Oncogene EVI1 drives acute myeloid leukemia via a targetable interaction with CTBP2.

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.

SOX4 regulates proliferation and apoptosis of human ovarian granulosa-like tumor cell line KGN through the Hippo pathway.

The proliferation and apoptosis of ovarian granulosa cells are important for folliculogenesis. As a transcription factor, SRY-box transcription factor 4 (SOX4) has important roles in regulating cellular proliferation and apoptosis. Nonetheless, the regulatory mechanisms of SOX4 on proliferation and apoptosis of granulosa cells remain elusive. Therefore, a stably overexpressed SOX4 ovarian granulosa cell line KGN was generated by lentivirus encapsulation. We observed that overexpression of SOX4 inhibits apoptosis, promotes proliferation and migration of KGN cells. Comparative analysis of the transcriptome revealed 868 upregulated and 696 downregulated DEGs in LV-SOX4 in comparison with LV-CON KGN cell lines. Afterward, further assessments were performed to explore the possible functions about these DEGs. The data showed their involvement in many biological processes, particularly the Hippo signaling pathway. Moreover, the expression levels of YAP1, WWTR1, WTIP, DLG3, CCN2, and AMOT, which were associated with the Hippo signaling pathway, were further validated by qRT-PCR. In addition, the protein expression levels of YAP1 were markedly elevated, while p-YAP1 were notably reduced after overexpression of SOX4 in KGN cells. Thus, these results suggested that SOX4 regulates apoptosis, proliferation and migration of KGN cells, at least partly, through activation of the Hippo signaling pathway, which might be implicated in mammalian follicle development.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

The fungal pathogen Ustilago maydis targets the maize corepressor RELK2 to modulate host transcription for tumorigenesis.

Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection. In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N-terminus of RELK2 through its two Ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of RELK2. We propose that Tip6 mimics the recruitment of RELK2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6. Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor RELK2 to disrupt the transcriptional networks of the host plant.

Intestinal-specific Hdac3 deletion increases susceptibility to colitis and small intestinal tumor development in mice fed a high-fat diet.

High-fat (HF) diets (HFDs) and inflammation are risk factors for colon cancer; however, the underlying mechanisms remain to be fully elucidated. The transcriptional corepressor HDAC3 has recently emerged as a key regulator of intestinal epithelial responses to diet and inflammation with intestinal-specific Hdac3 deletion (Hdac3IKO) in mice increasing fatty acid oxidation genes and the rate of fatty acid oxidation in enterocytes. Hdac3IKO mice are also predisposed to experimentally induced colitis; however, whether this is driven by the intestinal metabolic reprogramming and whether this predisposes these mice to intestinal tumorigenesis is unknown. Herein, we examined the effects of intestinal-specific Hdac3 deletion on colitis-associated intestinal tumorigenesis in mice fed a standard (STD) or HFD. Hdac3IKO mice were highly prone to experimentally induced colitis, which was further enhanced by an HFD. Hdac3 deletion also accelerated intestinal tumor development, specifically when fed an HFD and most notably in the small intestine where lipid absorption is maximal. Expression of proteins involved in fatty acid metabolism and oxidation (SCD1, EHHADH) were elevated in the small intestine of Hdac3IKO mice fed an HFD, and these mice displayed increased levels of lipid peroxidation, DNA damage, and apoptosis in their villi, as well as extensive expansion of the stem cell and progenitor cell compartment. These findings reveal a novel role for Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover.NEW & NOTEWORTHY We reveal a novel role for the transcriptional corepressor Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover. We also identify a unique mouse model for investigating the complex interplay between diet, metabolic reprogramming, and tumor predisposition in the intestinal epithelium.

GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage.

Post-translational modifications of histones are important regulators of the DNA damage response (DDR). By using affinity purification mass spectrometry (AP-MS) we discovered that genetic suppressor element 1 (GSE1) forms a complex with the HDAC1/CoREST deacetylase/demethylase co-repressor complex. In-depth phosphorylome analysis revealed that loss of GSE1 results in impaired DDR, ATR signalling and γH2AX formation upon DNA damage induction. Altered profiles of ATR target serine-glutamine motifs (SQ) on DDR-related hallmark proteins point to a defect in DNA damage sensing. In addition, GSE1 knock-out cells show hampered DNA damage-induced phosphorylation on SQ motifs of regulators of histone post-translational modifications, suggesting altered histone modification. While loss of GSE1 does not affect the histone deacetylation activity of CoREST, GSE1 appears to be essential for binding of the deubiquitinase USP22 to CoREST and for the deubiquitination of H2B K120 in response to DNA damage. The combination of deacetylase, demethylase, and deubiquitinase activity makes the USP22-GSE1-CoREST subcomplex a multi-enzymatic eraser that seems to play an important role during DDR. Since GSE1 has been previously associated with cancer progression and survival our findings are potentially of high medical relevance.

Normal cell cycle progression requires negative regulation of E2F1 by Groucho during S phase and its relief at G2 phase.

The cell cycle depends on a sequence of steps that are triggered and terminated via the synthesis and degradation of phase-specific transcripts and proteins. Although much is known about how stage-specific transcription is activated, less is understood about how inappropriate gene expression is suppressed. Here, we demonstrate that Groucho, the Drosophila orthologue of TLE1 and other related human transcriptional corepressors, regulates normal cell cycle progression in vivo. We show that, although Groucho is expressed throughout the cell cycle, its activity is selectively inactivated by phosphorylation, except in S phase when it negatively regulates E2F1. Constitutive Groucho activity, as well as its depletion and the consequent derepression of e2f1, cause cell cycle phenotypes. Our results suggest that Cdk1 contributes to phase-specific phosphorylation of Groucho in vivo. We propose that Groucho and its orthologues play a role in the metazoan cell cycle that may explain the links between TLE corepressors and several types of human cancer.

HHEX suppresses advanced thyroid cancer by interacting with TLE3.

Haematopoietically Expressed Homeobox (HHEX) gene is highly expressed in the thyroid gland and plays critical roles in the development and differentiation of the thyroid gland. While it has been indicated to be downregulated in thyroid cancer, its function and the underlying mechanism remain unclear. Herein, we observed low expression and aberrant cytoplasmic localization of HHEX in thyroid cancer cell lines. Knockdown of HHEX significantly enhanced cell proliferation, migration and invasion, while overexpression of HHEX showed the opposite effects in vitro and in vivo. These data provide evidence that HHEX is a tumor suppressor in thyroid cancer. Additionally, our results showed that HHEX overexpression upregulated the expression of sodium iodine symporter (NIS) mRNA and also enhanced NIS promoter activity, suggesting a favorable effect of HHEX in promoting thyroid cancer differentiation. Mechanistically, HHEX exerted a regulatory effect on the expression of transducin-like enhancer of split 3 (TLE3) protein, which inhibited the Wnt/ß-catenin signaling pathway. Nuclear localized HHEX bound to and upregulated TLE3 expression by preventing TLE3 protein from being distributed to the cytoplasm and being ubiquitinated. In conclusion, our study suggested that restoring HHEX expression has the potential to be a new strategy in the treatment of advanced thyroid cancer.

CDYL knockdown reduces glioma development through an antitumor immune response in the tumor microenvironment.

Gliomas are highly prevalent and aggressive brain tumors. Growing evidence shows that epigenetic changes are closely related to cancer development. Here we report the roles of Chromodomain Y-like (CDYL), an important epigenetic transcriptional corepressor in the central nervous system in glioma progression. We found that CDYL was highly expressed in glioma tissues and cell lines. CDYL knockdown decreased cell mobility in vitro and significantly reduced tumor burden in the xenograft mouse in vivo. RNA sequencing analysis revealed the upregulation of immune pathways after CDYL knockdown, as well as chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 12. The immunohistochemistry staining and macrophage polarization assays showed increased infiltration of M1-like tumor-associated macrophages/microglia (TAMs) while decreased infiltration of M2-like TAMs after CDYL knockdown in vivo and in vitro. Following the in situ TAMs depletion or CCL2 antibody neutralization, the tumor-suppressive role of CDYL knockdown was abolished. Collectively, our results show that CDYL knockdown suppresses glioma progression, which is associated with CCL2-recruited monocytes/macrophages and the polarization of M1-like TAMs in the tumor microenvironment, indicating CDYL as a promising target for glioma treatment.

JAZF1 safeguards human endometrial stromal cells survival and decidualization by repressing the transcription of G0S2.

Decidualization of human endometrial stromal cells (hESCs) is essential for the maintenance of pregnancy, which depends on the fine-tuned regulation of hESCs survival, and its perturbation contributes to pregnancy loss. However, the underlying mechanisms responsible for functional deficits in decidua from recurrent spontaneous abortion (RSA) patients have not been elucidated. Here, we observed that JAZF1 was significantly downregulated in stromal cells from RSA decidua. JAZF1 depletion in hESCs resulted in defective decidualization and cell death through apoptosis. Further experiments uncovered G0S2 as a important driver of hESCs apoptosis and decidualization, whose transcription was repressed by JAZF1 via interaction with G0S2 activator Purß. Moreover, the pattern of low JAZF1, high G0S2 and excessive apoptosis in decidua were consistently observed in RSA patients. Collectively, our findings demonstrate that JAZF1 governs hESCs survival and decidualization by repressing G0S2 transcription via restricting the activity of Purß, and highlight the clinical implications of these mechanisms in the pathology of RSA.

Somatostatin receptor activity assessed by 68Ga-DOTATOC PET can preoperatively predict DAXX/ATRX loss of expression in well-differentiated pancreatic neuroendocrine tumors.

PURPOSE: To evaluate the role of 68Ga-DOTATOC PET parameters in predicting DAXX/ATRX loss of expression in patients with Pancreatic neuroendocrine tumors (PanNET) candidate to surgery. METHODS: This retrospective study included 72 consecutive patients with PanNET (January 2018-March 2022) who underwent to 68Ga-DOTATOC PET for preoperative staging. Image analysis: qualitative assessment and extraction of SUVmax, SUV mean, somatostatin receptor density (SRD), and total lesion somatostatin receptor density (TLSRD) from primary PanNET. Radiological diameter and biopsy information (grade, Ki67) were collected. Loss of expression (LoE) of DAXX/ATRX was assessed by immunohistochemistry on surgical specimen. Student t-test, univariate and multivariate logistic regression and ROC curves have been used to investigate the predictive value of PET parameters on DAXX/ATRX LoE. RESULTS: Forty-two/72 patients had a G1, 28/72 a G2, and 2/72 a G3 PanNET. Seven/72 patients had DAXX LoE, 10/72 ATRX LoE, and 2/72 DAXX/ATRX LoE. SRD and TLSRD could predict DAXX LoE (p = 0.002, p = 0.018, respectively). By evaluating SRD in combination with radiological diameter, only SRD maintained statistical significance (multivariate logistic regression: p = 0.020, OR = 1.05), providing the best prediction (AUC-ROC = 79.01%; cut-off = 46.96; sensitivity = 77.78%; specificity = 88.89%). In the sub-analysis performed on 55 patients with biopsy availability, SRD demonstrated its role in providing useful and additional information (multivariate logistic regression: SRD p = 0.007; grade p = 0.040). CONCLUSION: SRD has a predictive role on DAXX LoE in PanNETs, with higher probability of LoE at increasing SRD values. SRD provides complementary/additional information to grade assessed on biopsy material, and the combined use of these approaches might support patients' management by preoperatively identifying subjects with more aggressive diseases.

DAXX drives de novo lipogenesis and contributes to tumorigenesis.

Cancer cells exhibit elevated lipid synthesis. In breast and other cancer types, genes involved in lipid production are highly upregulated, but the mechanisms that control their expression remain poorly understood. Using integrated transcriptomic, lipidomic, and molecular studies, here we report that DAXX is a regulator of oncogenic lipogenesis. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipogenesis, and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and DAXX mutants unable to bind SREBP1/2 have weakened activity in promoting lipogenesis and tumor growth. Remarkably, a DAXX mutant deficient of SUMO-binding fails to activate SREBP1/2 and lipogenesis due to impaired SREBP binding and chromatin recruitment and is defective of stimulating tumorigenesis. Hence, DAXX's SUMO-binding activity is critical to oncogenic lipogenesis. Notably, a peptide corresponding to DAXX's C-terminal SUMO-interacting motif (SIM2) is cell-membrane permeable, disrupts the DAXX-SREBP1/2 interactions, and inhibits lipogenesis and tumor growth. These results establish DAXX as a regulator of lipogenesis and a potential therapeutic target for cancer therapy.

ERN1 dependent impact of glucose and glutamine deprivations on PBX3, PBXIP1, PAX6, MEIS1, and MEIS2 genes expression in U87 glioma cells.

Objective. Homeobox genes play a fundamental role in the embryogenesis, but some of them have been linked to oncogenesis. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of homeobox genes such as PAX6 (paired box 6), PBX3 (PBX homeobox 3), PBXIP1 (PBX homeobox interacting protein 1), MEIS1 (MEIS homeobox 1), and MEIS2 in ERN1 knockdown U87 glioma cells with the intent to reveal the role of ERN1 (endoplasmic reticulum to nucleus signaling 1) signaling pathway on the endoplasmic reticulum stress dependent regulation of homeobox genes. Methods. The control (transfected by empty vector) and ERN1 knockdown (transfected by dominant-negative ERN1) U87 glioma cells were exposed to glucose and glutamine deprivations for 24 h. The cells RNA was extracted and reverse transcribed. The expression level of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes was evaluated by a real-time quantitative polymerase chain reaction analysis and normalized to ACTB. Results. It was found that glucose deprivation down-regulated the expression level of PAX6, MEIS1, and MEIS2 genes in control glioma cells, but did not significantly alter PBX3 and PBXIP1 genes expression. At the same time, ERN1 knockdown significantly modified the sensitivity of all studied genes to glucose deprivation. Other changes in gene expression were detected in control glioma cells under the glutamine deprivation. The expression of PBX3 and MEIS2 genes was down- while PAX6 and PBXIP1 genes up-regulated. Furthermore, ERN1 knockdown significantly modified the effect of glutamine deprivation on the majority of studied genes expression in U87 glioma cells. Conclusion. The results of the present study demonstrate that the exposure of U87 glioma cells under glucose and glutamine deprivations affected the expression of the majority of the studied homeobox genes and that the sensitivity of PAX6, PBX3, PBXIP1, MEIS1, and MEIS2 genes expression under these experimental conditions is mediated by ERN1, the major pathway of the endoplasmic reticulum stress signaling.

LMCD1-AS1 Facilitates Cell Proliferation and EMT by Sponging miR-873-3p in Cervical Cancer.

Long non-coding RNA LMCD1 antisense RNA 1 (LMCD1-AS1) has recently been reported to participate in the pathogenesis of several tumors, including thyroid cancer and osteosarcoma. However, the clinical significance of LMCD1-AS1 and the related biological function have not been reported in cervical cancer (CC). In this study, we observed that LMCD1-AS1 expression was highly expressed in CC specimens compared with adjacent normal specimens using quantitative real-time PCR. Chi-square test showed that high LMCD1-AS1 expression was correlated with FIGO stage and lymph node metastasis. Kaplan-Meier survival analysis showed poor prognosis with high LMCD1-AS1 expression. Moreover, FIGO stage, lymph node metastasis and high LMCD1-AS1 expression could be independent prognostic factors for the patients with CC. Functionally, knockdown of LMCD1-AS1 suppressed the proliferation, migration and invasion of two CC cell lines (HeLa and CaSki) cells by CCK-8 assay, colony formation assay, and Transwell assay. Knockdown of LMCD1-AS1 upregulated E-cadherin expression and downregulated the expression of PCNA, N-cadherin, and imentin in HeLa and CaSki cells. Luciferase reporter assay and RIP assay were conducted to evaluate the downstream molecular mechanisms of LMCD1-AS1. LMCD1-AS1 possesses a putative miR-873-3p-binding site and confirmed the negative correlation between them in CC tissues. Moreover, overexpression of LMCD1-AS1 promoted CC cell proliferation and EMT process through the regulation of miR-873-3p. In addition, depletion of LMCD1-AS1 reduced tumor growth and Ki-67 protein expression. In summary, our findings indicate that LMCD1-AS1 might exert an oncogenic role in CC and targeting LMCD1-AS1 might be a promising therapeutic target for CC treatment.

Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT).

Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.

Fibroblast-derived Extracellular Vesicles Induce Lung Cancer Progression in the Idiopathic Pulmonary Fibrosis Microenvironment.

Idiopathic pulmonary fibrosis (IPF) is a progressive aging-related lung disease associated with increased lung cancer risk. Although previous studies have shown that IPF worsens the survival of patients with lung cancer, whether IPF independently affects cancer malignancy and prognosis remains inconclusive. Extracellular vesicles (EVs) have recently emerged as active carriers of molecular biomarkers and mediators of intercellular communication in lung homeostasis and pathogenesis. EV cargo-mediated fibroblast-tumor cell communication might participate in the development and progression of lung cancer by modulating various signaling pathways. In this study, we examined the impact of lung fibroblast (LF)-derived EVs on non-small cell lung cancer (NSCLC) malignancy in the IPF microenvironment. Here, we showed that LFs derived from patients with IPF have phenotypes of myofibroblast differentiation and cellular senescence. Furthermore, we found that IPF LF-derived EVs have markedly altered microRNA compositions and exert proproliferative functions on NSCLC cells. Mechanistically, the phenotype was attributed mainly to the enrichment of miR-19a in IPF LF-derived EVs. As a downstream signaling pathway, mir-19a in IPF LF-derived EVs regulates ZMYND11-mediated c-Myc activation in NSCLC, potentially contributing to the poor prognosis of patients with NSCLC with IPF. Our discoveries provide novel mechanistic insights for understanding lung cancer progression in the IPF microenvironment. Accordingly, blocking the secretion of IPF LF-derived EV miR-19a and their signaling pathways is a potential therapeutic strategy for managing IPF and lung cancer progression.

Aldolase B attenuates clear cell renal cell carcinoma progression by inhibiting CtBP2.

Aldolase B (ALDOB), a glycolytic enzyme, is uniformly depleted in clear cell renal cell carcinoma (ccRCC) tissues. We previously showed that ALDOB inhibited proliferation through a mechanism independent of its enzymatic activity in ccRCC, but the mechanism was not unequivocally identified. We showed that the corepressor C-terminal-binding protein 2 (CtBP2) is a novel ALDOB-interacting protein in ccRCC. The CtBP2-to-ALDOB expression ratio in clinical samples was correlated with the expression of CtBP2 target genes and was associated with shorter survival. ALDOB inhibited CtBP2-mediated repression of multiple cell cycle inhibitor, proapoptotic, and epithelial marker genes. Furthermore, ALDOB overexpression decreased the proliferation and migration of ccRCC cells in an ALDOB-CtBP2 interaction-dependent manner. Mechanistically, our findings showed that ALDOB recruited acireductone dioxygenase 1, which catalyzes the synthesis of an endogenous inhibitor of CtBP2, 4-methylthio 2-oxobutyric acid. ALDOB functions as a scaffold to bring acireductone dioxygenase and CtBP2 in close proximity to potentiate acireductone dioxygenase-mediated inhibition of CtBP2, and this scaffolding effect was independent of ALDOB enzymatic activity. Moreover, increased ALDOB expression inhibited tumor growth in a xenograft model and decreased lung metastasis in vivo. Our findings reveal that ALDOB is a negative regulator of CtBP2 and inhibits tumor growth and metastasis in ccRCC.

Structures of human TR4LBD-JAZF1 and TR4DBD-DNA complexes reveal the molecular basis of transcriptional regulation.

Testicular nuclear receptor 4 (TR4) modulates the transcriptional activation of genes and plays important roles in many diseases. The regulation of TR4 on target genes involves direct interactions with DNA molecules via the DNA-binding domain (DBD) and recruitment of coregulators by the ligand-binding domain (LBD). However, their regulatory mechanisms are unclear. Here, we report high-resolution crystal structures of TR4DBD, TR4DBD-DNA complexes and the TR4LBD-JAZF1 complex. For DNA recognition, multiple factors come into play, and a specific mutual selectivity between TR4 and target genes is found. The coactivators SRC-1 and CREBBP can bind at the interface of TR4 originally occupied by the TR4 activation function region 2 (AF-2); however, JAZF1 suppresses the binding through a novel mechanism. JAZF1 binds to an unidentified surface of TR4 and stabilizes an α13 helix never reported in the nuclear receptor family. Moreover, the cancer-associated mutations affect the interactions and the transcriptional activation of TR4 in vitro and in vivo, respectively. Overall, our results highlight the crucial role of DNA recognition and a novel mechanism of how JAZF1 reinforces the autorepressed conformation and influences the transcriptional activation of TR4, laying out important structural bases for drug design for a variety of diseases, including diabetes and cancers.

ZFX-mediated upregulation of CEBPA-AS1 contributes to acute myeloid leukemia progression through miR-24-3p/CTBP2 axis.

Emerging reports demonstrated that long non-coding RNAs (lncRNAs) play a role in the pathogenesis and metastasis of cancers. However, the biological functions and underlying mechanisms of LncRNA CEBPA-AS1 in acute myeloid leukemia (AML) remain largely elusive. The level of CEBPA-AS1 was examined in AML clinical tissues and cell lines via fluorescence in situ hybridization (FISH) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In vivo and in vitro functional tests were applied to identify the pro-oncogenic role of CEBPA-AS1 in AML development. The overexpressed CEBPA-AS1 was linked to poor survival in AML patients. Moreover, the relationships among CEBPA-AS1, Zinc Finger Protein X-Linked (ZFX), and miR-24-3p were predicted by bioinformatics and validated by RNA immunoprecipitation (RIP) and luciferase reporter assays. Our findings unveiled that transcription factor ZFX particularly interacted with the promoter of CEBPA-AS1 and activated CEBPA-AS1 transcription. Downregulation of CEBPA-AS1 inhibited the proliferation and invasion while promoted apoptosis of AML cells in in vitro, as well as in vivo, xenograft tumor growth was modified. However, overexpression of CEBPA-AS1 observed the opposite effects. Furthermore, CEBPA-AS1 acted as a competitive endogenous RNA (ceRNA) of miR-24-3p to attenuate the repressive effects of miR-24-3p on its downstream target CTBP2. Taken together, this study emphasized the pro-oncogenic role of CEBPA-AS1 in AML and illustrated its connections with the upstream transcription factor ZFX and the downstream regulative axis miR-24-3p/CTBP2, providing important insights to the cancerogenic process in AML.

KCTD1 and Scalp-Ear-Nipple ('Finlay-Marks') syndrome may be associated with myopia and Thin basement membrane nephropathy through an effect on the collagen IV α3 and α4 chains.

INTRODUCTION: Scalp-Ear-Nipple syndrome is caused by pathogenic KCTD1 variants and characterised by a scalp defect, prominent ears, and rudimentary breasts. We describe here further clinical associations in the eye and kidney. METHODS: Fifteen affected members from two unrelated families with p.(Ala30Glu) or p.(Pro31Leu) in KCTD1 were examined for ocular and renal abnormalities. The relevant proteins were studied in the eye and kidney, and the mutation consequences determined from mouse knockout models. RESULTS: Five males and 10 females with a median age of 40 years (range 1-70) with pathogenic variants p.(Ala30Glu) (n = 12) or p.(Pro31Leu) (n = 3) in KCTD1 were studied. Of the 6 who underwent detailed ophthalmic examination, 5 (83%) had low myopic astigmatism, the mean spherical equivalent of 10 eyes was 2.38D, and one (17%) had hypermetropic astigmatism. One female had a divergent strabismus.Five individuals had renal cysts (5/15, 33%), with renal biopsy in one demonstrating a thinned glomerular basement membrane identical to that seen in Thin basement membrane nephropathy (AD Alport syndrome).In the eye, KCTD1 and its downstream targets, TFAP2, and the collagen IV α3 and α4 chains localised to the cornea and near the retinal amacrine cells. In the kidney, all these proteins except TFAP2 were expressed in the podocytes and distal tubules. TFAP2B and COL4A4 knockout mice also had kidney cysts, and COL4A3 and COL4A4 knockout mice had myopia. CONCLUSION: Individuals with a pathogenic KCTD1 variant may have low myopic astigmatism and represent a further rare genetic cause for a thinned glomerular basement membrane.