Ovol2 promoter mutations in mice and human illuminate species-specific phenotypic divergence.

Pathogenic variants in the highly conserved OVOL2 promoter region cause posterior polymorphous corneal dystrophy (PPCD) 1 by inducing an ectopic expression of the endothelial OVOL2 mRNA. Here we produced an allelic series of Ovol2 promoter mutations in the mouse model including the heterozygous c.-307T>C variant (RefSeq NM_021220.4) causing PPCD1 in humans. Despite the high evolutionary conservation of the Ovol2 promoter, only some alterations of its sequence had phenotypic consequences in mice. Four independent sequence variants in the distal part of the Ovol2 promoter had no significant effect on endothelial Ovol2 mRNA level or caused any ocular phenotype. In contrast, the mutation c.-307T>C resulted in increased Ovol2 expression in the corneal endothelium. However, only a small fraction of adult mice c.-307T>C heterozygotes developed ocular phenotypes such as irido-corneal adhesions, and corneal opacity. Interestingly, phenotypic penetrance was increased at embryonic stages. Notably, c.-307T>C mutation is located next to the Ovol1/Ovol2 transcription factor binding site. Mice carrying an allele with a deletion encompassing the Ovol2 binding site c.-307_-320del showed significant Ovol2 gene upregulation in the cornea endothelium and exhibited phenotypes similar to the c.-307T>C mutation. In conclusion, although the mutations c.-307T>C and -307_-320del lead to a comparably strong increase in endothelial Ovol2 expression as seen in PPCD1 patients, endothelial dystrophy was not observed in the mouse model, implicating species-specific differences in endothelial cell biology. Nonetheless, the emergence of dominant ocular phenotypes associated with Ovol2 promoter variants in mice implies a potential role of this gene in eye development and disease.

Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis.

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that OVOL2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) that drives gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and, consequently, induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation. This article has an associated 'The people behind the papers' interview.

Ovol1/2 loss-induced epidermal defects elicit skin immune activation and alter global metabolism.

Skin epidermis constitutes the outer permeability barrier that protects the body from dehydration, heat loss, and myriad external assaults. Mechanisms that maintain barrier integrity in constantly challenged adult skin and how epidermal dysregulation shapes the local immune microenvironment and whole-body metabolism remain poorly understood. Here, we demonstrate that inducible and simultaneous ablation of transcription factor-encoding Ovol1 and Ovol2 in adult epidermis results in barrier dysregulation through impacting epithelial-mesenchymal plasticity and inflammatory gene expression. We find that aberrant skin immune activation then ensues, featuring Langerhans cell mobilization and T cell responses, and leading to elevated levels of secreted inflammatory factors in circulation. Finally, we identify failure to gain body weight and accumulate body fat as long-term consequences of epidermal-specific Ovol1/2 loss and show that these global metabolic changes along with the skin barrier/immune defects are partially rescued by immunosuppressant dexamethasone. Collectively, our study reveals key regulators of adult barrier maintenance and suggests a causal connection between epidermal dysregulation and whole-body metabolism that is in part mediated through aberrant immune activation.

"Pulsed Hypoxia" Gradually Reprograms Breast Cancer Fibroblasts into Pro-Tumorigenic Cells via Mesenchymal-Epithelial Transition.

Hypoxia arises in most growing solid tumors and can lead to pleotropic effects that potentially increase tumor aggressiveness and resistance to therapy through regulation of the expression of genes associated with the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). The main goal of the current work was to obtain and investigate the intermediate phenotype of tumor cells undergoing the hypoxia-dependent transition from fibroblast to epithelial morphology. Primary breast cancer fibroblasts BrC4f, being cancer-associated fibroblasts, were subjected to one or two rounds of "pulsed hypoxia" (PH). PH induced transformation of fibroblast-shaped cells to semi-epithelial cells. Western blot analysis, fluorescent microscopy and flow cytometry of transformed cells demonstrated the decrease in the mesenchymal markers vimentin and N-cad and an increase in the epithelial marker E-cad. These cells kept mesenchymal markers αSMA and S100A4 and high ALDH activity. Real-time PCR data of the cells after one (BrC4f_Hyp1) and two (BrC4f_Hyp2) rounds of PH showed consistent up-regulation of TWIST1 gene as an early response and ZEB1/2 and SLUG transcriptional activity as a subsequent response. Reversion of BrC4f_Hyp2 cells to normoxia conditions converted them to epithelial-like cells (BrC4e) with decreased expression of EMT genes and up-regulation of MET-related OVOL2 and c-MYC genes. Transplantation of BrC4f and BrC4f_Hyp2 cells into SCID mice showed the acceleration of tumor growth up to 61.6% for BrC4f_Hyp2 cells. To summarize, rounds of PH imitate the MET process of tumorigenesis in which cancer-associated fibroblasts pass through intermediate stages and become more aggressive epithelial-like tumor cells.

An NF-κB/OVOL2 circuit regulates glucose import and cell survival in non-small cell lung cancer.

BACKGROUND: Tumor cells tend to utilize glycolysis rather than aerobic respiration even under aerobic conditions. OVOL2, an inhibitory C2H2 zinc finger transcription factor, is a potential tumor suppressor in cancers. However, the association between OVOL2 and tumor energy metabolism is unknown. METHODS: Western blotting was used to determine the expression of OVOL2 in different non-small cell lung cancer (NSCLC) cell lines and mouse models. The metabolic parameters in NSCLC cells following overexpression or knockdown OVOL2 were examined. To define the mechanism by which OVOL2 regulates aerobic glycolysis, interacting protein of OVOl2 and downstream molecular events were identified by luciferase assay and co-immunoprecipitation. We documented the regulatory mechanism in mouse xenograft models. Finally, clinical relevance of OVOL2, NF-κB signaling and GLUT1 was measured by immunostaining. RESULTS: OVOL2 is downregulated in NSCLC and overexpression of OVOL2 inhibits the survival of cancer cells. Moreover, OVOL2 directly binds to P65 and inhibits the recruitment of P300 but facilitates the binding of HDAC1 to P65, which in turn negatively regulates NF-κB signaling to suppress GLUT1 translocation and glucose import. In contrast, OVOL2 expression is negatively regulated by NF-κB signaling in NSCLC cells via the ubiquitin-proteasome pathway. Re-expression of OVOL2 significantly compromise NF-κB signaling-induced GLUT1 translocation, aerobic glycolysis in NSCLC cells and mouse models. Immunostaining revealed inverse correlations between the OVOL2 and phosphorylated P65 levels and between the OVOL2 and membrane GLUT1 levels, and a strong correlation between the phosphorylated P65 and membrane GLUT1 levels. CONCLUSIONS: These results suggest a regulatory circuit linking NF-κB and OVOL2, which highlights the role of NF-κB signaling and OVOL2 in the modulation of glucose metabolism in NSCLC. Video Abstract.

OVOL1/2: Drivers of Epithelial Differentiation in Development, Disease, and Reprogramming.

OVOL proteins (OVOL1 and OVOL2), vertebrate homologs of Drosophila OVO, are critical regulators of epithelial lineage determination and differentiation during embryonic development in tissues such as kidney, skin, mammary epithelia, and testis. OVOL can inhibit epithelial-mesenchymal transition and/or can promote mesenchymal-epithelial transition. Moreover, they can regulate the stemness of cancer cells, thus playing an important role during cancer cell metastasis. Due to their central role in differentiation and maintenance of epithelial lineage, OVOL overexpression has been shown to be capable of reprogramming fibroblasts to epithelial cells. Here, we review the roles of OVOL-mediated epithelial differentiation across multiple contexts, including embryonic development, cancer progression, and cellular reprogramming.

Altered Epithelial-mesenchymal Plasticity as a Result of Ovol2 Deletion Minimally Impacts the Self-renewal of Adult Mammary Basal Epithelial Cells.

Stem-cell containing mammary basal epithelial cells exist in a quasi-mesenchymal transcriptional state characterized by simultaneous expression of typical epithelial genes and typical mesenchymal genes. Whether robust maintenance of such a transcriptional state is required for adult basal stem cells to fuel self-renewal and regeneration remains unclear. In this work, we utilized SMA-CreER to direct efficient basal cell-specific deletion of Ovol2, which encodes a transcription factor that inhibits epithelial-to-mesenchymal transition (EMT), in adult mammary gland. We identified a basal cell-intrinsic role of Ovol2 in promoting epithelial, and suppressing mesenchymal, molecular traits. Interestingly, Ovol2-deficient basal cells display minimal perturbations in their ability to support tissue homeostasis, colony formation, and transplant outgrowth. These findings underscore the ability of adult mammary basal cells to tolerate molecular perturbations associated with altered epithelia-mesenchymal plasticity without drastically compromising their self-renewal potential.

An Ovol2-Zeb1 transcriptional circuit regulates epithelial directional migration and proliferation.

Directional migration is inherently important for epithelial tissue regeneration and repair, but how it is precisely controlled and coordinated with cell proliferation is unclear. Here, we report that Ovol2, a transcriptional repressor that inhibits epithelial-to-mesenchymal transition (EMT), plays a crucial role in adult skin epithelial regeneration and repair. Ovol2-deficient mice show compromised wound healing characterized by aberrant epidermal cell migration and proliferation, as well as delayed anagen progression characterized by defects in hair follicle matrix cell proliferation and subsequent differentiation. Epidermal keratinocytes and bulge hair follicle stem cells (Bu-HFSCs) lacking Ovol2 fail to expand in culture and display molecular alterations consistent with enhanced EMT and reduced proliferation. Live imaging of wound explants and Bu-HFSCs reveals increased migration speed but reduced directionality, and post-mitotic cell cycle arrest. Remarkably, simultaneous deletion of Zeb1 encoding an EMT-promoting factor restores directional migration to Ovol2-deficient Bu-HFSCs. Taken together, our findings highlight the important function of an Ovol2-Zeb1 EMT-regulatory circuit in controlling the directional migration of epithelial stem and progenitor cells to facilitate adult skin epithelial regeneration and repair.

Ovol2, a zinc finger transcription factor, is dispensable for spermatogenesis in mice.

Ovol2, a mouse homolog of Drosophila ovo, was identified as a zinc finger transcription factor predominantly expressed in testis. However, the function of Ovol2 in postnatal male germ cell development remains enigmatic. Here, we firstly examined the mRNA and protein levels of Ovol2 in developing mouse testes by RT-qPCR and western blot and found that both mRNA and protein of Ovol2 are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to adult testes (P56) and exhibits its higher level at adult testis. Further testicular immuno-staining revealed that OVOL2 is highly expressed in the spermatogonia, spermatocytes and round spermatids. Interestingly, our conditional ovol2 knockout mouse model show that loss of ovol2 in embryonic germ cells does not affect fecundity in mice. Our data also show that Ovol1 may have compensated for the loss of Ovol2 functions in germ cells. Overall, our data indicate that ovol2 is dispensable for germ cell development and spermatogenesis.

Alterations in GRHL2-OVOL2-ZEB1 axis and aberrant activation of Wnt signaling lead to altered gene transcription in posterior polymorphous corneal dystrophy.

Mutations associated with posterior polymorphous corneal dystrophy (PPCD) have been identified in three genes: ZEB1 (zinc-finger E-box binding homeobox 1) associated with sub-type PPCD3; OVOL2 (ovol-like zinc finger 2) associated with sub-type PPCD1; and GRHL2 (grainyhead like transcription factor 2) associated with sub-type PPCD4. Each of these genes encodes a transcription factor that regulates cell-state transitions. While the discovery of these PPCD-associated genes has greatly expanded our knowledge of the genetic basis of PPCD, the molecular mechanisms via which mutations in these genes lead to indistinguishable disease phenotypes have yet to be elucidated. To characterize the gene expression profiles of the genetic sub-types of PPCD, RNA-seq was performed on corneal endothelium derived from an individual with PPCD1 who harbors a c.-307T > C OVOL2 promoter mutation. Transcriptomic analysis of this and previously-reported RNA-seq data from two individuals with PPCD (the first with PPCD3 associated with a ZEB1 truncating mutation (c.1381delinsGACGAT) and the second with genetically unresolved PPCD in which ZEB1 coding region, OVOL2 promoter and GRHL2 promoter, exon 1, and intron 1 mutations were excluded) revealed: OVOL2 expression increased in PPCD1 (259 fold), unchanged in PPCD3 and slightly increased in genetically unresolved PPCD (from 0 TPM to 0.86 TPM, undefined fold change); ZEB1 expression decreased in PPCD1 (-5.9 fold), PPCD3 (-3.95 fold) and genetically unresolved PPCD (-3.96 fold); and GRHL2 expression increased in PPCD1 (333.5 fold), slightly increased (from 0 TPM to 0.67 TPM, undefined fold change) in PPCD3 and increased in genetically unresolved PPCD (1853 fold). Additionally, as the majority of pedigrees affected with PPCD remain genetically unresolved, we screened the promoter, exon 1, and intron 1 regions of GRHL2 in 24 PPCD probands who do not harbor a ZEB1 or OVOL2 mutation. GRHL2 screening did not identify any novel or rare GRHL2 variant in these 24 individuals. As ZEB1 can act as an activator or repressor of downstream target gene expression depending on Wnt signaling pathway activation or deactivation, we also sought to determine whether or not Wnt signaling is active in PPCD by performing immunohistochemistry in corneal tissue sections derived from an individual affected with PPCD3 and from an individual with genetically unresolved PPCD. Immunohistochemistry results demonstrated corneal endothelial nuclear accumulation of S552 phos-ß-catenin and cytosolic localization of S33/37/T42 non-phosphorylated ß-catenin in PPCD, indicating aberrant activation of Wnt signaling, which was not observed in control corneal endothelium. These findings suggest that alterations in the ZEB1-OVOL2-GRHL2 axis (caused by PPCD-associated mutations) lead to changes in corneal endothelial cell state and molecular pathways, including the aberrant activation of the Wnt signaling pathway.

The presence of androgen receptor elements regulates ZEB1 expression in the absence of androgen receptor.

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that plays a central role in the epithelial to mesenchymal transition (EMT) of cancer cell lines. Studies on its regulation have mostly focused on the negative 3'UTR binding of miR200c. Interestingly, it has been previously reported that androgen receptor (AR) regulates ZEB1 expression in breast and prostate cancers. In order to validate this, various ZEB1 promoter deletions were cloned into a luciferase reporter system to elucidate the contribution of two putative androgen response elements (AREs). The in vivo contribution of AR was also assessed in cell lines after R1881 treatment using qPCR with prostate specific antigen (PSA) as the positive control. We discovered that AR upregulates the levels of expression of ZEB1 10-fold on a luciferase promoter that only contains the distal ARE. However, when the proximal ARE is included, no additional activation is apparent with AR or its hormone independent variant, AR-V7. Furthermore, we demonstrate here that a promoter construct containing both AREs activates transcription of ZEB1 even in the AR-null cell lines DU145 and PC3. Incubation of the AR-positive cell line, LNCaP with R1881, failed to substantially increase the expression levels of ZEB1. Despite the presence of AREs in the promoter region, it appears that ZEB1 expression can be induced even without AR. In addition, the region around the distal ARE is a potent repressor in AR-null cell lines.

Loss of OVOL2 in Triple-Negative Breast Cancer Promotes Fatty Acid Oxidation Fueling Stemness Characteristics.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, has a poor prognosis and lacks effective treatment strategies. Here, the study discovered that TNBC shows a decreased expression of epithelial transcription factor ovo-like 2 (OVOL2). The loss of OVOL2 promotes fatty acid oxidation (FAO), providing additional energy and NADPH to sustain stemness characteristics, including sphere-forming capacity and tumor initiation. Mechanistically, OVOL2 not only suppressed STAT3 phosphorylation by directly inhibiting JAK transcription but also recruited histone deacetylase 1 (HDAC1) to STAT3, thereby reducing the transcriptional activation of downstream genes carnitine palmitoyltransferase1 (CPT1A and CPT1B). PyVT-Ovol2 knockout mice develop a higher number of primary breast tumors with accelerated growth and increased lung-metastases. Furthermore, treatment with FAO inhibitors effectively reduces stemness characteristics of tumor cells, breast tumor initiation, and metastasis, especially in OVOL2-deficient breast tumors. The findings suggest that targeting JAK/STAT3 pathway and FAO is a promising therapeutic strategy for OVOL2-deficient TNBC.

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

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 have suggested some noncanonical roles of OVOL2 that do not rely on the DNA binding of zinc-finger domains or regulation of EMT. 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. Last, we propose potential applications of targeting OVOL2 to reduce human obesity.

OVOL2 inhibits macrophage M2 polarization by regulating IL-10 transcription, and thus inhibits the tumor metastasis by modulating the tumor microenvironment.

Invasion and metastasis of breast cancer cells is an important cause of death in breast cancer patients. In the tumor microenvironment, M2 polarization of macrophages can promote the invasion and metastasis of tumor cells. OVOL2 is an evolutionarily conserved transcription regulator, but its effect in macrophages has not been described previously. The aim of this study was to investigate the effects of OVOL2 on macrophage polarity and the role of these effects in the tumor metastasis. We found that overexpression of OVOL2 in macrophages significantly inhibited M2 polarization and thus inhibits breast cancer metastasis. We propose a novel mechanism in which OVOL2 inhibits M2 polarization of macrophages and thus reduces their ability to induce invasion and metastasis of breast cancer. By shedding new light on the regulation of metastasis in cancers, our study provides a new strategy for the targeted therapy of cancer.

Posterior corneal vesicles are not associated with the genetic variants that cause posterior polymorphous corneal dystrophy.

PURPOSE: Posterior corneal vesicles (PCVs) have clinical features that are similar to posterior polymorphous corneal dystrophy (PPCD). To help determine whether there is a shared genetic basis, we screened 38 individuals with PCVs for changes in the three genes identified as causative for PPCD. METHODS: We prospectively recruited patients for this study. We examined all individuals clinically, with their first-degree relatives when available. We used a combination of Sanger and exome sequencing to screen regulatory regions of OVOL2 and GRHL2, and the entire ZEB1 coding sequence. RESULTS: The median age at examination was 37.5 years (range 4.7-84.0 years), 20 (53%) were male and in 19 (50%) the PCVs were unilateral. Most individuals were discharged to optometric review, but five had follow-up for a median of 12 years (range 5-13 years) with no evidence of progression. In cases with unilateral PCVs, there was statistically significant evidence that the change in the affected eye was associated with a lower endothelial cell density (p = 0.0003), greater central corneal thickness (p = 0.0277) and a steeper mean keratometry (p = 0.0034), but not with a higher keratometric astigmatism or a reduced LogMAR visual acuity. First-degree relatives of 13 individuals were available for examination, and in 3 (23%), PCVs were identified. No possibly pathogenic variants were identified in the PPCD-associated genes screened. CONCLUSION: We found no evidence that PCVs share the same genetic background as PPCD. In contrast to PPCD, we confirm that PCVs is a mild, non-progressive condition with no requirement for long-term review. However, subsequent cataract surgery can lead to corneal oedema.

Ovo Like Zinc Finger 2 (OVOL2) Suppresses Breast Cancer Stem Cell Traits and Correlates with Immune Cells Infiltration.

Background: Breast cancer stem cells (BCSCs) are associated with tumor initiation, invasion, metastasis and drug resistance. It is known that many proteins and signaling pathways are involved in the regulation of BCSCs, however, much more efforts are needed to understand BCSCs comprehensively. Tumor-infiltrating immune cells are important in cancer treatment efficacy and patient prognosis. We tried to identify potential suppressor of BCSCs and analyze its correlation with various immune cells infiltration by bioinformatic and experimental methods. Methods: Expression level and methylation state of OVOL2 were analyzed by tools from bc-GenExMiner v4.8 and UALCAN databases. The Kaplan-Meier plotter was applied to evaluate the prognostic values of OVOL2. Gene expression datasets (GSE7515, GSE15192) were selected to analyze differentially expressed genes (DEGs) related to BCSCs. GO and KEGG pathway analyses of DEGs were conducted. MCODE app plugin of Cytoscape was used to screen modules in PPI network of downregulated DEGs. Correlation of OVOL2 expression with infiltrating immune cells was evaluated by TIMER 2.0. Experiments were conducted to verify whether OVOL2 could inhibit stemness traits of breast cancer cell MDA-MB-231. Results: The expression level of OVOL2 in basal/TNBC was significantly lower than that of other subtypes. Survival analyses indicated that high expression of OVOL2 was associated with favorable prognosis. GO and KEGG pathway analyses for upregulated and downregulated DEGs were conducted. The top three clusters of downregulated DEGs showed that tight junction and chemokines may play important roles in BCSCs. OVOL2 is one module of clusters. OVOL2 expression is correlated with various immune cells infiltration. Experiments showed that OVOL2 suppresses CD44+/CD24- ratio and mammospheres formation of MDA-MB-231. Conclusion: OVOL2 may play an important role in the regulation of breast cancer stemness and immune cell infiltration, and is likely to be a target for the treatment of breast cancer.

Transcriptional Repression of Aerobic Glycolysis by OVOL2 in Breast Cancer.

Aerobic glycolysis (Warburg effect), a hallmark of cancer, plays a critical role in cancer cell growth and metastasis; however, direct inhibition of the Warburg effect remains largely unknown. Herein, the transcription factor OVO-like zinc finger 2 (OVOL2) is demonstrated to directly repress the expression of several glycolytic genes, blocking the Warburg effect and breast tumor growth and metastasis in vitro and in vivo. OVOL2 inhibits glycolysis by recruiting the nuclear receptor co-repressor (NCoR) and histone deacetylase 3 (HDAC3). The tumor suppressor p53, a key regulator of cancer metabolism, activates OVOL2 by binding to the oncoprotein mouse double minute 2 homolog (MDM2) and inhibiting MDM2-mediated ubiquitination and degradation of OVOL2. OVOL2 expression is negatively correlated with glycolytic gene expression and can be a good predictor of prognosis in patients with breast cancer. Therefore, targeting the p53/MDM2/OVOL2 axis provides a potential avenue for cancer treatment, especially breast cancer.

OVOL2 impairs RHO GTPase signaling to restrain mitosis and aggressiveness of Anaplastic Thyroid Cancer.

BACKGROUND: Anaplastic Thyroid Cancer (ATC) is an undifferentiated and aggressive tumor that often originates from well-Differentiated Thyroid Carcinoma (DTC) through a trans-differentiation process. Epithelial-to-Mesenchymal Transition (EMT) is recognized as one of the major players of this process. OVOL2 is a transcription factor (TF) that promotes epithelial differentiation and restrains EMT during embryonic development. OVOL2 loss in some types of cancers is linked to aggressiveness and poor prognosis. Here, we aim to clarify the unexplored role of OVOL2 in ATC. METHODS: Gene expression analysis in thyroid cancer patients and cell lines showed that OVOL2 is mainly associated with epithelial features and its expression is deeply impaired in ATC. To assess OVOL2 function, we established an OVOL2-overexpression model in ATC cell lines and evaluated its effects by analyzing gene expression, proliferation, invasion and migration abilities, cell cycle, specific protein localization through immunofluorescence staining. RNA-seq profiling showed that OVOL2 controls a complex network of genes converging on cell cycle and mitosis regulation and Chromatin Immunoprecipitation identified new OVOL2 target genes. RESULTS: Coherently with its reported function, OVOL2 re-expression restrained EMT and aggressiveness in ATC cells. Unexpectedly, we observed that it caused G2/M block, a consequent reduction in cell proliferation and an increase in cell death. This phenotype was associated to generalized abnormalities in the mitotic spindle structure and cytoskeletal organization. By RNA-seq experiments, we showed that many pathways related to cytoskeleton and migration, cell cycle and mitosis are profoundly affected by OVOL2 expression, in particular the RHO-GTPase pathway resulted as the most interesting. We demonstrated that RHO GTPase pathway is the central hub of OVOL2-mediated program in ATC and that OVOL2 transcriptionally inhibits RhoU and RhoJ. Silencing of RhoU recapitulated the OVOL2-driven phenotype pointing to this protein as a crucial target of OVOL2 in ATC. CONCLUSIONS: Collectively, these data describe the role of OVOL2 in ATC and uncover a novel function of this TF in inhibiting the RHO GTPase pathway interlacing its effects on EMT, cytoskeleton dynamics and mitosis.

Obesity caused by an OVOL2 mutation reveals dual roles of OVOL2 in promoting thermogenesis and limiting white adipogenesis.

Using random germline mutagenesis in mice, we identified a viable hypomorphic allele (boh) of the transcription-factor-encoding gene Ovol2 that resulted in obesity, which initially developed with normal food intake and physical activity but decreased energy expenditure. Fat weight was dramatically increased, while lean weight was reduced in 12-week-old boh homozygous mice, culminating by 24 weeks in massive obesity, hepatosteatosis, insulin resistance, and diabetes. The Ovol2boh/boh genotype augmented obesity in Lepob/ob mice, and pair-feeding failed to normalize obesity in Ovol2boh/boh mice. OVOL2-deficient mice were extremely cold intolerant. OVOL2 is essential for brown/beige adipose tissue-mediated thermogenesis. In white adipose tissues, OVOL2 limited adipogenesis by blocking C/EBPα engagement of its transcriptional targets. Overexpression of OVOL2 in adipocytes of mice fed with a high-fat diet reduced total body and liver fat and improved insulin sensitivity. Our data reveal that OVOL2 plays dual functions in thermogenesis and adipogenesis to maintain energy balance.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.