Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development

Abstract

Eggs and oocytes have a remarkable ability to induce transcription of sperm after normal fertilization and in somatic nuclei after somatic cell nuclear transfer. This ability of eggs and oocytes is essential for normal development. Nuclear actin and actin-binding proteins have been shown to contribute to transcription, although their mode of action is elusive. Here, we find that Xenopus Wave1, previously characterized as a protein involved in actin cytoskeleton organization, is present in the oocyte nucleus and is required for efficient transcriptional reprogramming. Moreover, Wave1 knockdown in embryos results in abnormal development and defective hox gene activation. Nuclear Wave1 binds by its WHD domain to active transcription components, and this binding contributes to the action of RNA polymerase II. We identify Wave1 as a maternal reprogramming factor that also has a necessary role in gene activation in development.

Fig. 1. Wave1 is present in the Xenopus oocyte nuclei and transplanted mouse nuclei

(A) Western blot analysis revealed that Wave1 is accumulated in the GV of the Xenopus oocyte. (B) Mouse C2C12 nuclei were injected into the GV overexpressing HA-NLS-Wave1 (fig. S1A). Immunofluorescence analysis was performed 24 hours after NT. HA-NLS-Wave1 localization was visualized using antibody against HA (αHA). DAPI, 4′,6′-diamidino-2-phenylindole. Control staining is shown in fig. S3.

Fig. 2. Nuclear Wave1 is required for transcriptional reprogramming in oocytes

All error bars represent SEM. *P < 0.05. (A) Injection of antibody against Wave1 into GVs inhibits the transcriptional activation of Oct4 in NT oocytes (n = 12). Immunoglobulin G was injected as control (n = 13). Relative fold increases of gene transcripts to control NT were measured by QPCR. (B) Three transcript variants expressed in Xenopus oocytes were identified (A, B, and C), and each protein domain is represented by different colors. (C) Knockdown of endogenous Wave1 proteins in the GV by antisense oligonucleotide injection (AS) as checked by Western blot. SC control means scrambled oligonucleotides–injected control. Cyto represents the oocyte cytoplasm. (D) Knockdown of Wave1 by AS inhibits transcriptional activation of many embryonic genes in NT oocytes (blue bars). n = 6.

Fig. 3. Nuclear Wave1 is required for embryonic development and hox gene expression

**P < 0.01. (A) Cleavage and development of ICSI embryos are significantly impaired by Wave1 knockdown with AS. Actual numbers of embryos treated and developed are indicated above the corresponding graphs. (B) Abnormal cleavage caused by Wave1 knockdown is rescued by overexpression of NLS–Wave1-B, but not by NLS–Wave1-C. (C) Heat-map analysis shows lower expression of hox genes in Wave1-MO embryos (MO1-4) than control embryos. (High expression in red and low expression in blue.) (D) RNA-seq analysis shows that down-regulated hoxc5 expression by Wave1 MO injection is partially rescued by overexpressing NLS–Wave1-B. Black boxes in hoxc5 gene represent exons.

Fig. 4. Nuclear Wave1 binds to active transcriptional components through the WHD domain and is important for their activities in embryos

(A) Coimmunoprecipitation analysis in embryo extracts revealed that the WHD domain (Fig. 2B) is sufficient to bind to active Pol II and Mll. Five percent input was loaded. (B) Binding of elongating Pol II(phoS2) and H3K4me3 to hoxb1 and hoxc5 genes was examined by ChIP analyses. The levels of binding were compared in gastrula embryos with or without Wave1 MO injection (MO and Control, respectively). Relative fold increases of precipitated DNA over input DNA were measured by QPCR. *P < 0.05. n = 4 (Pol II) and 8 (H3K4me3). Error bars represent SEM.