Abstract
Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.
Highlights
Our results suggest that the Notably, Zscan[4] expression is accompanied by absence of the functional full-length Rif[1] due to upregulation of a group of genes including Tcstv[1] its instability or alternative splicing and potential and Usp17l, endogenous retrovirus (ERV) and inactivation of Rif[1] through dominant inhibition non-coding RNA of major satellite, all of which by N-terminally truncated Rif[1] polypeptides, is transcribed in embryos at 2-cell may be involved in 2C-specific transcription stage (9)
Rif[1] depletion alters were fractionated into the soluble and the insoluble profiles are diversely different among species and cell types
Most of the short forms in 2-cell genes and non-coding transcripts are likely embryo were detected in the soluble fraction, restricted to mouse embryonic stem (ES) cells and not appreciable in other types of cells ((3,4,20); our unpublished which suppress broad targets, and the depletion of result in HCT116 cells)
Summary
Rif[1] is dispensable for self-renewal and caused by non-specific protein degradation maintenance of undifferentiated state of ES during sample preparation, since other proteins, cells, but is required for regulation of Zscan4-. Journal Pre-proof plasmid DNA containing Rif[1] cDNA full-length and the N-terminal coding regions were highly unstable in bacterial cells for unknown reasons, transformation and culture has been carried out at 25 to 30 °C in XL10-Gold (Stratagene) and expanded at least 3 clones per constructs. For bacterial expression of GST-fused mouse Rif[1] N206 aa protein, N-terminal Rif[1] coding region (AY428571) was amplified by PCR with mRif1_N_BamHI and mRif1_R1137_XhoI primers (shown in Table S2) using pBluescript II SK(+)-full length Rif[1] cDNA derived from R1 mouse ES cells Microarray data are available in GEO with accession number GSE158220
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