Identification of novel CDKL5 molecular targets/pathways associated with skeletal defects present in CDKL5 associated disorders

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Functional analysis of two novel TBX5 variants present in individuals with Holt-Oram syndrome with different clinical manifestations

Publication . Varela, Debora; Varela, Tatiana; Conceição, Natércia; Ferreira, Angela; Marques, Nuno; Silva, Ana Paula; Azevedo, Pedro; Pereira, Salome; Camacho, Ana; de Jesus, Ilidio; Cancela, M. Leonor

Holt-Oram syndrome (HOS) is a rare disorder characterized by cardiac and upper-limb defects. Pathogenic variants in TBX5-a gene encoding a transcription factor important for heart and skeletal development-are the only known cause of HOS. Here, we present the identification and functional analysis of two novel TBX5 pathogenic variants found in two individuals with HOS presenting distinct phenotypes. The individual with the c.905delA variant has a severe cardiac phenotype but mild skeletal defects, unlike the individual with the c.246_249delGATG variant who has no cardiac problems but severe upper limbs malformations, including phocomelia. Both frameshift variants, c.246_249delGATG and c.905delA, generate mRNAs harbouring premature stop codons which, if not degraded by nonsense mediated decay, will lead to the production of shorter TBX5 proteins, p.Gln302Argfs*92 and p.Met83Phefs*6, respectively. Immunocytochemistry results suggest that both mutated proteins are produced and furthermore, like the wild-type protein, p.Gln302Argfs*92 mutant appears to be mainly localized in the nucleus, in contrast with p.Met83Phefs*6 mutant that displays a higher level of cytoplasmic localization. In addition, luciferase activity analysis revealed that none of the TBX5 mutants are capable of transactivating the NPPA promoter. In conclusion, our results provide evidence that both pathogenic variants cause a severe TBX5 loss-of-function, dramatically reducing its biological activity. The absence of cardiac problems in the individual with the p.Met83Phefs*6 variant supports the existence of other mechanisms/genes underlying the pathogenesis of HOS and/or the existence of an age-related delay in the development of a more serious cardiac phenotype. Further studies are required to understand the differential effects observed in the phenotypes of both individuals.

2021Journal article

Restricted

Cdkl5 mutant zebrafish shows skeletal and neuronal alterations mimicking human CDKL5 deficiency disorder

Publication . Varela, Tatiana; Varela, Débora; Martins, Gil; Conceição, Natércia; Cancela, M. Leonor

CDKL5 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.

2022Journal article

Open Access

Regulation of human ZNF687, a gene associated with Paget's disease of bone

Publication . Varela, Débora; Varela, Tatiana; Conceição, Natércia; Cancela, M. Leonor

Mutations 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.

2023-01Journal article

Open Access

Transcriptional regulation of human DUSP4 gene by cancer‐related transcription factors

Publication . Varela, Tatiana; Conceição, Natércia; Laizé, Vincent; Cancela, M. Leonor

Dual 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.

2021Journal article

Open Access