Epigenetics of adipocyte commitment. Regulation of Pparγ and Zfp423 expression

Obesity is the most frequent metabolic disease worldwide and the major risk factor for metabolic disorders such as insulin resistance and type 2 diabetes. In response to overfeeding, excess lipids are stored in the adipocytes, leading to inappropriate adipose cell expansion (hypertrophic obesity), which is associated with local inflammation and dysregulated and insulin resistant adipose tissue. Hypertrophic obesity is also characterized by an inability to recruit and differentiate precursor cells into mature adipocytes. This is not due to lack of these precursor cells but instead is a consequence of an impaired ability to induce commitment and promote differentiation of available precursor cells, by inactivating inhibitory pathways, and/or activating pathways needed to commit and/or differentiate. Very recently, the zinc finger protein Zfp423 has been identified as a transcriptional regulator of preadipocyte determination. Zfp423 protein, indeed, directly binds the peroxisome proliferator-activated receptor gamma (Pparγ) promoter, thus promoting the gene transcription of the master regulator of adipocyte differentiation. Furthermore, Zfp423 protein is the point of convergence between the anti-adipogenic Wnt1-inducible-signaling pathway protein 2 (Wisp2) pathway and the pro-adipogenic bone morphogenetic protein 4 (Bmp4) pathway. In light of this, it is now clear that alterations impacting on Zfp423 and Pparγ protein and/or gene expressions are at least in part responsible for the restricted adipogenesis observed in hypertrophic obesity. Increasing evidence sustains that even adipogenesis might be regulated by an epigenetically induced gene transcription reprogramming. Thus, in light of all these observations, I aimed to establish which are the molecular mechanisms regulating the gene expression of Zfp423 and of its downstream target Pparγ looking at epigenetic changes involvement. Experiments were performed in two cell models: 3T3-L1 and NIH-3T3 cells, which are committed to and uncommitted to adipocyte lineage, respectively. The mRNA expression of both Zfp423 and Pparγ genes are increased in 3T3-L1 compared with NIH-3T3 cells. Furthermore, the expression differences among the two cellular models are not dependent on differences in the promoter DNA sequence of both genes, but rather to epigenetic mechanisms and chromatin remodeling. Indeed, performing methylation studies by bisulfite sequencing and nucleosomes positioning and occupancy analysis by micrococcal nuclease (MNase) digestion, I revealed that the DNA methylation status and the nucleosomes occupancy of Zfp423 and Pparγ promoter regions are increased in NIH-3T3 compared with 3T3-L1 cells. Finally, chemically-induced demethylation of the Zfp423 and Pparγ promoters by DNA-methyltransferase (Dnmt) inhibitor 5-Azacyditine (AZA) treatment promotes adipocyte terminal differentiation in the uncommitted NIH-3T3 cell line. 9 In conclusion, I demonstrated that Zfp423 and Pparγ genes, which are involved in adipocyte commitment and differentiation, are transcriptionally regulated by DNA methylation and dynamic chromatin remodeling, and that the modulation of the methylation status of the promoter region of these two genes is relevant to the regulation of adipocyte commitment and terminal differentiation in vitro

Twenty years of the Italian Fanconi Anemia Registry: where we stand and what remains to be learned

The natural history of Fanconi anemia remains hard to establish because of its rarity and its heterogeneous clinical presentation; since 1994, the Italian Fanconi Anemia Registry has collected clinical, epidemiological and genetic data of Italian Fanconi Anemia patients. This registry includes 180 patients with a confirmed diagnosis of Fanconi anemia who have either been enrolled prospectively, at diagnosis, or later on. After enrollment, follow-up data were periodically collected to assess the clinical course, possible complications and long-term survival; the median follow up was 15.6 years. The main goal of the study was to describe the natural history of Fanconi anemia, focusing on the following variables: family history, disease presentation, development of hematological manifestations, development of malignancies, occurrence of hematopoietic stem cell transplantation and survival. Typical morphological and/or hematological abnormalities and/or growth retardation were the most common manifestations at diagnosis; the majority of patients (77%) exhibited hematological abnormalities at the initial presentation, and almost all (96%) eventually developed hematological manifestations. More than half of the patients (57%) underwent a bone-marrow transplant. The occurrence of cancer was quite rare at diagnosis, whereas the cumulative incidence of malignancies at 10, 20 and 30 years was 5%, 8% and 22%, respectively, for hematological cancers and 1%, 15% and 32%, respectively, for solid tumors. Overall survival at 10, 20 and 30 years were 88%, 56% and 37%, respectively; the main causes of death were cancer, complications of the hematological presentation and complications of transplantation. These data clearly confirm the detrimental outcome of Fanconi anemia, with no major improvement in the past decades

Whole gene deletion of EBF3 supporting haploinsufficiency of this gene as a mechanism of neurodevelopmental disease

Mutations in early B cell factor 3 (EBF3) were recently described in patients with a neurodevelopmental disorder (NDD) that includes developmental delay/intellectual disability, ataxia, hypotonia, speech impairment, strabismus, genitourinary abnormalities, and mild facial dysmorphisms. Several large 10q terminal and interstitial deletions affecting many genes and including EBF3 have been described in the literature. However, small deletions (<1 MB) affecting almost exclusively EBF3 are not commonlyreported. We performed array comparative genomic hybridization (aCGH) (Agilent 180K) and quantitative PCR analysis in a female patient with intellectual disability. A clinical comparison between our patient and overlapping cases reported in the literature was also made. The patient carries a de novo 600 Kb deletion at 10q26.3 affecting the MGMT, EBF3, and GLRX genes. The patient has severe intellectual disability, language impairment, conductive hearing loss, hypotonia, vision alterations, triangular face, short stature, and behavior problems. This presentation overlaps that reported for patients carrying EBF3 heterozygous point mutations, as well as literature reports of patients carrying large 10qter deletions. Our results and the literature review suggest that EBF3 haploinsufficiency is a key contributor to the common aspects of the phenotype presented by patients bearing point mutations and indels in this gene, given that deletions affecting the entire gene (alone or in addition to other genes) are causative of a similar syndrome, including intellectual disability (ID) with associated neurological symptoms and particular facial dysmorphisms.FCT—Fundação para a Ciência e a Tecnologia within the projects and scholarships (PIC/IC/83026/2007, PIC/IC/83013/2007, SFRH/BD/90167/2012). This article has been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnershi p Agreement, through the European Regional Development Fund (FEDER).info:eu-repo/semantics/publishedVersio

Current Experience in Testing Mitochondrial Nutrients in Disorders Featuring Oxidative Stress and Mitochondrial Dysfunction: Rational Design of Chemoprevention Trials

An extensive number of pathologies are associated with mitochondrial dysfunction (MDF) and oxidative stress (OS). Thus, mitochondrial cofactors termed mitochondrial nutrients (MN), such as alpha-lipoic acid (ALA), Coenzyme Q10 (CoQ10), and L-carnitine (CARN) (or its derivatives) have been tested in a number of clinical trials, and this review is focused on the use of MN-based clinical trials. The papers reporting on MN-based clinical trials were retrieved in MedLine up to July 2014, and evaluated for the following endpoints: (a) treated diseases; (b) dosages, number of enrolled patients and duration of treatment; (c) trial success for each MN or MN combinations as reported by authors. The reports satisfying the above endpoints included total numbers of trials and frequencies of randomized, controlled studies, i.e., 81 trials testing ALA, 107 reports testing CoQ10, and 74 reports testing CARN, while only 7 reports were retrieved testing double MN associations, while no report was found testing a triple MN combination. A total of 28 reports tested MN associations with classical antioxidants, such as antioxidant nutrients or drugs. Combinations of MN showed better outcomes than individual MN, suggesting forthcoming clinical studies. The criteria in study design and monitoring MN-based clinical trials are discussed

Oxidative Stress and Mitochondrial Dysfunction across Broad-Ranging Pathologies: Toward Mitochondria-Targeted Clinical Strategies

Beyond the disorders recognized as mitochondrial diseases, abnormalities in function and/or ultrastructure of mitochondria have been reported in several unrelated pathologies. These encompass ageing, malformations, and a number of genetic or acquired diseases, as diabetes and cardiologic, haematologic, organ-specific (e.g., eye or liver), neurologic and psychiatric, autoimmune, and dermatologic disorders. The mechanistic grounds for mitochondrial dysfunction (MDF) along with the occurrence of oxidative stress (OS) have been investigated within the pathogenesis of individual disorders or in groups of interrelated disorders. We attempt to review broad-ranging pathologies that involve mitochondrial-specific deficiencies or rely on cytosol-derived prooxidant states or on autoimmune-induced mitochondrial damage. The established knowledge in these subjects warrants studies aimed at elucidating several open questions that are highlighted in the present review. The relevance of OS and MDF in different pathologies may establish the grounds for chemoprevention trials aimed at compensating OS/MDF by means of antioxidants and mitochondrial nutrients

Mutation spectrum of MLL2 in a cohort of kabuki syndrome patients

ABSTRACT: BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management

The E1A-Associated p400 Protein Modulates Cell Fate Decisions by the Regulation of ROS Homeostasis

The p400 E1A-associated protein, which mediates H2A.Z incorporation at specific promoters, plays a major role in cell fate decisions: it promotes cell cycle progression and inhibits induction of apoptosis or senescence. Here, we show that p400 expression is required for the correct control of ROS metabolism. Depletion of p400 indeed increases intracellular ROS levels and causes the appearance of DNA damage, indicating that p400 maintains oxidative stress below a threshold at which DNA damages occur. Suppression of the DNA damage response using a siRNA against ATM inhibits the effects of p400 on cell cycle progression, apoptosis, or senescence, demonstrating the importance of ATM–dependent DDR pathways in cell fates control by p400. Finally, we show that these effects of p400 are dependent on direct transcriptional regulation of specific promoters and may also involve a positive feedback loop between oxidative stress and DNA breaks since we found that persistent DNA breaks are sufficient to increase ROS levels. Altogether, our results uncover an unexpected link between p400 and ROS metabolism and allow deciphering the molecular mechanisms largely responsible for cell proliferation control by p400