OVOL2: an epithelial lineage determiner with emerging roles in energy homeostasis

Abstract

Ovo like zinc finger 2 (OVOL2) is an evolutionarily conserved regulator of epithelial lineage determination and differentiation during embryogenesis. OVOL2 binds to DNA using zinc-finger domains to suppress epithelial-mesenchymal transition (EMT), which is critical for tumor metastasis. However, recent studies suggest some noncanonical roles of OVOL2 that do not rely on the DNA binding of zinc-finger domains or regulation of EMT. On the other hand, OVOL2 and EMT regulators have emerging roles in adipogenesis, thermogenesis, and lipid metabolism. Here, we review different roles of OVOL2 from embryo development to adult tissue homeostasis and discuss how OVOL2 and other EMT regulators orchestrate a regulatory network to control energy homeostasis. Lastly, we propose potential applications of targeting OVOL2 to reduce human obesity.

OVOL2 and OVO family proteins

OVOL2 is a zinc-finger transcription factor that belongs to the OVO family. The ovo gene was first characterized as a germline sex determination gene in Drosophila. Later three mammalian homologs (OVOL1/2/3) were identified in mice and humans [1–4]. All OVO proteins contain four zinc-finger domains that are highly conserved among species. As transcription factors, OVOL1 and OVOL2 bind to similar consensus DNA sequences as Drosophila Ovo, containing CC/TGTTA [5–7] (Figure 1A). Both Ovol1 and Ovol2 harbor a CCGTTA sequence in their promoter regions, and the expression of Ovol2 is upregulated in Ovol1 knockout mice, implying that OVOL1 can suppress Ovol2 expression and/or that compensatory upregulation of Ovol2 expression occurs in the absence of OVOL1 [8, 9]. The overlapping roles of OVOL1 and OVOL2 are well discussed previously [10, 11], while the function of OVOL3 remains largely unknown.

Figure 1. The versatile roles of OVOL2.

(A) In addition to the four zinc-finger domains in the C-terminus of OVOL2 protein, there is a SNAG domain at the very N-terminus of OVOL2 which functions as a repressor domain, following by a TA domain. OVOL2 is an epithelial lineage determiner that inhibits the EMT. (B) During embryogenesis and development, OVOL2 is critical for neural tube development, vascularization and hematopoiesis, keratinocytes and hair follicle cell development, and mammary gland morphogenesis. (C) In humans, aberrant expression of OVOL2 is linked to posterior corneal dystrophy and various cancers. (D) Recently, OVOL2 has been reported to regulate adipogenesis and thermogenesis to maintain energy homeostasis in adult mice. Abbreviations: SNAG, SNAIL/GFI; TA, transactivation; ZnF, zinc finger; OVOL2, ovo like zinc finger 2; EMT, epithelial-mesenchymal transition.

Functional studies using global Ovol2 knockout mice suggest that OVOL2 participates in the process of extraembryonic and embryonic vascularization, cranial neural tube development, and heart formation, showing the critical role of OVOL2 in embryonic development [12, 13] (Figure 1B). One important function of OVOL2 is the inhibition of epithelial-mesenchymal transition (EMT) (Figure 1A). During development, loss of OVOL2 induces EMT-like behavior in the mammary gland terminal end buds and embryonic epidermis isolated in vitro [9, 14]. Overexpression of OVOL2 in mouse skin epithelia causes premature differentiation in embryonic epidermal progenitor cells and adult hair follicle bulge stem cells and compromised progenitor cell compartments [9] (Figure 1B). The inhibitory role of OVOL2 during EMT has also been implicated in tumor metastasis [11] (Figure 1C). OVOL2 regulates the process of EMT by directly binding to the promoter region of major EMT inducers (ZEB1, ZEB2, TWIST1, SNAIL1, and SNAIL2) and repressing their expression [14–18]. Additionally, OVOL2 also binds to the promoter regions of other genes that do not directly regulate EMT (See Table 1 for reported downstream target genes of OVOL2). In humans, non-coding mutations in the OVOL2 promoter are involved in the pathogenesis of posterior polymorphous corneal dystrophy (PPCD), which results from dysfunction of the corneal endothelium and Descemet’s membrane (Figure 1C). Moreover, a recent study revealed that OVOL2 plays dual roles in promoting thermogenesis and limiting adipogenesis to control energy homeostasis [19] (Figure 1D). In this opinion, we look at both the classic roles of OVOL2 in embryonic development and cancer and the emerging roles of OVOL2 in energy homeostasis. In addition to OVOL2, other EMT regulators have been reported to involve in adipogenesis, thermogenesis, and lipid metabolism. We discuss the potential direct links between OVOL2 and EMT regulators in energy homeostasis which represents an exciting area of future investigation.

Table 1.

Summary of OVOL2 downstream target genes and their related functions.

Gene	Cell type	Function	Ref
c-Myc, Notch1	HaCaT human keratinocytes	Suppress keratinocyte proliferation and terminal differentiation	[5]
histone H1t	Mouse germ cells	Participate in the XY body formation during spermatogenesis	[30]
Zeb1	Human breast cancer MDA-MB-231 cells	Restrict the EMT of mammary epithelial cells, suppress breast cancer invasion and metastasis.	[14,17]
	Human corneal epithelial cells	Suppress EMT	[15]
	Bovine embryonic stem cells	Suppress EMT	[24]
Zeb2	Mouse HC11 Mammary Epithelium	Suppress EMT	[14]
	Human corneal epithelial cells	Suppress EMT	[15]
Vim, Snail1, Snail2, Twist1, Cdh2	Mouse HC11 Mammary Epithelium	Suppress EMT	[14]
Smad4	Mouse breast cancer 4T1 cells	Inhibit TGF-β signaling induced EMT and mammary tumor metastasis	[16]
Id2	Mouse trophoblast stem cells (F4 line)	Drive trophoblast stem cells differentiation and placental development	[20]
RhoU, RhoJ	8505c cells (human undifferentiated thyroid carcinomas)	Restrain mitosis and anaplastic thyroid cancer aggressiveness	[39]
Blimp1	Mouse embryonic stem cells	Participate in primordial germ cell specification	[33]
IL-10	Raw264.7 cells (mouse macrophage cell line)	Inhibit macrophage M2 polarization and tumor metastasis	[36]
GLUT1, HK2, GPI, PFKL, ALDOA, PGK1, PGAM1, ENO1, PKM2, LDHA	Human breast cancer MDA-MB-231 cells	Inhibit aerobic glycolysis in breast cancer	[37]

OVOL2 in embryonic development, epithelial tissue morphogenesis, and germline development

During embryogenesis, the expression of Ovol2 was observed in placenta, yolk sac, and embryo at E8.5-E10.5 [13]. Ovol2 knockout mice died at E9.5-E10.5 with severe defects in the development of cranial neural tube, gut, heart, blood vessels, and placenta, which strongly indicates the critical role of OVOL2 in embryonic development. There are some pieces of evidence that OVOL2 is required for the maintenance and migration of neural crest cells and angiogenesis by endothelial cells and OVOL2 influences the generation of the mammalian placenta by controlling the differentiation and migration of trophoblast stem cells [12, 13, 20, 21]. Nevertheless, the detailed mechanism and how OVOL2 affects the formation of gut and heart during embryonic development remain largely unknown. Other studies have linked OVOL2 to germ layer development, which occurs earlier than organ development. In human corneal epithelium cells, which are derived from surface ectoderm, OVOL2 inhibits the expression of mesenchymal genes to maintain corneal epithelium cells [15]. In contrast, in neuroectoderm derivatives, OVOL2 represses mesenchymal genes to induce the transcriptional program of corneal epithelium cells. This suggests the importance of OVOL2 and EMT in the neuroectoderm and surface ectoderm differentiation [15]. It is also reported that the expression of Ovol2 is directly regulated by bone morphogenetic protein (BMP) signaling, and OVOL2 promotes mesendodermal differentiation at the expense of neural differentiation [22]. However, another group finds that OVOL2 alone is not sufficient for the formation of FLK1⁺ mesoderm, which is essential for both hematopoiesis and vessel development, in a serum-free condition [23]. When OVOL2 interacts with ER71, it enhances the ER71-mediated activation of Flk1 and regulates the generation of FLK1⁺ cells in developing embryos [23] (Figure 2A). Thus, it’s worth trying to specifically knockout Ovol2 in different germ layer cells in vivo, and more work is needed to fully understand the complex regulation of OVOL2 and OVOL2 interacting proteins during gastrulation and embryonic germ layers development. In embryo implantation, OVOL2 expression is decreased at the post-implantation stage to ensure the proper EMT of bovine trophectoderm, suggesting the involvement of OVOL2 and EMT in embryo implantation and pregnancy establishment [24].

Figure 2. Summary of OVOL2 interacting proteins.

Besides the classic role of OVOL2 as a transcription factor that binds with DNA and regulates target gene expression, OVOL2 also directly interacts with other proteins. During hematopoietic cell generation, OVOL2 binds with transcription factor ER71 and functions as a co-activator to induce Flk1 expression and generate FLK1+ cells. In several tumors, OVOL2 interacts with NCoR and HDAC1/3 to repress target gene expression. (B) In some cases, OVOL2 interacts with transcription factors (SMAD4, C/EBPα) and prevents transcription factors from binding with DNA, thus blocks downstream gene activation. (C) Some interaction proteins add post-translational modifications onto OVOL2, including PARylation by PARP1 and ubiquitination by MDM2. Abbreviations: OVOL2, ovo like zinc finger 2; ER71, ETS variant transcription factor 2 (ETV2), aka ER71; Flk1, kinase insert domain receptor (Kdr), aka Flk1; NCoR, nuclear receptor corepressor; HDAC3, histone deacetylase 3; p300, E1A binding protein p300, aka EP300; p65, RELA proto-oncogene, NF-κB subunit, aka RELA; HDAC1, histone deacetylase 1; NF-κKB, nuclear factor kappa B; GLUT1, glucose transporter 1; TCF4, transcription factor 4; PARP1, poly(ADP-ribose) polymerase 1; SKP2, S-phase kinase associated protein 2; p53, tumor protein p53, aka TP53; MDM2, mouse double minute 2.

During epithelial tissue morphogenesis, OVOL2 is identified as a transcriptional gatekeeper in skin keratinocytes, hair follicle cells, mammary epithelial cells, and duct epithelial cells. In human keratinocytes, OVOL2 represses the expression of c-Myc and Notch1, resulting in transient cell proliferation and loss of long-term differentiation potential of cells [5]. Consistent with this, Ovol2-deficient newborn primary keratinocytes show decreased proliferative ability and increased expression of Zeb1 and EMT genes [25]. In hair follicle cells, loss of OVOL2 inhibits the expansion of bulge hair follicle stem cells, and adult hair follicle cells with high aberrant expression of OVOL2 undergo premature differentiation [9, 25]. Inhibition of EMT by OVOL2 is also required for the proper morphogenesis and regeneration of the mammary gland during puberty and pregnancy [14]. Ovol2-deficient mammary stem/progenitor cells are prone to undergo EMT and lose epithelial identity, partially through the activation of Zeb1 and transforming growth factor β (TGF-β) signaling [14]. On the other hand, overexpression of OVOL2 during embryogenesis causes skin blistering at birth [26]. In duct epithelial cells, GRHL2 activates OVOL2 and then jointly controls the expression of Cldn4 and Rab25, participating in renal epithelial morphogenesis, but a direct correlation between OVOL2 and kidney development is missing [27]. Collectively, these data clearly indicate the importance of OVOL2 to the maintenance of epithelial identity.

Since the identification of the ovo locus in Drosophila, it becomes clear that Drosophila Ovo only regulates germline development in females, but not in males [3]. Among all three Drosophila Ovo isoforms (Ovo-A, Ovo-B, and Svb), Ovo-A serves as a negative transcriptional regulator and Ovo-B serves as a positive transcriptional regulator in the female germline, respectively [28]. However, the expression of Ovol1 (movol1) was only detected in mouse testis other than in mouse ovary. Loss of Ovol1 in mice causes abnormal morphology of testis with diminished sperm production [4]. Different from Drosophila Ovo and mouse OVOL1, the function of OVOL2 in germline development and spermatogenesis seems controversial. Despite the facts that OVOL2 is predominantly expressed in adult mouse testis and spermatogenic cells [1, 29], and OVOL2 expression is restricted in the XY body of both mouse and human spermatocytes at the pachytene stage [30, 31], germ cell-specific Ovol2 knockout mice have no defect in fertility, germ cell development, and spermatogenesis [29]. This may be due to the compensation effect of OVOL1 in germ cells [29]. Another possibility is that the Vasa-cre used to generate germ cell-specific Ovol2 knockout mice only expresses at E15 while OVOL2 is reported to be involved in the specification of primordial germ cells at around E6 [32, 33]. Thus, OVOL2 might be only involved in the early embryonic stage of germline development.

OVOL2 in cancer and corneal endothelial dystrophy

EMT is a critical step during cancer cell metastasis and invasion [34, 35]. The role of OVOL2 in cancer by repressing EMT has been widely reported and reviewed previously [11]. It is worth noting that examination of EMT markers does not provide rigorous evidence for EMT and a role of OVOL2 in cancer is not equivalent to a role of EMT in cancer. Indeed, several recent studies have elucidated novel mechanisms by which OVOL2 affects cancer development. In mammary cancer, OVOL2 inhibits the TGF-β signaling by directly interacting with SMAD4 [16] (Figure 2B), modulates the tumor microenvironment by inhibiting macrophage M2 polarization [36], and blocks the Warburg effect of cancer cells by directly binding to promoters of glycolytic genes and repressing their transcription [37]. In colorectal cancer, OVOL2 acts as a tumor repressor that inhibits Wnt signaling by promoting the recruitment of histone deacetylases (HDACs) to the TCF4/β-catenin complex [38] (Figure 2A). In anaplastic thyroid cancer, OVOL2 not only restrains EMT but also blocks GTPase signaling to prevent the mitosis and aggressiveness of cancer [39]. In non-small cell lung cancer, OVOL2 interacts with P65, blocks the binding of P65 to P300, and promotes the binding of P65 to HDAC1, thus inhibiting NF-κB signaling and regulating aerobic glycolysis [40] (Figure 2A). Additionally, it is also reported that OVOL2 can be PARylated by PARP1 in human embryonic kidney cell line HEK 293T cells and human cervical cancer cell line Hela cells. PARylated OVOL2 induces aneuploidy and cell death in Hela cells and suppresses the growth of Hela cells and human breast cancer cells (MCF7) in vivo [41] (Figure 2C). It is important to note that although many studies suggest the function of OVOL2 in various cancers, the majority of them are limited to cancer cells in vitro, and mouse models are generally lacking.

There are also no reports of OVOL2 mutations being found in human cancers. In vivo experiments are needed to emphasize the role of OVOL2 in cancer and to figure out whether OVOL2 participates in cancer development through EMT. One other thing to note is that most of these “non-EMT” roles of OVOL2 in cancer require other interacting proteins, and the classification of OVOL2 as a zinc-finger transcription factor should not limit the functional studies of OVOL2 to DNA binding. For example, OVOL2 protein can undergo PARylation by PARP1 [41] and ubiquitination by MDM2 [37] (Figure 2C). OVOL2 safeguards many developmental processes and adult homeostasis, thus its activity must be tightly controlled. Searching for other possible posttranslational modifications of OVOL2 (such as phosphorylation, acetylation, methylation, O-GlcNAcylation, SUMOylation, etc.) might shed new light on the regulation of OVOL2 activity.

PPCD is an autosomal dominant disorder of the corneal endothelium and Descemet’s membrane, producing a wide clinical variability [42]. Depending on the mutant genes, PPCD is further characterized into four different subtypes (PPCD1–4). Truncating mutations or deletions of ZEB1 account for about 30% of PPCD cases (PPCD3) [43], non-synonymous variations in COL8A2 are associated with minor PPCD cases (PPCD2) [44], and GRHL2 variants are linked to PPCD4 [45]. Several different non-coding mutations in the promoter of OVOL2 are linked to PPCD1 [46–48]. Although OVOL2 expression is not detected in the normal corneal endothelium, these mutations cause increased promoter activity and aberrant OVOL2 hyper-activation which may contribute to PPCD1 development [46, 47]. A complex fact about PPCD is that different PPCD causative genes can regulate the expression of each other, including ZEB1 as a downstream gene of OVOL2 and OVOL2 is a direct target of GRHL2 [27]. Further study shows that alterations in the ZEB1-OVOL2-GRHL2 axis caused by PPCD-associated mutations lead to aberrant activation of the Wnt signaling pathway and changes in corneal endothelial cell state [49]. Together, these data suggest that aberrant activation of OVOL2, or inactivation of ZEB1, may lead to abnormalities of the corneal epithelium and the majority of PPCD cases. However, whether OVOL2 mutations in PPCD directly decrease the expression of ZEB1 in corneal endothelium and how ZEB1/OVOL2/GRHL2 causes PPCD remain to be fully understood.

OVOL2 and EMT regulators in adipogenesis

The importance of OVOL2 in regulating adipogenesis has been recently discovered [19]. Mice with a viable mutation of Ovol2 (boh) develop severe obesity with hyperplasia of white adipose tissue, and overexpression of OVOL2 in adipocytes reduces diet-induced obesity. Mechanistic study shows that OVOL2 suppresses adipogenesis through direct interaction with C/EBPα to inhibit its transcriptional activity [19] (Figure 2B). Meanwhile, accumulated studies have also linked the key transcription factors that regulate EMT with adipogenesis and obesity, such as ZEB1, TWIST1, SNAI1, and SNAI2. In mouse pre-adipocyte 3T3-L1 cells and mouse stromal-vascular fraction (SVF) cells, ZEB1 cooperatively binds to adipogenic regulatory regions with C/EBPβ and mediates adipogenesis [50]. Although retroviral overexpression of TWIST1 in 3T3-L1 cells has no influence on adipogenic differentiation, knockdown of Twist1 decreases the expression of PPARγ in 3T3-L1 cells, and PPARγ agonist and antagonist also influence Twist1 expression, implying a possible regulatory link between TWIST1 and PPARγ in adipocytes [51–53]. In addition, SNAIL1 inhibits adipocyte differentiation by binding to the promoters of several adipogenic genes (Adipoq, Pparγ, and Nr2f6) and represses their expression [54–56]. SNAIL2 inhibits adipocyte differentiation of 3T3-L1 cells by interacting with the orphan nuclear receptor TR4 (NR2C2) and repressing its transcriptional activity [57]. It is noteworthy that the majority of these in vitro experiments are performed in 3T3-L1 cells, which may not recapitulate the whole process of adipogenesis as a later-stage model of adipocyte differentiation. In mouse models, it is reported that Zeb1^+/− female mice gained more weight on a high fat-diet than wild type controls [58]. Besides, a non-coding nucleotide substitution in the intron of Zeb1 causes obesity in twirler mice, as well as multiple developmental malformations [59]. SNAIL2 deficient mice exhibit decreased white adipose tissue size, and SNAIL2 overexpression mice have increased white adipose tissue size [60]. In human studies, genomic variation in the ZEB1 locus is associated with body fat distribution and obesity [61, 62]. Additionally, the mRNA level of ZEB1 in human SVF cells is correlated with fat cell differentiation potential [50]. TWIST1 regulates inflammation and fatty acid oxidation in human white adipose tissues, and its expression is negatively correlated with obesity and insulin resistance [63–65]. The involvement of OVOL2 and some EMT regulators in adipogenesis or obesity is clearly emerging from these data. However, it seems that all the reported EMT regulators don’t participate in adipogenesis through influencing the EMT process. It is unclear whether there is a direct connection between EMT and adipogenesis, or whether the function of these transcription factors in adipogenesis is independent of EMT. Also, the complex regulation between these EMT factors should be taken into account during investigations. It would be interesting to understand the role of these different EMT transcription factors in the regulatory network of adipogenic genes, the detailed mechanism, and the association of EMT and adipogenesis.

OVOL2 and EMT regulators in thermogenesis and lipid metabolism

OVOL2 deficient mice are extremely cold intolerant and display reduced brown and beige thermogenic adipose tissues with decreased thermal energy expenditure [19]. Among three different types of adipose tissues, white adipose tissues function as an energy reservoir, while brown and beige adipose tissues are specialized for heat production [66, 67]. On top of the role of OVOL2 in inhibiting white adipogenesis, OVOL2 is essential for promoting brown/beige adipose tissue-mediated thermogenesis [19]. Nevertheless, how OVOL2 functions in brown/beige adipose tissues is poorly understood. Besides OVOL2, other key EMT transcription factors are also involved in regulating brown/beige adipose tissues. TWIST1 plays an important role in brown adipose tissues by interacting with the transcriptional coactivator PGC-1α, which is a central regulator in brown fat thermogenesis [53]. Lack of TWIST1 in Twist1 haploinsufficiency (Twist1^+/−) mice leads to hyperactivation of PGC-1α target genes and an obesity-resistant phenotype with increased brown fat thermogenesis. In line with this, fat-specific overexpression of TWIST1 inhibits the expression of PGC-1α target genes and leads to the development of obesity on a high fat-diet with decreased brown fat thermogenesis [53]. Furthermore, the knockdown of Zeb1 decreases the level of the placental growth factor that is released from endothelial cells and the expression of Ucp1 in 3T3-L1 cells [68]. The studies of OVOL2 [19] and TWIST1 [53] focus on whole body level or fat cells, while the study of ZEB1 [68] focuses on endothelial cells within the microvasculature of adipose tissues, suggesting that OVOL2 and EMT regulators might work differently in separate cell populations of brown/beige adipose tissues to regulate thermogenesis. Besides thermogenesis, EMT transcription factors are also reported to regulate lipid metabolism in different tissues. In adipocytes, SNAIL1 inhibits the expression of adipose triglyceride lipase (ATGL) to regulate lipolysis and lipid partitioning between adipose and non-adipose tissues [69]. In hepatocytes, insulin stimulates a noncanonical pathway to upregulate the level of SNAIL1, which epigenetically suppresses the expression of lipogenic genes [70]. Different from SNAIL1, insulin-stimulated SNAIL2 (SLUG) in hepatocytes recruits lysine-specific demethylase-1 (LSD1) to bind to the promoter of fatty acid synthase (FASN) and stimulate its expression and lipogenesis [71]. In the Drosophila heart, the Snail family of transcription factors regulates lipogenesis and lipolysis gene expression and controls systemic lipid homeostasis [72]. Same as in adipogenesis, the relationship between the function of these EMT regulators in thermogenesis and lipid metabolism and the EMT process is also elusive. Brown/beige-mediated thermogenesis and lipid metabolism interact with each other to maintain a balanced energy metabolism. Is it a coincidence that these transcription factors simultaneously play a role in the EMT process and energy metabolism? Further investigation will hopefully yield insights into the complex regulation of OVOL2 and other EMT transcription factors in energy homeostasis.

Concluding remarks

Since the identification of OVOL2 as a mammal homolog of the Drosophila ovo family of transcription factors, numerous studies have revealed the critical role of OVOL2 in embryonic development and epithelial tissue morphogenesis with whole-body Ovol2 knockout mice and tissue-specific Ovol2 knockout mice. However, the lethality of homozygous Ovol2 knockout embryos before E10.5 has largely limited the exploration of OVOL2’s role in adult animals at the whole-body level. Recently, random mutagenesis in mice identified a hypomorphic mutation of Ovol2 (boh) that allows the development and survival of Ovol2^boh/boh into adulthood while presenting a massive obesity phenotype [19]. The boh is a missense mutation (C120Y in OVOL2-A) that does not affect the protein stability of OVOL2 while severely reducing the function of OVOL2, at least in energy metabolism. We believe this Ovol2^boh mouse will present a unique model to revisit previous studies regarding the role of OVOL2 in embryogenesis and epithelial tissue morphogenesis, as well as further studies of the role of OVOL2 in energy homeostasis. While the classic roles of OVOL2 in embryonic development and cancers are beginning to be understood, the emerging roles of OVOL2 and other EMT factors in energy homeostasis raise several outstanding questions (see Outstanding questions).

Outstanding questions.

How to understand the versatile roles of OVOL2 in different tissues with relatively low Ovol2 expression? Is the level of Ovol2 dynamically regulated under different physiological or pathological conditions?

What is the role of OVOL2 beyond a transcription factor and an inhibitor of EMT? What are the target organ (s) of OVOL2 in regulating energy metabolism?

Can OVOL2 affect angiogenesis and vascular remodeling in brown/beige adipose tissues? What is the role of the OVOL2-TWIST1 axis in brown/beige thermogenesis?

Is that possible that OVOL2 functions in adipocyte precursors to balance different pools of adipocytes?

How do OVOL2 and other EMT transcription factors coordinate with each other to control energy homeostasis?

How to study and understand the human relevance of OVOL2 in energy homeostasis?

What are the potential applications of OVOL2 in reducing human obesity?

In adult mice, OVOL2 is predominantly expressed in the testis. However, the expression of OVOL2 has also been found at relatively low levels in other tissues (such as skin, stomach, intestine, ovary, heart, skeletal muscle, and fat) [1, 11, 19, 73]. It is possible that OVOL2 is only highly expressed in a small subset of cells in these tissues. Considering the aberrant upregulation of OVOL2 expression in several PPCD non-coding mutants, the expression of OVOL2 itself might be dynamically regulated. Further studies are needed to thoroughly check the expression pattern of OVOL2 in different cell populations under various physiological or pathological conditions. Due to the existence of four zinc-finger domains in the OVOL2 protein, OVOL2 has long been thought of as a transcription factor that directly binds with DNA. Although this is largely true for the role of OVOL2 in EMT, several studies have shown that OVOL2 functions as a co-factor through directly interacting with other proteins, including ER71/ETV2, p65, TCF4, HDACs, SMAD4, and C/EBPα [16, 19, 23, 38, 40] (Figure 2). Further investigations are needed to define these “non-classic” roles of OVOL2 in the absence of DNA binding.

Although OVOL2 deficient mice are extremely cold intolerant with defective brown/beige adipose tissues, the exact role of OVOL2 in maintaining brown/beige thermogenesis is unknown. Further studies with tissue/cell type-specific deletion of Ovol2 would be important to define the target organ (s) of OVOL2 in regulating energy metabolism. Brown/beige adipose tissues are highly vascularized. Angiogenesis and vascular remodeling are tightly regulated with adipogenesis and thermogenesis [74]. Given the importance of OVOL2 in angiogenesis, the impact of OVOL2’s role in adipose tissue angiogenesis on brown/beige adipose tissues needs to be characterized. It is also important to identify the molecular link between OVOL2 and brown/beige fat thermogenesis, including a possible candidate – TWIST1, a direct target that is repressed by OVOL2 [14, 18]. The possible involvement of the OVOL2-TWIST1 axis in brown/beige thermogenesis needs to be carefully examined in the future. Distinct adipocyte precursors control the pool of white, brown, and beige adipocytes, which is critical for maintaining energy homeostasis [75]. Since the OVOL2 deficient mice present with excessive white adipose tissues with loss of brown/beige adipose tissues [19], further studies are needed to check the possible roles of OVOL2 in respective adipocyte precursors to balance the adipocyte pool. Besides OVOL2, other key EMT transcription factors have also been reported to regulate adipogenesis, thermogenesis, and lipid metabolism. How do OVOL2 and other EMT factors operate in healthy individuals and respond to energy imbalance and whether there is a potential direct connection between EMT and energy metabolism would be of great interest for further investigations.

Mouse OVOL2 protein is 83% identical to human OVOL2 and highly similar homologs are also found in other vertebrates [19]. We believe studying the human relevance of OVOL2 in energy metabolism as a potential therapeutic target to reduce obesity is a particularly exciting area for future research. Genome-wide association studies (GWAS) of body weight/body mass index (BMI) for OVOL2 mutations, the expression level of OVOL2 mRNA in individuals with high/low BMIs, and the different modifications of OVOL2 proteins in individuals with high/low BMIs should provide hints for study OVOL2 in humans. The potential applications of OVOL2 in reducing human obesity would be increased OVOL2 transcription, improved OVOL2 protein stability, or enhanced OVOL2 protein activity in adipose tissues. Understanding the transcriptional regulation of OVOL2 in human fat cells could lead to new therapeutic targets. Further characterization of OVOL2 interacting partners would allow for disruption of protein complexes that either directly interfere with OVOL2 activity or result in protein level change.

Highlights.

Ovo like zinc finger 2 (OVOL2) is a transcription factor that is mainly considered as a repressor of epithelial-to-mesenchymal transition (EMT).

OVOL2 determines the epithelial identity during embryogenesis and inhibits tumor metastasis during cancer development, many of these processes rely on the function of OVOL2 as a EMT inhibitor.

In addition to binding DNA with zinc-finger domains, recent studies show that OVOL2 interacts directly with other transcription factors to regulate downstream gene expression.

Recent advances of OVOL2 and other EMT factors in adipogenesis, thermogenesis, and lipid metabolism call attention to investigate the function of the EMT gene network in energy homeostasis.

Glossary

Descemet’s membrane

A basement membrane that lies between the posterior corneal stroma and the corneal endothelium. It is a dense, thick, relatively transparent, and cell-free matrix composed of different kinds of collagens.

Epithelial-mesenchymal transition (EMT)

A process by which epithelial cells lose their polarity and their cell-cell adhesion, and gain migratory, proliferation and differentiation properties to become mesenchymal cells.

Gastrulation

A key process in embryonic development that occurs after fertilization and before organogenesis. It is a complex process that involves the formation of three germ layers: ectoderm, mesoderm, and endoderm.

Haploinsufficiency

In diploid organisms, a single copy of a gene is insufficient to produce the wild-type phenotype.

Hypomorphic mutation

A type of loss-of-function mutation that can cause a partial loss of function in the protein it encodes.

Neural crest cells

A temporary group of cells that arise from the embryonic ectoderm germ layer, and in turn give rise to a diverse cell lineage—including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons, and glia.

NF-κB

A protein complex that controls transcription of DNA, cytokine production and cell survival. It is involved in a wide range of cellular processes such as inflammation, immune responses, cell proliferation and apoptosis.

Pachytene stage

A stage of meiosis that occurs during prophase I. It is characterized by the pairing of homologous chromosomes and crossing over between them.

PARylation

Poly(ADP-ribosyl)ation (RARylation) is a posttranslational modification of proteins by linear or branched chains of ADP-ribose residues from NAD+.

Primordial germ cells

The precursors of gametes in mammals. They are the first cells that differentiate into male or female germ cells.

Stromal Vascular Fraction (SVF)

The SVF includes several stem cell types which are precursors of fat tissue cells, as well as immune cells, fibroblasts, pericytes, endothelial cells, and others.

Transforming growth factor β (TGF-β)

A multifunctional cytokine that plays a crucial role in cell differentiation, proliferation, migration, and apoptosis.

Trophectoderm

Trophectoderm cells form extraembryonic tissues that act in a supporting role for the embryo proper.

Trophoblast stem cells

The precursors of the differentiated cells of the placenta.

Warburg effect

A phenomenon where cancer cells preferentially use glycolysis instead of oxidative phosphorylation to generate energy even in the presence of oxygen.

Wnt signaling

A group of signal transduction pathways that begin with proteins that pass signals into a cell through cell surface receptors. Wnt signals are active in numerous contexts, initially in early development and later during the growth and maintenance of various tissues.

XY body

A specialized nuclear territory formed by the sex chromosomes of mammalian spermatocytes during meiosis. It is a special subnuclear domain that is distinct from the rest of the nucleus and is in distinct contrast to the autosomes.