CXXC5: A novel regulator and coordinator of TGF‐β, BMP and Wnt signaling

Abstract CXXC5 is a member of the CXXC‐type zinc‐finger protein family. Proteins in this family play a pivotal role in epigenetic regulation by binding to unmethylated CpG islands in gene promoters through their characteristic CXXC domain. CXXC5 is a short protein (322 amino acids in length) that does not have any catalytic domain, but is able to bind to DNA and act as a transcription factor and epigenetic factor through protein‐protein interactions. Intriguingly, increasing evidence indicates that expression of the CXXC5 gene is controlled by multiple signaling pathways and a variety of transcription factors, positioning CXXC5 as an important signal integrator. In addition, CXXC5 is capable of regulating various signal transduction processes, including the TGF‐β, Wnt and ATM‐p53 pathways, thereby acting as a novel and crucial signaling coordinator. CXXC5 plays an important role in embryonic development and adult tissue homeostasis by regulating cell proliferation, differentiation and apoptosis. In keeping with these functions, aberrant expression or altered activity of CXXC5 has been shown to be involved in several human diseases including tumourigenesis. This review summarizes the current understanding of CXXC5 as a transcription factor and signaling regulator and coordinator.


| CXXC-TYPE ZINC-FINGER PROTEIN FAMILY
The combined pattern of DNA and histone modifications confers an epigenetic code that can profoundly affect both chromatin structure and gene expression. 13 In vertebrates, DNA methylation mainly occurs on cytosine within the CpG dinucleotides, which are frequently found in mammalian gene promoters and are associated with a compacted chromatin structure and transcriptional repression. 14 Previous studies have demonstrated that proteins harbouring methyl-CpG-binding domains (MBDs) play a significant role in this process. 14,15 However, as mentioned above, methylation-resistant CpG islands are prevalent in many promoters, up to 60% of human gene promoters. 1,2,13 It is reasonable to assume that these unmethylated CpG islands are also targeted by epigenetic factors and are associated with an open chromatin state and transcriptional activation. Indeed, the specialized CXXC-type zinc-finger domain has been shown to bind unmethylated CpG in vitro. 16 Further, ChIP-sequencing (ChIP-Seq) analyses have demonstrated that some of the CXXC domain-containing proteins are capable of binding to CpG at the genomic scale in various in vivo contexts. 1,4,17 Twelve CXXC zinc-finger protein family members have been identified in mammalians (Table 1). [1][2][3]18 The CXXC-type zinc-finger domain is characterized by two conserved cysteine-rich motifs (ie, CxxCxxCx [4][5] CxxCxxC and CxxRxC, wherein x indicates residues other than cysteine), with three cysteines from the first motif and one from the latter together coordinating one Zn 2+ ion, therefore forming two zinc-finger structures. 2,14 However, the linker region between the two motifs is less conserved and classifies the family members into different subgroups. CFP1/CXXC1, KDM2A/CXXC8, KDM2B/CXXC2, FBXL19/CXXC11, DNMT1/CXXC9, MLL1/CXXC7, MLL2/CXXC10 and MBD1/CXXC3 share a conserved KFGG motif and fall into the same subgroup. Meanwhile, TET1/CXXC6, TET3, IDAX/CXXC4 and CXXC5 are shorter, lack the KFGG motif, and constitute a second subgroup. The majority of CXXC family members exhibit chromatin-modifying activity due to the existence of functional domains. CXXC4 and CXXC5, though structurally similar, lack these types of domains. 11,19 The CXXC5 protein is 322 amino acids in length, has a molecular weight of 32.98 kDa, and contains a typical CXXC zinc-finger domain (amino acids 257-302) near the C-terminus ( Figure 1) Several other reports have provided additional evidence that CXXC5 could act as a transcription factor and/or epigenetic factor by binding to DNA via its CXXC domain (Table 2). In neural stem cells, Wnt3a-stimulated CXXC5 directly interacts with the promoter region (−720 to −511 bp) and promotes the expression of the myelin basic protein gene, which is a molecular marker for oligodendrocyte differentiation. 20 In endothelial cells, the CXXC5 protein interacts with the Flk-1 promoter through its CXXC domain, activating Flk-1 expression and promoting endothelial cell differentiation, migration and vessel formation. 7 In C2C12 myoblasts, CXXC5 is able to increase the activities of gene promoters that drive the expression of muscle creatine kinase, minimal thymidine kinase and myosin heavy chain, thus promoting skeletal muscle differentiation. 21 In other contexts, CXXC5 may repress gene expression. For instance, in CD8+ Th cells, CXXC5 could associate with the histone- was also suggested to interact with Smad2/3 and Smad4 to promote TNF-α-induced apoptosis of primary cortical neurons and normal heart development and function in zebrafish. 49,50

| CXXC5 in Wnt/β-catenin signaling
The Not surprisingly, the Wnt/β-catenin pathway is also subject to intense regulation. 54 FoxL2 is a transcription factor belonging to the large family of winged-helix forkhead transcription factors. This protein has been implicated in numerous cellular processes including apoptosis, cell cycle control, steroidogenesis and reactive oxygen species (ROS) detoxification. 69 Both CXXC4 and CXXC5 can interact with FoxL2, attenuating its transcriptional activity in luciferase reporter assays while facilitating its pro-apoptosis activity via unknown mechanisms. 70 The known interacting partners of CXXC5 are summarized in Table 3.

CROSSROADS OF MULTIPLE SIGNALING PATHWAYS
The CXXC5 gene is evolutionarily conserved from Xenopus and zebrafish to mammals. It is located on chromosome 5q31.3 in human, varies. 49 Another study examined CXXC5 expression in human tissues and found that it is highly expressed in placenta, heart and kidney, and moderately expressed in liver, lung and testis. 3 As mentioned above, the expression of the CXXC5 gene is transcriptionally induced by TGF-β, BMP4 and Wnt3a, which rely on Smad proteins and β-catenin respectively. 7,9,10  Nd., not determined; ATM, ataxia telangiectasia mutated; VDR, vitamin D receptor; TGF-β, transforming growth factor beta.  83 TGF-β, transforming growth factor beta; BMPs, bone and morphogenetic proteins.

Cytokine or transcription factor CXXC5 expression Cell type and context References
function as a crucial coordinator of multiple cellular signaling pathways ( Figure 2).

OF CXXC5
Considering its roles in regulating gene expression and coordinating cellular signaling, it is not surprising that CXXC5 regulates a diverse array of cellular events including cell proliferation, differentiation and apoptosis in developmental processes, adult tissue maintenance and pathological circumstances.
With respect to cell differentiation, retinoid-induced CXXC5 has been shown to play a role in normal primary leukaemia cell differentiation and tumoral myelopoiesis. 3 BMP4-activated CXXC5 promotes the differentiation of mESCs into endothelial cells. 7 In mice, both BMP4-and Wnt-induced CXXC5 can promote the differentiation of neural stem cells into oligodendrocytes, contributing to brain development and maturation. 11,20 In addition, CXXC5 facilitates skeletal myogenic differentiation in vitro by promoting the expression of related genes. 21 Conversely, CXXC5 inhibits Wnt-induced myofibroblast differentiation of dermal fibroblasts in cutaneous wound healing, and osteoblast differentiation and bone formation in mice. 8,9 Interestingly, a peptide that competitively inhibits the CXXC5-Dvl interaction is capable of alleviating CXXC5-mediated inhibition of Wnt signaling and accelerating both osteoblast differentiation and cutaneous wound healing. 8,9 In CD8+ cytotoxic T cells, CXXC5 represses CD40L expression, thus preventing the differentiation of helper-lineage T cells. 22 Importantly, gene-targeting studies in mice have verified the in vivo functions of CXXC5 in regulating cell differentiation. [7][8][9]20 CXXC5 is involved in regulating cell proliferation and death in various cell types. In primary leukaemia cells, CXXC5 not only promotes differentiation, as mentioned above, but it also contributes to retinoid-induced cell cycle arrest and cell death. 3 CXXC5 is capable F I G U R E 2 CXXC5 is an integrator and coordinator of cellular signaling networks. TGF-β, BMP4 and Wnt3a induce CXXC5 gene expression in different contexts. The protein product of this gene can in turn inhibit Wnt signaling or facilitate TGF-β signaling, forming distinct regulatory feedback loops. The expression of CXXC5 is also transcriptionally induced by Retinoid/RARα and E2/ERα signaling as well as by some transcription factors, including WT1, KANK1 and GATA2. Conversely, the transcription factor ThPOK and the RNA cleavage factor NUDT21 are capable of repressing CXXC5 gene expression. In addition to TGF-β and Wnt signaling, CXXC5 promotes ATM-mediated activation of the p53 protein and DNA damage response. CXXC5 also enhances the transcriptional activity of VDR and ameliorates that of FoxL2. TGF-β, transforming growth factor beta; BMPs, bone and morphogenetic proteins; ATM, ataxia telangiectasia mutated; VDR, vitamin D receptor of promoting cell cycle arrest, DNA repair or apoptosis by activating the ATM-p53 signaling axis. 12 In addition, CXXC5 associates with FoxL2 to enhance its pro-apoptotic activity in mammalian cells, 70 and may mediate TNF-α-induced apoptosis of primary cortical neurons by associating with Smad proteins. 49 Aberrant expression or altered activity of CXXC5 is closely linked to multiple human diseases, especially tumourigenesis ( Figure 3). The CXXC5 gene locates in a chromosomal DNA region that is frequently deleted in myeloid leukaemia (AML and myelodysplasia). 3 Indeed, CXXC5 is often downregulated in clinical AML samples and may serve as an independent prognostic factor in patients. 71,72 CXXC5 downregulation leads to augmentation of Wnt signaling and impairment of DNA damage-induced and p53-dependent cell cycle arrest. 71 We found that CXXC5 is also downregulated in the majority of HCC tissue samples, leading to breakdown of CXXC5-mediated positive feedback regulation of TGF-β signaling and attenuation of its anti-proliferative effects. 10 In addition, CXXC5 is involved in KANK1-induced cell growth inhibition and apoptosis in MPNSTs. 79 However, it has also been shown that CXXC5 is overexpressed in ER+ breast cancer and associated with poor prognosis in patients. 84 This is in agreement with the observation that CXXC5 is upregulated by E2-ERα. 82 Together, these findings suggest that the role of CXXC5 in cancer initiation and progression could rely on cancer types.
CXXC5 has also been observed to play a role in some other pathological processes. Aberrant Wnt signaling has been found to be associated with hair loss (alopecia) and anabolic osteoporosis. A previous study demonstrated that expression of CXXC5 is upregulated and is inversely correlated with that of β-catenin in both haired and bald individuals. 85 In mice, CXXC5 knockout or competitive peptide-mediated disruption of the CXXC5-Dvl interaction restores Wnt signaling and accelerates hair regrowth and wound-induced hair follicle neogenesis. 85 Similarly, targeting the CXXC5-Dvl interaction with small molecules has been suggested as a new therapeutic approach for the treatment of anabolic osteoporosis. 86 Fourth, targeting of CXXC5, for example through inhibition of its interaction with the Dvl protein using competitive peptides or small molecules, is emerging as a promising means of therapeutic intervention in animal models. Given that aberrant CXXC5 expression is involved in various human diseases, especially cancer, it is reasonable to believe that targeting CXXC5 could be an effective clinical approach to treat these diseases.

GUIDELINES
The authors declare that they have no conflicts of interest. This is a review of previous studies and it does not contain any original human or animal studies performed by any of the authors.