ZHX2, a member of the ZHX (Zinc fingers and homeoboxes) family, is a ubiquitous transcriptional factor that was first identified as a negative regulator of murine postnatal alpha fetal protein (AFP) (1). In 1977, Roushlatti and colleagues compared serum AFP in different mouse strains and found a gene which they called Regulator of Alpha-fetoprotein (Raf), subsequently renamed Alpha-fetoprotein regulator 1 (Afr1), negatively regulated the AFP expression in adult mice (1, 2). In 2005, Perincheri et al. further refined and identified Zhx2 as the homologous gene of Afr1 by positional cloning (3). Human ZHX2 was first cloned by Nagase et al. from a size-fractionated brain cDNA library in 1998 (2). In 2003, human ZHX2 was then identified as a ZHX1-interacting protein by Kawata et al. (4).

ZHX2 has been extensively studied in cancer development. ZHX2 suppresses the transcription of oncofetal genes AFP ( 1, 3, 5) and glypican 3 (GPC3), and works as a tumor suppressor gene in HCC (5, 6). Subsequent studies have found that ZHX2 is widely expressed and participates in many types of cancer. Consistent with findings in HCC, low ZHX2 expression correlates with poor prognosis of thyroid cancer (7), multiple myeloma (8–10), and chronic lymphocytic leukemia (11, 12). On the contrary, ZHX2 promotes the development of ccRCC (13–15), TNBC (16), and gastric cancer (17, 18). Beyond regulating cancer development, the latest reports have shown that ZHX2 involves in several other physiological or pathological processes, including cell differentiation and development (19–21), lipid metabolism (22–24), and viral replication (25, 26). Especially, ZHX2 is abundantly expressed in the thymus and spleen (2) and there is clear evidence supporting the involvement of ZHX2 in regulating B cell development (27), NK cell maturation (28), and macrophage polarization (29–31).

In this review, we outline these new advances in ZHX2 mediated regulation in health and diseases. We also discuss the multiple mechanisms involved in controlling ZHX2 expression and transcription.

The human ZHX2 gene is localized on chromosome 8q24.13 and consists of 4 exons (4). The third exon is the sole coding exon of ZHX2 which encodes a protein of 837 amino acid residues (4). Human ZHX2 protein, like the other two family members ZHX1 and ZHX3, contains two Cys-Xaa₂-Cys-Xaa₁₂-His-Xaa₄-His-type zinc finger domains (Znf) and four homeodomains (HD) (originally thought as five HDs) (4). Besides, ZHX2 contains a proline-rich region (PRR) at position 408 to 440 between HD1 and HD2 (4). The homology of ZHX2 protein in humans and mice is as high as 87%. Kawata et al., in 2003, identified ZHX2 as a ubiquitous transcription factor. ZHX2 interacts with nuclear transcription factor Y subunit alpha (NF-YA) and forms homodimers or heterodimers with ZHX1 or ZHX3 to exert transcriptional inhibitory function (5). The amino acid sequence between residues 195 and 358 containing HD1 is required for homodimerization of ZHX2, and ZHX2 interacts with NF-YA via the region between 263 and 497 residues (4). Similar to full-length ZHX2, truncated ZHX2 containing residues 242-446 (ZHX2(242-446)) but not ZHX2(242-439) maintain the capability to localize in the nuclei and suppress the expression of Cyclin A/E in HCC (6). The decreased nucleic ZHX2 expression significantly correlates with poor survival of HCC patients (6). However, how ZHX2 loses its nuclear localization is completely unknown. More studies are required to define the exact nuclear localization signal (NLS) and the molecules or mechanisms regulating the nuclei translocation of ZHX2.

A growing number of genes have been identified as the ZHX2 targets, most of which are cancer-related. ZHX2 not only negatively controls the transcription of liver cancer marker genes AFP and GPC3, but also inhibits cell proliferation-related genes such as Cdc25 ( 4), Cyclin A/E ( 6), and Notch1 ( 32). In addition, ZHX2 represses transcription of multidrug resistance mutation 1 (MDR1) (33), lipoprotein lipase (LPL) (34), lysine demethylase 2A (KMD2A) (35), and S100 calcium binding protein A14 (S100A14) (7) in HCC and thyroid cancer cells. Although ZHX2 was originally reported to be a ubiquitous transcriptional repressor, recent reports uncover another face of ZHX2 as a transcriptional activator (36, 37). Jiang et al. found that Zhx2 binds Mup promoters and is required for high levels of Mup expression in adult mouse liver (36). ZHX2 also binds to the promoter of phosphatase and tensin homolog (PTEN) and subsequently promotes the transcription of PTEN ( 37). Strikingly, several non-coding RNAs have been elucidated as the ZHX2 targets, either enhanced or inhibited. ZHX2 represses transcription of H19 ( 3, 38, 39), the first imprinted non-coding transcript to be identified. In glioma cells, ZHX2 binds to the promoter region of linc00707 and negatively regulates its expression, leading to glioma cells proliferation, migration and invasion, and vasculogenic mimicry (VM) formation (40). On the contrary, ZHX2 increases transcription of miR-24-3p and linc01431, which targets SREBP1c ( 24) and PRMT1 (26) in hepatocytes respectively.

The mechanism by which ZHX2 controls target gene transcription is not fully understood. ZHX2 was originally known as an NF-YA interacting protein (4) and therefore represses transcription of MDR1, Cdc25, and Notch1 by interacting with NF-YA (4, 6, 32, 33). However, there is no evidence for the presence of NF-YA binding sites in promoter of some other ZHX2-targeted genes, such as Cyclin E, or AFP ( 5, 6). A global analysis of Zhx2 targets using ChIP-seq in a murine macrophage cell line shows a significant overlap with two known apoptosis regulators Jun (41) and Bcl6 (42), which suggest a strong involvement of Zhx2 in cell apoptosis (30). In ccRCC, ChIP-seq data indicate that the genome-wide chromatin occupancy of ZHX2 overlaps with 75% of p65-binding motifs (13). ZHX2 and RelA/p65 overlapping sites also display a strong enrichment for H3K4me3 and H3K27ac, indicating that ZHX2 colocalizes with NF-κB to active gene promoters (13). In TNBC, the integrated ChIP-seq and gene expression profiling show that ZHX2 and HIF1α co-occupy transcriptional active promoters to promote gene expression (16). These studies suggest that ZHX2 may mainly serve as a transcriptional cofactor, interacting with different coactivators/repressors in different physiological circumstance to control its localization in the genome and downstream transcriptional activity. In addition, the Znf domains of ZHX2 process potential DNA-binding activity, however, whether ZHX2 can bind DNA directly and its consensus binding motif still need to be investigated.

ZHX2 expression is tightly regulated under different circumstances. A computational network study indicates ZHX2 as one of the most regulated transcription factors in myeloid cells to avoid an avalanche of transcriptional events (31). In Hodgkin lymphoma (HL), a chromosomal rearrangement far upstream region of ZHX2 gene results in the transcriptional silence of ZHX2, and two transcription factors, homeodomain protein MSX1 and bZIP protein XBP1, are identified to directly regulate ZHX2 expression (11). Furthermore, human ZHX2 is lower expressed in fetal liver, increased after birth, and silenced in HCC (43–45). Consequently, multiple mechanisms are revealed to control ZHX2 expression at different levels ( Figure 1 ):

At the ZHX2 gene transcription level- Lv et al. found that ZHX2 promoter region is hypermethylated in HCC, suggesting that the hypermethylation-mediated silencing of ZHX2 is an epigenetic event involved in HCC (45). In addition, copy number analysis showed that ZHX2 gene is amplified in various cancers, including ovarian cancer (~40%) and breast cancer (~15%). The ZHX2 copy number significantly correlates with enhanced ZHX2 expression (16). Wu et al. (34) and Zhao et al. (37)found that Zhx2 expression can be repressed by free fatty acid in hepatocytes. Constantly, hepatic Zhx2 is reduced in mice with fatty liver, indicating that ZHX2 could be regulated by the metabolic microenvironment. This is consistent with a previous computational network study indicating ZHX2 as one of the most regulated transcription factors in myeloid cells (31). The detailed mechanisms regulating ZHX2 expression in different circumstances need to be further studied.

At the post-transcription level- microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. They generally bind to the 3’-UTR (untranslated region) of their target mRNAs and reduce protein production by destabilizing mRNA or translational silencing (46, 47). HBV-encoded proteins, particularly a well-known oncogenic protein HBx, drive the high expression of miR-155, which binds to seed sites in the 3’-UTR of the ZHX2 mRNA and inhibit its translation (48). Similarly, HBx promotes CREB-mediated activation of miR-3188 to repress ZHX2 expression, leading to activated Notch signaling in HCC (32). While in TNBC, lncRNA PART1 promotes ZHX2 transcription (49).

At the posttranslational modifications (PTMs) level- Zhang et al. report that inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase in ccRCC leads to the accumulation of ZHX2 protein and its nuclear localization. ZHX2 protein hydroxylation at proline 427, 440, and 464 allows VHL to bind and promote its protein degradation (13). However, a deubiquitinase USP13 inhibits the ubiquitination of ZHX2 and enhances its stability (15). A recent study found that the N-terminal methylation (Nα-methylation) of ZHX2 by the methyltransferase NRMT1 regulates its transcription factor activity and its occupancy on targeted promoters (50). Up to now, whether there are other PTMs and their roles in ZHX2 trafficking, stability, and transcriptional activity are less clear.

ZHX2 is initially identified as an AFP repressor and a tumor repressor in HCC (3, 5). Whereafter, abnormal expression of ZHX2 is reported in multiple types of tumor (6, 8, 11). Furthermore, ZHX2 expression is closely related to the malignancy and poor prognosis of B-cell lymphoma (11, 12), myeloma (8–10), lung cancer (51), and thyroid cancer (7), suggesting that ZHX2 plays an important role in tumorigenesis and cancer development. Interestingly, latest studies reported that ZHX2 functions as an oncogene in ccRCC (13, 14) and TNBC (16). Likewise, Jiang et al. reported that the whole-body knockout of Zhx2 results in reduced liver tumors in diethylnitrosamine (DEN)-induced HCC mice (52). Therefore, ZHX2 is abnormally expressed in multiple tumors and plays different roles, either acting as a tumor suppressor or oncogene in a context-dependent manner ( Figure 2 ). Here, we outline the role of ZHX2 in multiple tumors.

Besides the complicated roles in tumors, recent studies suggest the involvement of ZHX2 in the regulation of cell differentiation, HBV replication, lipid homeostasis, and immune responses ( Figure 3 ). In agreement, ZHX2 has been reported in the occurrence of chronic hepatitis B (CHB) (32, 48), metabolism-related diseases (30, 37), nerve-related diseases (19, 60, 61), and immune-related diseases (29) ( Figure 3 ). We will discuss the role of ZHX2 in different physiological and pathological processes here.

As a transcription factor, ZHX2 transcriptionally regulates the expression of a series of genes that participate in cell proliferation, differentiation, and metabolism homeostasis. Accordingly, ZHX2 has a broader role in regulating multiple physiological and pathological processes, including cell development, immune regulation, cancer development, and metabolism-related diseases. Significantly, ZHX2 exerts its roles in a context-dependent manner. The exact mechanisms controlling the switch of ZHX2 function in health and diseases are still not clear. Nevertheless, it remains uncertain whether ZHX2 interacts with DNA directly or indirectly via other transcription factors to exert its transcriptional regulation role. Future research needs to be focused on ZHX2 structure, protein interactome, and high throughput screening to clarify its transcriptional regulation and identify new targeted genes. Equally important, the mechanisms that regulate ZHX2 expression are still uncertain. Accumulated studies have suggested that different stimuli regulate ZHX2 expression at different levels including transcription, post-transcription, and posttranslational modification levels. However, the mechanisms are not yet precisely understood. Moreover, in addition to hydroxylation, ubiquitination, and Nα-methylation, other PTMs that determine the biological function and nucleocytoplasmic shuttling of ZHX2 under different circumstances need to be further explored.

NL, ZW, and CM designed and prepared the manuscript and the figures. CM gave guidance on the outline and revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by grants from the National key research and development program (2021YFC2300603), the National Science Foundation of China (Key program 81830017, and 81902051), Taishan Scholarship (No.tspd20181201), Major Basic Research Project of Shandong Natural Science Foundation (No.ZR2020ZD12), Key Research and Development Program of Shandong (2019GSF108238).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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