BACH1 recruits STAT3 to enhance leukemia inhibitory factor receptor activity and augments the self-renewal capacity of mouse embryonic stem cells

Abstract

Background Genomic studies have linked single nucleotide variants in the enhancer region of the leukemia inhibitory factor receptor (Lifr) gene to chromatin accessibility and the regulation of self-renewal in mouse embryonic stem cells (mESCs). However, the underlying mechanisms remain unclear. This study investigates the role of the transcription factor BTB and CNC homology 1 (BACH1) in regulating the Lifr enhancer and its impact on mESC pluripotency. Methods We performed RNA-sequencing (RNA-seq) to assess the impact of Bach1 knockout on gene expression in mESCs. Additionally, chromatin immunoprecipitation (ChIP), co-immunoprecipitation (co-IP), and luciferase reporter gene analysis were employed to investigate the mechanism by which BACH1 regulates Lifr expression. Results Genomic analyses identified BACH1 binding at the Lifr enhancer proximal to rs50454566 in mESCs. Integrated single-cell RNA sequencing (scRNA-seq) data revealed co-upregulation of Bach1 and Lifr in inner cell mass (ICM) cells. RNA-seq analyses demonstrated that Bach1 depletion attenuated Lifr expression and impeded LIFR-signal transducer and activator of transcription 3 (STAT3) signaling. Mechanistically, BACH1 recruited STAT3 to the Lifr enhancer, driving Lifr transcription and facilitating the LIFR-STAT3 signaling pathway, thereby enhancing mESC self-renewal. Conclusion Our findings demonstrate that BACH1 enhances Lifr enhancer activity by recruiting STAT3 and activates the LIFR-STAT3 signaling pathway by promoting the LIFR expression, thereby maintaining mESC self-renewal.

1. Introduction

Embryonic stem cells (ESCs) are characterized by their ability to self-renew and differentiate into multiple cell lineages [1]. These cells are derived from the inner cell mass (ICM) of blastocysts—human ESCs (hESCs) from human blastocysts and mouse ESCs (mESCs) from mouse pre-implantation blastocysts [2]. The regulation of ESCs’ self-renewal is governed by a multitude of factors, including signaling pathways, transcription factors, epigenetic regulators, cytokines, and small molecular compounds [3]. Core transcription factors such as SRY-box transcription factor 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and nanog homeobox (NANOG) are essential for maintaining pluripotency in both hESCs and mESCs. These transcription factors play a pivotal role in sustaining the ground state of ESCs [4, 5]. However, distinct signaling pathways regulate the pluripotency state and self-renewal in both mESCs and hESCs. Specifically, hESCs require fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) and Activin/Nodal signaling for the maintenance of self-renewal and pluripotency [6]. Unlike hESCs, the self-renewal of mESCs is dependent on signaling from leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) [7]. However, it is still little known how the pluripotency signaling is fine-tuned.

LIF, a critical member of the interleukin-6 (IL-6) cytokine family, stimulates downstream signaling pathways through binding to its heterodimeric receptor comprising leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130) [8, 9]. As a transmembrane receptor, LIFR is implicated in cancer progression, angiogenesis, stem cell fate determination, and embryonic development [10]. Upon binding of LIF, the LIFR/gp130 receptor complex enhances the kinase activity of Janus kinases (JAK), leading to the phosphorylation of transcription factor signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 subsequently forms homodimers, translocates into the nucleus, and initiates the transcription of target genes [11, 12]. LIFR is also crucial for maintaining the pluripotency of mESCs. Prior studies have elucidated that the LIFR/STAT3 pathway directly modulates the expression of krüppel-like factor 4 (KLF4), which in turn promotes the transcription of SOX2 in mESCs [13]. Under hypoxic conditions, mESCs lose their self-renewal capacity and initiate differentiation due to the down-regulation of the LIFR-STAT3 pathway.