Transcription factor-mediated gene regulatory networks in the formation of oocytes
Di Wu
A , Zifan Liang B , Ziqi Li B , Boyang Zhang B , Qiwen Li B , Kesong Shi B C * and Shu Fang B *
A
B
C
Abstract
The induction of oocytes from embryonic stem cells (ESCs) in vitro provides a promising tool for the treatment of female infertility. Various molecules are involved in this complex process, which requires further elucidation.
This study aims to screen for factors that induce the differentiation of ESCs into oocytes in vitro by constructing transcription factor (TF)-mediated gene regulatory networks (GRNs) during the formation of oocytes.
Based on publicly available multi-omics data, the weighted gene co-expression network analysis (WGCNA) method identified oocyte-specific TFs and key oocyte-specific genes. Additionally, chromatin immunoprecipitation (ChIP) sequencing data and ChIP-qPCR analysis were used to examine GRNs mediated by oocyte-specific TFs.
First, by analyzing assay for transposase-accessible chromatin sequencing (ATAC-seq) and DNase I hypersensitive site sequencing (DNase-seq) data from human and mouse ESCs, primordial germ cells (PGCs), and oocytes, we identified five and three oocyte-specific TFs, respectively. RNA sequencing and WGCNA further revealed 38 key oocyte-specific genes. Subsequently, when comparing cell-specific TFs in mouse and human oocytes, we identified three overlapping oocyte-specific TFs (NFYA, NFYB, and NFYC). Notably, NFYA exhibited significantly elevated expression levels in oocytes compared to ESCs and PGCs. Additionally, ChIP-qPCR results demonstrated that NFYA was relatively enriched at the promoter region of the key oocyte-specific gene, m6A demethylase Alkbh5.
This study provides preliminary insights into the role of cell-specific TFs and TF-mediated GRNs in oocyte formation by identifying oocyte-specific genes and key oocyte-specific TFs.
The findings indicate that their intricate regulatory mechanisms may significantly contribute to enhancing the efficiency of differentiating ESCs into oocytes.
Keywords: Alkbh5, embryonic stem cells, female infertility, gene regulatory networks, NFYA, oocytes, primordial germ cells, transcription factors.
References
Bai L, Xiang Y, Tang M, Liu S, Chen Q, Chen Q, Zhang M, Wan S, Sang Y, Li Q, Wang S, Li Z, Song Y, Hu X, Mao L, Feng G, Cui L, Ye Y, Zhu Y (2023) ALKBH5 controls the meiosis-coupled mRNA clearance in oocytes by removing the N6-methyladenosine methylation. Nature Communications 14(1), 6532.
| Crossref | Google Scholar |
Bar-Joseph H, Ben-Aharon I, Rizel S, Stemmer SM, Tzabari M, Shalgi R (2010) Doxorubicin-induced apoptosis in germinal vesicle (GV) oocytes. Reproductive Toxicology 30(4), 566-572.
| Crossref | Google Scholar |
Bharti D, Jang S-J, Lee S-Y, Lee S-L, Rho G-J (2020) In vitro generation of oocyte like cells and their in vivo efficacy: how far we have been succeeded. Cells 9(3), 557.
| Crossref | Google Scholar |
Chen E, He Y, Jiang J, Yi J, Zou Z, Song Q, Ren Q, Lin Z, Lu Y, Liu J, Zhang J (2023) CDCA8 induced by NF-YA promotes hepatocellular carcinoma progression by regulating the MEK/ERK pathway. Experimental Hematology & Oncology 12(1), 9.
| Crossref | Google Scholar |
Dai H, Jin Q-Q, Li L, Chen L-N (2020) Reconstructing gene regulatory networks in single-cell transcriptomic data analysis. Zoological Research 41(6), 599-604.
| Crossref | Google Scholar |
Datta J, Palmer MJ, Tanton C, Gibson LJ, Jones KG, Macdowall W, Glasier A, Sonnenberg P, Field N, Mercer CH, Johnson AM, Wellings K (2016) Prevalence of infertility and help seeking among 15 000 women and men. Human Reproduction 31(9), 2108-2118.
| Crossref | Google Scholar |
Dolfini D, Gnesutta N, Mantovani R (2024) Expression and function of NF-Y subunits in cancer. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1879(2), 189082.
| Crossref | Google Scholar |
Dolfini D, Imbriano C, Mantovani R (2025) The role(s) of NF-Y in development and differentiation. Cell Death & Differentiation 32, 195-206.
| Crossref | Google Scholar |
Guo J, Kong LM, Peng AF, Long XH, Zhou Y, Shu Y (2016) Transcription factor NF-YA promotes a malignant phenotype by upregulating fatty acid synthase expression. Molecular Medicine Reports 14(6), 5007-5014.
| Crossref | Google Scholar |
Hamazaki N, Kyogoku H, Araki H, Miura F, Horikawa C, Hamada N, Shimamoto S, Hikabe O, Nakashima K, Kitajima TS, Ito T, Leitch HG, Hayashi K (2021) Reconstitution of the oocyte transcriptional network with transcription factors. Nature 589(7841), 264-269.
| Crossref | Google Scholar |
Lee Y, Trout A, Marti-Gutierrez N, Kang S, Xie P, Mikhalchenko A, Kim B, Choi J, So S, Han J, Xu J, Koski A, Ma H, Yoon JD, Van Dyken C, Darby H, Liang D, Li Y, Tippner-Hedges R, Xu F, Amato P, Palermo GD, Mitalipov S, Kang E (2022) Haploidy in somatic cells is induced by mature oocytes in mice. Communications Biology 5(1), 95.
| Crossref | Google Scholar |
Monteiro C, Marques PI, Cavadas B, Damião I, Almeida V, Barros N, Barros A, Carvalho F, Gomes S, Seixas S (2018) Characterization of microbiota in male infertility cases uncovers differences in seminal hyperviscosity and oligoasthenoteratozoospermia possibly correlated with increased prevalence of infectious bacteria. American Journal of Reproductive Immunology 79(6), e12838.
| Crossref | Google Scholar |
Mühlen D, Li X, Dovgusha O, Jäckle H, Günesdogan U (2023) Recycling of parental histones preserves the epigenetic landscape during embryonic development. Science Advances 9(5), eadd6440.
| Crossref | Google Scholar |
Myers ZA, Holt III BF (2018) NUCLEAR FACTOR-Y: still complex after all these years? Current Opinion in Plant Biology 45(Pt A), 96-102.
| Crossref | Google Scholar |
Nardini M, Gnesutta N, Donati G, Gatta R, Forni C, Fossati A, Vonrhein C, Moras D, Romier C, Bolognesi M, Mantovani R (2013) Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination. Cell 152(1), 132-143.
| Crossref | Google Scholar |
Oqani RK, So S, Lee Y, Ko JJ, Kang E (2022) Artificial oocyte: development and potential application. Cells 11(7), 1135.
| Crossref | Google Scholar |
Park IH, Lerou PH, Zhao R, Huo H, Daley GQ (2008) Generation of human-induced pluripotent stem cells. Nature Protocols 3(7), 1180-1186.
| Crossref | Google Scholar |
Qing T, Shi Y, Qin H, Ye X, Wei W, Liu H, Ding M, Deng H (2007) Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 75(10), 902-911.
| Crossref | Google Scholar |
Qu J, Yan H, Hou Y, Cao W, Liu Y, Zhang E, He J, Cai Z (2022) RNA demethylase ALKBH5 in cancer: from mechanisms to therapeutic potential. Journal of Hematology & Oncology 15(1), 8.
| Crossref | Google Scholar |
Saitou M, Hayashi K (2021) Mammalian in vitro gametogenesis. Science 374(6563), eaaz6830.
| Crossref | Google Scholar |
Shi K, Wang B, Dou L, Wang S, Fu X, Yu H (2022) Integrated bioinformatics analysis of the transcription factor-mediated gene regulatory networks in the formation of spermatogonial stem cells. Frontiers in Physiology 13, 949486.
| Crossref | Google Scholar |
Tsujikawa K, Koike K, Kitae K, Shinkawa A, Arima H, Suzuki T, Tsuchiya M, Makino Y, Furukawa T, Konishi N, Yamamoto H (2007) Expression and sub-cellular localization of human ABH family molecules. Journal of Cellular and Molecular Medicine 11(5), 1105-1116.
| Crossref | Google Scholar |
Unger Avila P, Padvitski T, Leote AC, Chen H, Saez-Rodriguez J, Kann M, Beyer A (2024) Gene regulatory networks in disease and ageing. Nature Reviews Nephrology 20(9), 616-633.
| Crossref | Google Scholar |
Wang B, Fang X, Sun X, Du C, Zhou L, Lv X, Li Y, Li H, Tang W (2021) m6A demethylase ALKBH5 suppresses proliferation and migration of enteric neural crest cells by regulating TAGLN in Hirschsprung’s disease. Life Sciences 278, 119577.
| Crossref | Google Scholar |
Wang H-F, Kuang M-J, Han S-J, Wang A-B, Qiu J, Wang F, Tan B-Y, Wang D-C (2020a) BMP2 modified by the m6A demethylation enzyme ALKBH5 in the ossification of the ligamentum flavum through the AKT signaling pathway. Calcified Tissue International 106(5), 486-493.
| Crossref | Google Scholar |
Wang J, Li Y, Wang P, Han G, Zhang T, Chang J, Yin R, Shan Y, Wen J, Xie X, Feng M, Wang Q, Hu J, Cheng Y, Zhang T, Li Y, Gao Z, Guo C, Wang J, Liang J, Cui M, Gao K, Chai J, Liu W, Cheng H, Li L, Zhou F, Liu L, Luo Y, Li S, Zhang H (2020b) Leukemogenic Chromatin alterations promote AML leukemia stem cells via a KDM4C-ALKBH5-AXL signaling axis. Cell Stem Cell 27(1), 81-97.e8.
| Crossref | Google Scholar |
Xu K, Mo Y, Li D, Yu Q, Wang L, Lin F, Kong C, Balelang MF, Zhang A, Chen S, Dai Q, Wang J (2020) N6-methyladenosine demethylases Alkbh5/Fto regulate cerebral ischemia-reperfusion injury. Therapeutic Advances in Chronic Disease 11, 2040622320916024.
| Crossref | Google Scholar |
Xu Z, Sun Y, Guo Y, Qin G, Mu S, Fan R, Wang B, Gao W, Wu H, Wang G, Zhang Z (2016) NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells. Oncology Reports 35(6), 3630-3638.
| Crossref | Google Scholar |
Yoshino T, Suzuki T, Nagamatsu G, Yabukami H, Ikegaya M, Kishima M, Kita H, Imamura T, Nakashima K, Nishinakamura R, Tachibana M, Inoue M, Shima Y, Morohashi K-I, Hayashi K (2021) Generation of ovarian follicles from mouse pluripotent stem cells. Science 373(6552), eabe0237.
| Crossref | Google Scholar |
Yu H, Wu H, Zheng F, Zhu C, Yin L, Dai W, Liu D, Tang D, Hong X, Dai Y (2020) Gene-regulatory network analysis of ankylosing spondylitis with a single-cell chromatin accessible assay. Scientific Reports 10(1), 19411.
| Crossref | Google Scholar |
Zhao X, Fan C, Qie T, Fu X, Chen X, Wang Y, Wu Y, Fu X, Shi K, Yan W, Yu H (2024) Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development. Reproductive Biology and Endocrinology 22(1), 82.
| Crossref | Google Scholar |
