fcell-09-705302 August 2, 2021 Time: 13:32 # 1 ORIGINAL RESEARCH published: 06 August 2021 doi: 10.3389/fcell.2021.705302 Choline Kinase Alpha Is a Novel Transcriptional Target of the Brg1 in Hepatocyte: Implication in Liver Edited by: Mauro Salvi, Regeneration University of Padua, Italy Reviewed by: Ming Kong1†, Wenhui Dong1†, Huihui Xu1†, Zhiwen Fan2,3, Xiulian Miao2,4, Yan Guo2,4, Claudia Banchio, Chengping Li2,4, Qing Ye5,6*, Yutong Wang7* and Yong Xu1,2* CONICET Instituto de Biología Molecular y Celular de Rosario (IBR), 1 Key Laboratory of Targeted Intervention of Cardiovascular Disease, Department of Pathophysiology, Collaborative Argentina Innovation Center for Cardiovascular Translational Medicine, Nanjing Medical University, Nanjing, China, 2 Institute Tahl Zimmerman, of Biomedical Research, Liaocheng University, Liaocheng, China, 3 Department of Pathology, Nanjing Drum Tower Hospital North Carolina Agricultural Affiliated with Nanjing University School of Medicine, Nanjing, China, 4 College of Life Sciences, Liaocheng University, and Technical State University, Liaocheng, China, 5 Division of Life Sciences and Medicine, Department of Pathology, The First Affiliated Hospital, University United States of Science and Technology of China, Hefei, China, 6 Division of Life Sciences and Medicine, Intelligent Pathology Institute, Juan Carlos Lacal, University of Science and Technology of China, Hefei, China, 7 Department of Cell Biology, The Municipal Laboratory of Liver Instituto de Investigaciones Protection and Regulation of Regeneration, School of Basic Medical Sciences, Beijing, China Biomédicas, Spain *Correspondence: Liver regeneration is a key compensatory process in response to liver injury serving Qing Ye [email protected] to contain damages and to rescue liver functions. Hepatocytes, having temporarily Yutong Wang exited the cell cycle after embryogenesis, resume proliferation to regenerate the injured [email protected] liver parenchyma. In the present study we investigated the transcriptional regulation Yong Xu [email protected] of choline kinase alpha (Chka) in hepatocytes in the context of liver regeneration. We †These authors have contributed report that Chka expression was significantly up-regulated in the regenerating livers in equally to this work the partial hepatectomy (PHx) model and the acetaminophen (APAP) injection model. In Specialty section: addition, treatment with hepatocyte growth factor (HGF), a strong pro-proliferative cue, This article was submitted to stimulated Chka expression in primary hepatocytes. Chka depletion attenuated HGF- Cellular Biochemistry, induced proliferation of hepatocytes as evidenced by quantitative PCR and Western a section of the journal Frontiers in Cell and Developmental blotting measurements of pro-proliferative genes as well as EdU incorporation into Biology replicating DNA. Of interest, deletion of Brahma-related gene 1 (Brg1), a chromatin Received: 05 May 2021 remodeling protein, attenuated Chka induction in the regenerating livers in mice and Accepted: 20 July 2021 Published: 06 August 2021 in cultured hepatocytes. Further analysis revealed that Brg1 interacted with hypoxia- Citation: inducible factor 1 alpha (HIF-1a) to directly bind to the Chka promoter and activate Chka Kong M, Dong W, Xu H, Fan Z, transcription. Finally, examination of human acute liver failure (ALF) specimens identified Miao X, Guo Y, Li C, Ye Q, Wang Y a positive correlation between Chka expression and Brg1 expression. In conclusion, our and Xu Y (2021) Choline Kinase Alpha Is a Novel Transcriptional Target of the data suggest that Brg1-dependent trans-activation of Chka expression may contribute Brg1 in Hepatocyte: Implication to liver regeneration. in Liver Regeneration. Front. Cell Dev. Biol. 9:705302. Keywords: transcriptional regulation, choline kinase, transcription factor, epigenetics, chromatin remodeling doi: 10.3389/fcell.2021.705302 protein, hepatocyte, liver regeneration Frontiers in Cell and Developmental Biology| www.frontiersin.org 1 August 2021| Volume 9| Article 705302 fcell-09-705302 August 2, 2021 Time: 13:32 # 2 Kong et al. Brg1 Activates Chka Transcription INTRODUCTION hepatitis, and lipopolysaccharide-induced acute liver injury in mice by regulating the transcription of pro-lipogenic genes, Acute or chronic loss of liver function following a wide range of chemokine nephronectin, and pattern recognition receptor injuries disrupts internal homeostasis and leads to liver failure TLR4, respectively (Fan et al., 2020; Dong et al., 2021; Hong et al., with life-threatening consequences (Arroyo et al., 2020). Liver 2021). On the other hand, Brg1 deletion in hepatocytes results in regeneration serves as a crucial compensatory mechanism by impairment of liver regeneration owing to dampened induction restoring the damaged liver mass and normalizing liver function of pro-proliferative genes downstream of the Wnt-b-catenin (Michalopoulos, 2017). Having temporarily exited the cell cycle, signaling (Li et al., 2019). In the present study we investigated the hepatocytes could resume proliferation to replenish the loss of transcriptional mechanism whereby Chka expression is regulated liver parenchyma and alleviate the decline of liver function. in hepatocyte in the context of liver regeneration. Our data Therefore, the robustness with which existing hepatocytes can suggest that Brg1 forms a transcriptional complex with hypoxia proliferate is observed to correlate with the prognosis of patients inducible factor to activate Chka transcription in hepatocytes. with end-stage liver diseases (Delhaye et al., 1999; Lanthier et al., 2015). Proliferation of quiescent hepatocyte and hence liver regeneration are guided by a complex signaling network. MATERIALS AND METHODS Hepatocyte growth factor (HGF) is one of the best characterized pro-proliferative cues residing at the center of this network. Animals Deletion of HGF (Phaneuf et al., 2004) or its receptor (c- All animal protocols were reviewed and approved the intramural Met) (Borowiak et al., 2004) impairs liver regeneration in Ethics Committee on Humane Treatment of Laboratory Animals mice. On the contrary, recombinant HGF appears to confer of Nanjing Medical University. The mice were maintained in protection against liver failure by boosting liver regeneration an SPF environment with 12 h light/dark cycles and libitum in mice (Kosai et al., 1999) and in humans (Cui et al., 2008). access to food and water. Hepatocyte conditional Brg1 knockout In accordance, serum HGF levels have been proposed as a (Brg1LKO) mice have been described previously (Kong et al., prognostic marker for patients with liver failure (Shiota et al., 2021). Liver regeneration by the partial hepatectomy (PHx) 1995; Ido and Tsubouchi, 2009). When stimulated by HGF, procedure or intraperitoneal injection of acetaminophen as hepatocytes undergo marked transcriptomic alterations with previously described (Li et al., 2019). multiple clusters of genes being up- or down-regulated (Kaposi- Novak et al., 2006). Regulatory mechanisms underlying these Cells, Transient Transfection, and transcription events and the implications in liver regeneration Reporter Assay have yet to be fully understood. Choline kinases catalyze the biosynthesis of Primary murine hepatocytes were isolated as previously phosphatidylcholine, a key component of cellular membranes described (Fang et al., 2020; Wang et al., 2020c). Mouse (McMaster, 2018). Two isoforms, choline kinase alpha (Chka) recombinant HGF was purchased from R&D. Brg1 expression and Chkb, have been identified with Chka being the predominant construct (Li et al., 2020c; Sun et al., 2020) and Chka promoter- isoform in mammals (Aoyama et al., 2004). The essentiality of luciferase constructs (Glunde et al., 2008) have been previously Chka in choline metabolism is evidenced by the observation described. Small interfering RNAs were purchased from that mice homozygous for Chka deletion die prematurely GenePharma. Transient transfections were performed with during embryogenesis (Wu et al., 2008). Recent studies have Lipofectamine 2000. Luciferase activities were assayed shown that over-expression/hyperactivation of Chka, but not 24–48 h after transfection using a luciferase reporter assay Chkb, is frequently observed in and associated with neoplastic system (Promega) as previously described (Li et al., 2020b; transformation of multiple cell lineages (Gallego-Ortega Mao et al., 2020). et al., 2011). For instance, Lin et al.(2017) have reported that Chka promotes aberrant proliferation of hepatocellular Protein Extraction and Western Blot carcinoma (HCC) cells by mediating the crosstalk between Whole cell lysates were obtained by re-suspending cell pellets the EGF signaling and the mTOR signaling. Chka levels in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% have also been shown to correlate positively with malignant Triton X-100) with freshly added protease inhibitor (Roche) expansion of liver tumors and inversely with patient outcome as previously described (Chen et al., 2020b,c). Western blot (Kwee et al., 2012; Carrillo-Reixach et al., 2020). Of interest, analyses were performed with anti-Chka (Proteintech, 13520-1), concurrent high expression of Chka and HGF has been anti-Brg1 (Abcam, Ab110641), and anti-b-actin (Sigma, A1978). noted in the HCC patients (Mok et al., 2016). It remains For densitometrical quantification, densities of target proteins undermined whether Chka expression can be regulated by were normalized to those of b-actin.