description:The goal of this study is to identify the differentially expressed genes in Gdf9-Cre and Zp3-Cre mediated Mtor oocyte-specific knockout (CKO) GV-stage fully-grown oocytes (FGO) by comparing their transcriptomes with that of the wild-type (WT) via RNA-Seq Analysis.
Project description:We performed mouse single oocyte RNA-seq and bulk oocyte CUT&Tag assays in the current project. In details, SMART-seq based single oocyte RNA-seq was performed at adult GV stage, GV3h stage and MII stage, using control and Dis3 oocyte-speicfic knockout (cKO) oocytes. RiboMinus-seq based single oocyte RNA-seq was performed at adult GV stage using control and Dis3 cKO oocytes, and at p20 GV stage using control, Dis3 cKO, Exosc10 cKO and Dis3/Exosc10 double cKO (dcKO) oocytes. Bulk CUT&Tag of anti-H3K27me3 was done in WT and Dis3 cKO oocytes at adult GV stage, and in WT and Dis3/Exosc10 dcKO oocytes at p20 stage. In addition, CUT&Tag of anti-RNA polymerase II (Ser2+Ser5) was performed in WT and Dis3 cKO oocytes at GV stage. All CUT&Tag experiments share the same rabbit-Igg negative control. All p20 stage oocytes were specified as p20. The non-specified GV oocytes were all adult GV oocytes.
