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- Cdkl5 mutant zebrafish shows skeletal and neuronal alterations mimicking human CDKL5 deficiency disorderPublication . Varela, Tatiana; Varela, Débora; Martins, Gil; Conceição, Natércia; Cancela, M. LeonorCDKL5 deficiency disorder (CDD) is a rare neurodevelopmental condition characterized primarily by seizures and impairment of cognitive and motor skills. Additional phenotypes include microcephaly, dysmorphic facial features, and scoliosis. Mutations in cyclin-dependent kinase-like 5 (CDKL5) gene, encoding a kinase essential for normal brain development and function, are responsible for CDD. Zebrafish is an accepted biomedical model for the study of several genetic diseases and has many advantages over other models. Therefore, this work aimed to characterize the phenotypic, behavioral, and molecular consequences of the Cdkl5 protein disruption in a cdkl5 mutant zebrafish line (sa21938). cdkl5(sa21938) mutants displayed a reduced head size, suggesting microcephaly, a feature frequently observed in CDD individuals. Double staining revealed shorter craniofacial cartilage structures and decrease bone mineralization in cdkl5 homozygous zebrafish indicating an abnormal craniofacial cartilage development and impaired skeletal development. Motor behavior analysis showed that cdkl5(sa21938) embryos had less frequency of double coiling suggesting impaired glutamatergic neurotransmission. Locomotor behavior analysis revealed that homozygous embryos swim shorter distances, indicative of impaired motor activity which is one of the main traits of CCD. Although no apparent spontaneous seizures were observed in these models, upon treatment with pentylenetetrazole, seizure behavior and an increase in the distance travelled were observed. Quantitative PCR showed that neuronal markers, including glutamatergic genes were dysregulated in cdkl5(sa21938) mutant embryos. In conclusion, homozygous cdkl5(sa21938) zebrafish mimic several characteristics of CDD, thus validating them as a suitable animal model to better understand the physiopathology of this disorder.
- Zebrafish: an interesting model to study CDKL5 deficiency disorderPublication . Varela, Tatiana; Varela, Débora; Vitorino, Marta; Conceição, Natércia; Cancela, M. Leonor; Martins, GilCDKL5 deficiency disorder is a rare X-linked condition that results in early onset of impairedmotor and cognitive skills such as motor rigidity, stereotypical hand movements and deficient language acquisition aswell as recurrent seizures. It is caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene, which encodes a serine/threonine kinase involved in important neuronal processes such as cell signaling and neuron morphogenesis.
- Transcriptional regulation of human DUSP4 gene by cancer‐related transcription factorsPublication . Varela, Tatiana; Conceição, Natércia; Laizé, Vincent; Cancela, M. LeonorDual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is responsible for the dephosphorylation and inactivation of ERK, JNK and p38, which are mitogen-activated protein kinases involved in cell proliferation, differentiation and apoptosis, but also in inflammation processes. Given its importance for cellular signalling, DUSP4 is subjected to a tight regulation and there is growing evidence that its expression is dysregulated in several tumours. However, the mechanisms underlying DUSP4 transcriptional regulation remain poorly understood. Here, we analysed the regulation of the human DUSP4 promoters 1 and 2, located upstream of exons 1 and 2, respectively, by the cancer-related transcription factors (TFs) STAT3, FOXA1, CTCF and YY1. The presence of binding sites for these TFs was predicted in both promoters through the in silico analysis of DUSP4, and their functionality was assessed through luciferase activity assays. Regulatory activity of the TFs tested was found to be promoter-specific. While CTCF stimulated the activity of promoter 2 that controls the transcription of variants 2 and X1, STAT3 stimulated the activity of promoter 1 that controls the transcription of variant 1. YY1 positively regulated both promoters, although to different extents. Through site-directed mutagenesis, the functionality of YY1 binding sites present in promoter 2 was confirmed. This study provides novel insights into the transcriptional regulation of DUSP4, contributing to a better comprehension of the mechanisms of its dysregulation observed in several types of cancer.
- Regulation of human ZNF687, a gene associated with Paget's disease of bonePublication . Varela, Débora; Varela, Tatiana; Conceição, Natércia; Cancela, M. LeonorMutations in Zinc finger 687 (ZNF687) were associated with Paget's disease of bone (PDB), a disease charac-terized by increased bone resorption and excessive bone formation. It was suggested that ZNF687 plays a role in bone differentiation and development. However, the mechanisms involved in ZNF687 regulation remain un-known. This study aimed to obtain novel knowledge regarding ZNF687 transcriptional and epigenetic regulation. Through in silico analysis, we hypothesized three ZNF687 promoter regions located upstream exon 1 A, 1B, and 1 C and denominated promoter regions 1, 2, and 3, respectively. Their functionality was confirmed by luciferase activity assays and positive/negative regulatory regions were identified using promoter deletions constructs. In silico analysis revealed a high density of CpG islands in these promoter regions and in vitro methylation sup-pressed promoters' activity. Using bioinformatic approaches, bone-associated transcription factor binding sites containing CpG dinucleotides were identified, including those for NF kappa B, PU.1, DLX5, and SOX9. By co-transfection in HEK293 and hFOB cells, we found that DLX5 specifically activated ZNF687 promoter region 1, and its methylation impaired DLX5-driven promoter stimulation. NF kappa B repressed and activated promoter regions 1 and 2, respectively, and these activities were affected by methylation. PU.1 induced ZNF687 promoter region 1 which was affected by methylation. SOX9 differentially regulated ZNF687 promoters in HEK293 and hFOB cells that were impaired after methylation. In conclusion, this study provides novel insights into ZNF687 regulation by demonstrating that NF kappa B, PU.1, DLX5, and SOX9 are regulators of ZNF687 promoters, and DNA methylation influences their activity. The contribution of the dysregulation of these mechanisms in PDB should be further elucidated.
- Analysis of transcriptional and post-transcriptional regulation of DUSP4, a human gene associated with colorectal cancer (CRC)Publication . Varela, Tatiana; Laizé, Vincent; Conceição, NatérciaColorectal cancer (CRC) is the third most common type of cancer and the fourth leading cause of death worldwide. Dual specificity phosphatase 4 (DUSP4) – a regulator of the mitogen-activated protein kinase activity – has been recently associated with CRC after its expression was found to be up-regulated in tumour tissue. However, expression data are limited and mechanisms underlying DUSP4 regulation poorly understood. Objectives of this work were to collect basic data on the expression of the different transcript variants in CRC samples and on the transcriptional and post-transcriptional regulation of human DUSP4. The expression of transcript variants 1, 2 and X1 was determined by qPCR in tumour and TAN tissues from 28 patients. All transcripts were overexpressed in tumour tissues, although to different extents. Variant X1 was the most up-regulated (>13 folds) and associated with several clinicopathological parameters (KRAS mutation and poorly differentiated tumour). DUSP4 transcript overexpression was also found to be possibly associated with a reduced overall survival and a more severe tumour stage. DUSP4 promoters activity was assessed using reporter promoter constructs and vectors expressing cancer-related transcription factors. While CTCF and FOXA1 regulated promoter#2 activity (i.e. transcription of variants 2 and X1) and STAT3 promoter#1 activity (i.e. the transcription of variant 1), SP1 and YY1 regulated both promoter activity, although to different extent. Preliminary data collected from the in silico analysis of DUSP4 3’-UTR revealed putative binding sites for miR-137, a microRNA acting as tumour suppressor in several cancers, including CRC. In conclusion, DUSP4 transcripts are all overexpressed in CRC tissues and have therefore the potential to serve as disease markers, especially variant X1. DUSP4 expression is regulated by cancer-related transcription factors in a promoter-specific manner, i.e. the expression of variant 1 and variants 2/X1 is controlled by different regulatory mechanisms, although up-regulated in a CRC context.
- Expression of DUSP4 transcript variants as a potential biomarker for colorectal cancerPublication . Varela, Tatiana; Laizé, Vincent; Conceição, Natércia; Caldeira, Paulo; Marreiros, Ana; Guerreiro, Horacio; Cancela, M. LeonorAim: To provide novel data on the expression of DUSP4 transcripts in colorectal cancer (CRC) tissues and to explore their potential as biomarkers. Materials & methods:DUSP4 transcripts expression was determined by quantitative real-time PCR in tissues from 28 CRC patients. Their association with clinicopathological factors and survival analysis was performed. Data from 380 CRC patients available at The Cancer Genome Atlas project were also analyzed. Results: All transcripts were overexpressed in CRC tissues. Variant X1 was the most upregulated and associated with KRAS mutations and poorly differentiated tumor. Overexpression of DUSP4 transcripts could distinguish all tumor stages from normal tissues. Similar results were found in The Cancer Genome Atlas cohort. Conclusion:DUSP4 transcripts have the potential to serve as diagnostic biomarkers for CRC, particularly variant X1.
- Epigenetic regulation of ZNF687 by miR-142a-3p and DNA methylation during osteoblast differentiation and mice bone development and agingPublication . Domingos Varela, Débora Cristina; Domingos Varela, Tatiana da Conceição; Conceição, Natércia; Cancela, M. LeonorZinc finger protein 687 (ZNF687), a transcription factor implicated in osteoblast/osteoclast differentiation and linked to Paget's disease of bone, has unclear mechanisms in bone metabolism. Epigenetic disruptions can affect bone cell activity and contribute to bone-related diseases. This work aimed to elucidate the regulatory role of epigenetics in modulating Zfp687 expression throughout osteoblast differentiation and bone growth/aging in mice. Differentiation of the mouse-derived osteoblast precursor cell line (MC3T3-E1) showed increased expression of osteogenic markers and decreased Zfp687 expression. In the hindlimb bones of C57BL/6J mice, the expression of most bone-forming genes decreased from youth to adulthood, while Zfp687 and Runx2 expression was maintained, being only significantly reduced in old mice in comparison to young mice. Bisulfite sequencing revealed hypomethylation of the Zfp687 promoter during MC3T3-E1 differentiation and bone growth/aging. Bioinformatics predicted miR-142a-3p, miR-122b-5p, and miR-124-3p binding sites in Zfp687 3 ' UTR, and RT-qPCR analysis showed higher expression of these miRNAs in mature osteoblasts. Transfection of a miR-142-3p mimic reduced luciferase activity in the wildtype Zfp687 3 ' UTR but not the mutant 3 ' UTR and downregulated the Zfp687 gene and protein levels. In conclusion, miR-142a-3p directly targets the Zfp687 3 ' UTR, promoting its downregulation during osteoblastogenesis. Furthermore, DNA methylation does not appear to regulate Zfp687 during osteoblast differentiation or bone development in mice.