Zebrafish Models to Study Ectopic Calcification and Calcium-Associated Pathologies.

Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio)-a well-established model for studying osteogenesis and mineralogenesis-has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.

Zebrafish optineurin: genomic organization and transcription regulation.

Optineurin (OPTN) is involved in a variety of mechanisms, such as autophagy, vesicle trafficking, and nuclear factor kappa-B (NF-κB) signaling. Mutations in the OPTN gene have been associated with different pathologies, including glaucoma, amyotrophic lateral sclerosis, and Paget's disease of bone. Since the relationship between fish and mammalian OPTN is not well understood, the objective of the present work was to characterize the zebrafish optn gene and protein structure and to investigate its transcriptional regulation. Through a comparative in silico analysis, we observed that zebrafish optn presents genomic features similar to those of its human counterpart, including its neighboring genes and structure. A comparison of OPTN protein from different species revealed a high degree of conservation in its functional domains and three-dimensional structure. Furthermore, our in vitro transient-reporter analysis identified a functional promoter in the upstream region of the zebrafish optn gene, along with a region important for its transcription regulation. Site-directed mutagenesis revealed that the NF-κB motif is responsible for the activation of this region. In conclusion, with this study, we characterize zebrafish optn and our results indicate that zebrafish can be considered as an alternative model to study OPTN's biological role in bone-related diseases.

Identification of a novel mutation in MEF2C gene in an atypical patient with frontotemporal lobar degeneration.

The MEF2C gene encodes a transcription factor known to play a crucial role in molecular pathways affecting neuronal development. MEF2C mutations were described as a genetic cause of developmental disease (MRD20), and several reports sustain its involvement in dementia-related conditions, such as Alzheimer's disease and amyotrophic lateral sclerosis. These pathologies and frontotemporal degeneration (FTLD) are thought to share common physiopathological pathways. In this exploratory study, we searched for alterations in the DNA sequence of exons and boundaries, including 5'- and 3'-untranslated regions (5'UTR, 3'UTR), of MEF2C gene in 11 patients with clinical phenotypes related with MRD20 or FTLD. We identified a heterozygous deletion of 13 nucleotides in the 5'UTR region of a 69 years old FTLD patient. This alteration was absent in 200 healthy controls, suggesting a contribution to this patient's disease phenotype. In silico analysis of the mutated sequence indicated changes in mRNA secondary structure and stability, thus potentially affecting MEF2C protein levels. Furthermore, in vitro functional analysis of this mutation revealed that the presence of this deletion abolished the transcriptional activity of the gene in human embryonic cells and rat brain neurons, probably by modifying MEF2C expression. Altogether, our results provide evidence for the involvement of MEF2C in FTLD manifesting with seizures.

Transcriptional regulation of human DUSP4 gene by cancer-related transcription factors.

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.

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

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.

Effect of genetic variants of OPTN in the pathophysiology of Paget's disease of bone.

Paget's disease of bone (PDB) is the second most frequent metabolic bone disease after osteoporosis. Genetic factors play an important role in PDB, but to date PDB causing mutations were identified only in the Sequestosome 1 gene at the PDB3 locus. OPTN has been recently associated with PDB, however little is known about the effect of genetic variants in this gene in PDB pathophysiology. By sequencing OPTN in SQSTM1 non-carriers PDB patients we found 16 SNPs in regulatory, coding and non-coding regions. One of those was found to be associated with PDB in our cohort - rs2234968. Our results show that rs2238968 effect may be explained by a change in OPTN splicing that give rise to a predicted truncated protein. We also performed functional studies on the variants located in OPTN promoter - rs3829923 and the rare variant -9906 - to investigate putative regulators of OPTN. Our results show that OPTN expression seems to be regulated by SP1, RXR, E47, and the E2F family. In conclusion, our work suggests a potential pathophysiological role of SNPs in OPTN, giving a new perspective about the regulatory mechanisms of this gene. Ultimately we discovered a new variant associated with PDB in OPTN, reinforcing the relevance of this gene for the development of this bone disease.

Expression pattern of cdkl5 during zebrafish early development: implications for use as model for atypical Rett syndrome.

Atypical Rett syndrome is a child neurodevelopmental disorder induced by mutations in CDKL5 gene and characterized by a progressive regression in development with loss of purposeful use of the hands, slowed brain and head growth, problems with walking, seizures, and intellectual disability. At the moment, there is no cure for this pathology and little information is available concerning animal models capable of mimicking its phenotypes, thus the development of additional animal models should be of interest to gain more knowledge about the disease. Zebrafish has been used successfully as model organism for many human genetic diseases; however, no information is available concerning the spatial and temporal expression of cdkl5 orthologous in this organism. In the present study, we identified the developmental expression patterns of cdkl5 in zebrafish by quantitative PCR and whole-mount in situ hybridization. cdkl5 is expressed maternally at low levels during the first 24 h of development. After that the expression of the gene increases significantly and it starts to be expressed mainly in the nervous system and in several brain structures, such as telencephalon, mesencephalon and diencephalon. The expression patterns of cdkl5 in zebrafish is in accordance with the tissues known to be affected in humans and associated to symptoms and deficits observed in Rett syndrome patients thus providing the first evidence that zebrafish could be an alternative model to study the molecular pathways of this disease as well as to test possible therapeutic approaches capable of rescuing the phenotype.

MEF2C orthologues from zebrafish: Evolution, expression and promoter regulation.

MEF2C is a crucial transcription factor for cranial neural crest cells development. An abnormal expression of this protein leads to severe abnormalities in craniofacial features. Recently, a human disease (MRD20) was described as a consequence of MEF2C haploinsufficiency. These patients show severe developmental delay, intellectual disability and dysmorphic features. Zebrafish presents two MEF2C orthologues, mef2ca and mef2cb. In this study we demonstrate a highly conserved pattern of chromosome localization for MEF2C between human and zebrafish, a similar protein sequence and tissue expression profile. We have focused our functional analysis on the zebrafish orthologue mef2cb. We identified three new exons through 5' RACE and described two new transcriptional start sites (TSS). These alternative TSS reflect the occurrence of two alternative promoters differentially regulated by nuclear factors related to craniofacial or neuronal development such as Sox9b, Sox10 and Runx2. We also predict that mef2cb gene may be post transcriptionally regulated by analysing the structure of its 5' UTR region, conserved throughout evolution. Our study provides new insights in MEF2C conservation and provides the first evidence of mef2cb regulation by both transcriptional and post transcriptional mechanisms, thus contributing to validate zebrafish as a good model for future studies concerning MEF2C dependent pathologies.

Matrix Gla protein and osteocalcin: from gene duplication to neofunctionalization.

Osteocalcin (OC or bone Gla protein, BGP) and matrix Gla protein (MGP) are two members of the growing family of vitamin K-dependent (VKD) proteins. They were the first VKD proteins found not to be involved in coagulation and synthesized outside the liver. Both proteins were isolated from bone although it is now known that only OC is synthesized by bone cells under normal physiological conditions, but since both proteins can bind calcium and hydroxyapatite, they can also accumulate in bone. Both OC and MGP share similar structural features, both in terms of protein domains and gene organization. OC gene is likely to have appeared from MGP through a tandem gene duplication that occurred concomitantly with the appearance of the bony vertebrates. Despite their relatively close relationship and the fact that both can bind calcium and affect mineralization, their functions are not redundant and they also have other unrelated functions. Interestingly, these two proteins appear to have followed quite different evolutionary strategies in order to acquire novel functionalities, with OC following a gene duplication strategy while MGP variability was obtained mostly by the use of multiple promoters and alternative splicing, leading to proteins with additional functional characteristics and alternative gene regulatory pathways.

Development and characterization of Xl1, a Xenopus laevis chondrocyte-like cell culture.

We describe the development and characterization of a new cell line, designated Xl1, derived from vertebra and long bones of Xenopus laevis. These cells can mineralize their extracellular matrix upon addition of an inorganic phosphate donor and vitamin C, as characterized by von Kossa staining. In addition they express genes such as matrix gla protein (mgp), alkaline phosphatase, type II collagen, and retinoic acid receptors, representing a valuable tool to analyze expression and regulation of Xenopus cartilage-associated genes. Continuous treatment with retinoic acid (RA) inhibited mineralization, alkaline phosphatase expression and its activity, suggesting that RA is a potential negative regulator of Xl1 cell differentiation. These cells are receptive to efficient transfer of DNA using conventional methods including calcium phosphate, liposome-mediated transfer or electroporation and were found to express basal levels of mgp at least 50-fold higher than the routinely used Xenopus A6 cell line, as seen by transcription assays with the distal X. laevis mgp promoter. Being the first amphibian cell line derived from bone tissue, the Xl1 culture provides an excellent in vitro tool for functional promoter studies, being suitable, among other uses, for identifying promoter elements mediating cartilage-expressed genes as shown here for mgp.

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

Mutations in Zinc finger 687 (ZNF687) were associated with Paget's disease of bone (PDB), a disease characterized 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 unknown. 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 suppressed promoters' activity. Using bioinformatic approaches, bone-associated transcription factor binding sites containing CpG dinucleotides were identified, including those for NFκB, 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. NFκB 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 NFκB, 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.

Assessment of MGP gene expression in cancer and contribution to prognosis.

Matrix Gla protein (MGP) was first identified as a calcification physiological inhibitor and the causal agent of the Keutel syndrome. MGP has been suggested to play a role in development, cell differentiation, and tumorigenesis. This study aimed to compare MGP expression and methylation status in different tumors and adjacent tissues, using The Cancer Genome Atlas (TCGA) data repository. We investigated if changes in MGP mRNA expression were correlated to cancer progression and whether the correlation coefficients could be used for prognosis. Strong correlations were observed between altered MGP levels and disease progression in breast, kidney, liver, and thyroid cancers, suggesting that it could be used to complement current clinical biomarker assays, for early cancer diagnosis. We have also analyzed MGP methylation and identified CpG sites in its promoter and first intron with clear differences in methylation status between healthy and tumoral tissue providing evidence for epigenetic regulation of MGP transcription. Furthermore, we demonstrate that these alterations correlate with the overall survival of the patients suggesting that its assessment can serve as an independent prognostic indicator of patients' survival.

mef2ca and mef2cb Double Mutant Zebrafish Show Altered Craniofacial Phenotype and Motor Behaviour.

The transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.

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

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). cdkl5sa21938 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 cdkl5sa21938 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 cdkl5sa21938 mutant embryos. In conclusion, homozygous cdkl5sa21938 zebrafish mimic several characteristics of CDD, thus validating them as a suitable animal model to better understand the physiopathology of this disorder.

Transcriptional regulation of human T-box 5 gene (TBX5) by bone- and cardiac-related transcription factors.

T-box 5 (TBX5) protein belongs to the T-box family whose members play a crucial role in cell-type specification, morphogenesis and organogenesis. TBX5 is a transcription factor important for cardiac development and upper limbs formation and its haploinsufficiency causes Holt-Oram syndrome (HOS). An increase in TBX5 dosage also leads to HOS, suggesting that TBX5 is a dose-sensitive transcription factor that needs to be tightly regulated but the molecular mechanisms involved remain unclear. In this work we report the cloning and functional analysis of human TBX5 promoter region 1 (upstream of exon 1) and promoter region 2 (upstream of exon 2), that probably regulate the transcription of the different transcript variants. In silico analysis showed several binding sites for cardiac and skeletal related transcription factors (TFs) and their functionality was assessed using promoter-luciferase constructions and TF-expressing vectors. MEF2A (Myocyte enhancer factor 2 A) was shown to positively regulate both TBX5 promoters, while EGR1 (early growth response 1) repressed both promoters. SOX9 (SRY (sex determining region Y)-box 9) repressed only the activity of promoter region 2. Interestingly, YY1 (Yin and yang 1) repressed promoter region 1 (that regulates the expression of variant 1 and 3), but activated promoter region 2 (that regulates the expression of variant 4). In conclusion, this work provides novel insights toward the better understanding of TBX5 transcriptional regulation by cardiac- and skeletal-related TFs.

Keutel Syndrome, a Review of 50 Years of Literature.

Keutel syndrome (KS) is a rare autosomal recessive genetic disorder that was first identified in the beginning of the 1970s and nearly 30 years later attributed to loss-of-function mutations in the gene coding for the matrix Gla protein (MGP). Patients with KS are usually diagnosed during childhood (early onset of the disease), and the major traits include abnormal calcification of cartilaginous tissues resulting in or associated with malformations of skeletal tissues (e.g., midface hypoplasia and brachytelephalangism) and cardiovascular defects (e.g., congenital heart defect, peripheral pulmonary artery stenosis, and, in some cases, arterial calcification), and also hearing loss and mild developmental delay. While studies on Mgp -/- mouse, a faithful model of KS, show that pathologic mineral deposition (ectopic calcification) in cartilaginous and vascular tissues is the primary cause underlying many of these abnormalities, the mechanisms explaining how MGP prevents abnormal calcification remain poorly understood. This has negative implication for the development of a cure for KS. Indeed, at present, only symptomatic treatments are available to treat hypertension and respiratory complications occurring in the KS patients. In this review, we summarize the results published in the last 50 years on Keutel syndrome and present the current status of the knowledge on this rare pathology.

Expression of DUSP4 transcript variants as a potential biomarker for colorectal cancer.

Aim: 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.

Data on the evaluation of FGF2 gene expression in Colorectal Cancer.

The data presented in this article is related with the research paper entitled "Evaluation of MGP gene expression in colorectal cancer", available on Gene journal [1]. From all the transcription factors known to regulate MGP, FGF2 is the most described in colon adenocarcinoma and colon tumor cell lines, where it was shown to: i) contribute for the invasiveness potential; and ii) promote proliferation and survival of colorectal cancer cells. These in vitro studies pose the hypothesis that FGF2 associated signaling pathways could be promoting the regulation of others genes, such as MGP, that may lead to tumor progression which ultimately could result in poor prognosis in colon adenocarcinoma.

Evaluation of MGP gene expression in colorectal cancer.

PURPOSE: Matrix Gla protein (MGP) is a vitamin K-dependent, γ-carboxylated protein that was initially found to be a physiological inhibitor of ectopic calcifications affecting mainly cartilage and the vascular system. Mutations in the MGP gene were found to be responsible for a human pathology, the Keutel syndrome, characterized by abnormal calcifications in cartilage, lungs, brain and vascular system. MGP was recently implicated in tumorigenic processes such as angiogenesis and shown to be abnormally regulated in several tumors, including cervical, ovarian, urogenital and breast. This fact has triggered our interest in analyzing the expression of MGP and of its regulator, the transcription factor runt related transcription factor 2 (RUNX2), in colorectal cancer (CRC). METHODS: MGP and RUNX2 expression were analyzed in cancer and non-tumor biopsies samples from 33 CRC patients and 9 healthy controls by RT-qPCR. Consequently, statistical analyses were performed to evaluate the clinical-pathological significance of MGP and RUNX2 in CRC. MGP protein was also detected by immunohistochemical analysis. RESULTS: Showed an overall overexpression of MGP in the tumor mucosa of patients at mRNA level when compared to adjacent normal mucosa and healthy control tissues. In addition, analysis of the expression of RUNX2 mRNA demonstrated an overexpression in CRC tissue samples and a positive correlation with MGP expression (Pearson correlation coefficient 0.636; p ≤ 0.01) in tumor mucosa. However correlations between MGP gene expression and clinical-pathological characteristics, such as gender, age and pathology classification did not provide relevant information that may shed light towards the differences of MGP expression observed between normal and malignant tissue. CONCLUSIONS: We were able to associate the high levels of MGP mRNA expression with a worse prognosis and survival rate lower than five years. These results contributed to improve our understanding of the molecular mechanism underlying MGP deregulation in cancer.

Retinoic acid is a negative regulator of matrix Gla protein gene expression in teleost fish Sparus aurata.

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues while accumulated only in bone and cartilage under physiological conditions. Although the precise molecular mechanism of action of MGP remains unknown, all available evidence indicates that it acts as a physiological inhibitor of mineralization. This work presents the cloning of gilthead seabream MGP gene (SaMGP) and the functional analysis of its promoter. SaMGP gene was found to be organized in five exons and to be under control of a distal and a proximal promoter, both, capable of activating SaMGP transcription in transient transfections. Furthermore, we present strong evidence that retinoic acid down-regulates SaMGP gene transcription by interacting, through binding of its receptor, with a specific region within distal promoter. Interestingly, the presence of repetitive motifs in the proximity of SaMGP gene regulatory regions suggests that they may modulate promoter accessibility to transcription machinery, as already seen for other genes. This work provides additional evidence of the usefulness of non-mammalian model systems to elucidate the complex regulation of MGP gene transcription.