Disruption of neural crest enhancer landscapes as an etiological mechanism for human neurocristopathies

The embryonic development of the human facial features is a highly complex mechanism which requires very exact spatial and temporal regulation of gene expression during neural crest (NC) development. NC cells (NCC) are a transient embryonic cell type with wide differentiation potential that contributes to the formation and morphogenesis of multiple tissues and organs, including many parts of the face. Just like any other cell type, NCC possess a characteristic set of enhancers that, by controlling the expression of specific genes, define cellular identity. Impairment of this regulation can lead to craniofacial malformations, such as orofacial cleft (OFC), which are frequently referred to as neurocristopathies and that represent a heavy burden on both the affected individuals and society. Understanding how genetic or structural disruption of enhancer activity during NC development can lead to human neurocristopathies is the central goal of this work. In the long term, the gained knowledge should serve to enable early detection and show potential therapeutic approaches. Here we investigate the pathomechanism of both syndromic (i.e. Branchiooculofacial Syndrome (BOFS)) and non-syndromic (i.e. OFC) neurocristopathies, by combining in vitro and in vivo NC developmental models with genetic engineering approaches and multiple genomic methods. First, we describe a unique patient with BOFS, who, in contrast to previously reported cases, does not present a heterozygous mutations within TFAP2A, a NC master regulator. Instead, the patient carries a de novo heterozygous 89 Mb inversion in which one of the breakpoints is located 40 kb downstream of TFAP2A. We first showed that this inversion separates TFAP2A from enhancers that are located within the same large topologically associating domain (TAD) and that are essential for TFAP2A expression in NCC. Importantly, using patient-specific human induced pluripotent stem cells (hiPSC) and a robust in vitro differentiation system towards NCC, we then showed that the inversion causes a loss of physical interactions between the inverted TFAP2A allele and its cognate enhancers, leading to TFAP2A monoallelic and haploinsufficient expression in human NCC. Overall, this first part provides a powerful approach to investigate the pathological mechanisms of structural variants predicted to disrupt 3D genome organization of gene regulatory landscapes and that, due to various reasons (i.e. limited access to relevant patient material, differences in gene dosage sensitivity between mice and humans, difficulties in recapitulating certain structural variants), cannot be properly evaluated in vivo. Second, we combined previously generated hNCC enhancer maps with OFC risk-loci identified through genome-wide association studies (GWAS) and, as a result, we revealed a highly conserved enhancer (i.e. Enh2p24.2) as a potential candidate harboring genetic variants involved in OFC. GWAS link common single nucleotide polymorphisms (SNPs) with quantitative traits and complex disorders. However, most disease-associated SNPs occur in non-coding regions of the human genome and consequently, the etiological relevance of these genetic variants cannot be easily connected to a gene. Nevertheless, accumulating evidences suggest that these disease-associated SNPs may contribute to human disease susceptibility by altering enhancers. Interestingly, SNPs associated with OFC are overrepresented in NCC enhancers. Therefore, we hypothesize that SNPs associated with OFC contribute to the etiology of the disorder by altering NCC enhancers and, consequently, the expression of relevant genes. Using Enh2p24.2 as a bait in circularized chromosome conformation capture sequencing (4C-seq) experiments, we identified two distally located genes, MYCN and DDX1, as its potential targets. Using in vitro and in vivo NCC developmental models, we then demonstrated that both genes are essential for normal facial development. While MYCN was not a surprising candidate to be involved in the etiology of OFC, the identification of DDX1 as a novel regulator of facial development might provide new insights into the molecular processes (e.g. transcription-coupled DNA repair) implicated in OFC and, potentially, other human neurocristopathies (e.g. neuroblastoma)

The characterization of genetic risk factors associated with autism

Autism is a severe neurodevelopmental disorder. Development of a molecular diagnostic screen is an imperative step towards personalized treatments. Gene expression profiling using buccal samples was employed to identify susceptibility genes and dysregulated signaling pathways. Analyses of differentially regulated genes revealed numerous genes that were associated with development and function of the nervous and immune systems, circadian rhythm, and ERBB signaling. Amongst the affected participants there was a patient with a 3p14.1-p13 deletion, where FOXP1 is located. FOXP2 mutations are responsible for human speech and language disorders. Since FOXP1, FOXP2, and FOXP4 require dimerization for transcriptional activity, investigating the FOXP1/2/4 molecular network provides insight into the neural mechanisms behind language impairments in autism. HEK293 cells were transfected with FOXP1/2/4 constructs. QRT-PCR was used to evaluate mRNA expression of FOXP2 target genes. Results suggest that specific combinations of FOXP1/2/4 dimers may influence the transcription of target genes involved in language acquisition

28th Fungal Genetics Conference

Full abstracts from the 28th Fungal Genetics Conference Asilomar, March 17-22, 2015

Characterization of the cargo of circulating extracellular vesicles in patients affected with idiopathic inflammatory myopathies and evaluation of clinical correlates in a cross-sectional comparative analysis from a monocentric cohort.

openBackground Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune disorders that comprise different clinical entities, characterized by different features. Although several myositis-specific and myositis-associated antibodies have been characterized, the molecular mechanisms underlying these conditions require further exploration. The research field on extracellular vescicles (EVs) is rapidly evolving, highlighting their role in intercellular communication. EVs convoy a cargo of proteins and nucleic acids, such as microRNA (miRNAs), that mediate immune-response regulation in autoimmune diseases. miRNAs regulate gene-expression post-transcriptionally and are involved in multiple molecular pathways of human disease. Evidence of EVs and miRNAs in IIM is still limited and undefined. Aim of the study This study aims to quantify the circulating EVs and characterize their cargo, with a specific focus on miRNAs content to propose novel biomarkers of IIM. Materials and methods A monocentric study was conducted including adult IIM patients (≥18 years old) followed at the Rheumatology Unit of Padua University Hospital, and age- and sex- matched healthy controls (HD). EVs were isolated from platelet-free plasma through size exclusion chromatography followed by ultrafiltration. EVs were quantified by nanoparticle tracking analysis (NTA) and. EV-miRNA cargo was investigated through Next-Generation Sequencing (NGS). Statistical analysis was performed with parametric Student-T test and one-way Anova (Bonferroni correction). Results Sixty-four consecutive IIM patients and sixty-five HDs were included in the study. NTA measurements of EVs concentration showed a significantly higher mean concentration of circulating EVs in IIM patients than in HD (p=0.0073). Across IIM subsets, patients affected with cancer associated myositis (CAM) displayed the highest levels of circulating EVs compared to no CAM patients (p=0.0060) and to HD (p=0.0004). Patients with circulating myositis-associated autoantibodies displayed significantly higher EV levels than HD (p=0.0363). Patients in clinical remission displayed higher levels of circulating EVs compared to those with active disease (p=0.0087). EVs levels were significantly reduced in IIM patients treated with rituximab (RTX) than in patients receiving other treatments (p<0.0001). NGS analysis detected EV-miRNAs with different expression profiles between IIM (n=47) and HDs (n=49): miR-223-3p (p=0.019), miR-15a-5p (p=0.0189), miR-451a (p=0.0074), miR-486-5p (p=0.0052), miR-32-5p (p=0.0146), and miR-222-3p (p=0.0282) were up-regulated in IIM, while miR-141-3p (p=0.0313), miR-142-3p (p=0.0244), and let-7a-5p (p=0.0003) were down-regulated in IIM patients vs. HDs. Other EV-miRNAs expression varied across IIM subsets: CAM patients displayed up-regulated expression of miR-143-3p compared to non-CAM patients (p=0.0085), while miR-148a-3p (p=0.0171) and miR-335-5p (p=0.0171) were up-regulated in dermatomyositis vs. polymyositis/ inclusion body myositis/anti-synthetase syndrome patients. Patients characterized by active disease displayed an up-regulated expression of miR-222-3p (p=0.002) and miR-151-3p (p=0.0233) and down-regulated expression of miR-363-3p (p=0.0001), miR-374a-5p (p=0.0258), miR-144-3p (p=0.0170), miR-181a-5p (p=0.0037) compared to those in clinical remission. Moreover, IIM patients receiving only glucocorticoids (GC) reported up-regulated expression of miR-4433b-5p (p=0.0439), miR-92a-3p (p=0.0111), let-7f-5p (p=0.0304), and down-regulated expression of miR-27a-3p (p=0.0486) compared to patients receiving GC in combination with immunosuppressants (IS). Conclusions Our results showed significantly increased concentration of circulating EVs in IIM patients. That is confirmed within specific disease phenotypes and pharmacological treatments. EV miRNAs exhibited a differential expression profile between IIM and HD, and significant differences were outlined among IIM subsets.Background Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune disorders that comprise different clinical entities, characterized by different features. Although several myositis-specific and myositis-associated antibodies have been characterized, the molecular mechanisms underlying these conditions require further exploration. The research field on extracellular vescicles (EVs) is rapidly evolving, highlighting their role in intercellular communication. EVs convoy a cargo of proteins and nucleic acids, such as microRNA (miRNAs), that mediate immune-response regulation in autoimmune diseases. miRNAs regulate gene-expression post-transcriptionally and are involved in multiple molecular pathways of human disease. Evidence of EVs and miRNAs in IIM is still limited and undefined. Aim of the study This study aims to quantify the circulating EVs and characterize their cargo, with a specific focus on miRNAs content to propose novel biomarkers of IIM. Materials and methods A monocentric study was conducted including adult IIM patients (≥18 years old) followed at the Rheumatology Unit of Padua University Hospital, and age- and sex- matched healthy controls (HD). EVs were isolated from platelet-free plasma through size exclusion chromatography followed by ultrafiltration. EVs were quantified by nanoparticle tracking analysis (NTA) and. EV-miRNA cargo was investigated through Next-Generation Sequencing (NGS). Statistical analysis was performed with parametric Student-T test and one-way Anova (Bonferroni correction). Results Sixty-four consecutive IIM patients and sixty-five HDs were included in the study. NTA measurements of EVs concentration showed a significantly higher mean concentration of circulating EVs in IIM patients than in HD (p=0.0073). Across IIM subsets, patients affected with cancer associated myositis (CAM) displayed the highest levels of circulating EVs compared to no CAM patients (p=0.0060) and to HD (p=0.0004). Patients with circulating myositis-associated autoantibodies displayed significantly higher EV levels than HD (p=0.0363). Patients in clinical remission displayed higher levels of circulating EVs compared to those with active disease (p=0.0087). EVs levels were significantly reduced in IIM patients treated with rituximab (RTX) than in patients receiving other treatments (p<0.0001). NGS analysis detected EV-miRNAs with different expression profiles between IIM (n=47) and HDs (n=49): miR-223-3p (p=0.019), miR-15a-5p (p=0.0189), miR-451a (p=0.0074), miR-486-5p (p=0.0052), miR-32-5p (p=0.0146), and miR-222-3p (p=0.0282) were up-regulated in IIM, while miR-141-3p (p=0.0313), miR-142-3p (p=0.0244), and let-7a-5p (p=0.0003) were down-regulated in IIM patients vs. HDs. Other EV-miRNAs expression varied across IIM subsets: CAM patients displayed up-regulated expression of miR-143-3p compared to non-CAM patients (p=0.0085), while miR-148a-3p (p=0.0171) and miR-335-5p (p=0.0171) were up-regulated in dermatomyositis vs. polymyositis/ inclusion body myositis/anti-synthetase syndrome patients. Patients characterized by active disease displayed an up-regulated expression of miR-222-3p (p=0.002) and miR-151-3p (p=0.0233) and down-regulated expression of miR-363-3p (p=0.0001), miR-374a-5p (p=0.0258), miR-144-3p (p=0.0170), miR-181a-5p (p=0.0037) compared to those in clinical remission. Moreover, IIM patients receiving only glucocorticoids (GC) reported up-regulated expression of miR-4433b-5p (p=0.0439), miR-92a-3p (p=0.0111), let-7f-5p (p=0.0304), and down-regulated expression of miR-27a-3p (p=0.0486) compared to patients receiving GC in combination with immunosuppressants (IS). Conclusions Our results showed significantly increased concentration of circulating EVs in IIM patients. That is confirmed within specific disease phenotypes and pharmacological treatments. EV miRNAs exhibited a differential expression profile between IIM and HD, and significant differences were outlined among IIM subsets

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin

Beyond transcription : a post-transcriptional role of 3D chromatin crosstalk in oncogene regulation

This thesis explores how stochastic chromatin fibre interactions, chromatin organization in the 3D nuclear architecture, and environmental signals collaborate to regulate MYC oncogene expression in human colon cancer cells. In Paper I, we employ the ultra-sensitive Nodewalk technique to uncover the dynamic and stochastic nature of chromatin networks impinging on MYC. The analyses revealed that the MYC interactome mainly consists of stochastic pairwise interactions between MYC and its flanking enhancers in two neighbouring topologically associated domains (TADs), which are insulated self-interacting genomic domains. The limits of Nodewalk were also pushed to enable the detection of interactions in very small cell populations, corresponding to the genomic content of ~7 cells. Comparing the frequency of interactions detected in such small input samples with ensemble interactomes of large cell populations uncovered that the enhancer hubs of the ensemble interactomes that appear to simultaneously interact with MYC likely represent virtual events, which are not present in reality at the single cell level. These data support a model where MYC interacts with its enhancers in a mutually exclusive way, with MYC screening for enhancer contacts, rather than the other way around. Paper II provides a detailed understanding of a novel post-transcriptional mechanism of enhancer action on MYC expression. We have thus uncovered that the cancer-specific recruitment of the MYC gene to nuclear pores and ensuing rapid nuclear export of MYC transcripts - a process that increases MYC expression by enabling the escape of MYC mRNAs from rapid decay in the nucleus - require a CTCF binding site positioned within the colorectal oncogenic super-enhancer. Genetic editing by CRISPR-Cas9 was thus commissioned to establish two clones of human colon cancer cells with a mutated sequence in the OSE-specific CTCFBS. Comparing the mutant cells to the parental cell line, we uncovered that the WNT-dependent increase in the nuclear export rate of MYC transcripts was abrogated in the CTCFBS mutant clones, providing the first genetic evidence of super- enhancer-mediated gene gating in human cells. In line with this finding, the OSE-specific CTCFBS thus conferred a significant growth advantage to the parental colon cancer cells, compared to the mutant clones. Moreover, we found that WNT-dependent CCAT1 eRNA transcription is mediated by the OSE-specific CTCFBS that is required for recruitment of AHCTF1 to the OSE to mediate the positioning of the OSE to the nuclear periphery, enabling the subsequent facilitation of MYC mRNA export. A multistep molecular process including WNT signalling and the OSE-specific CTCFBS thus underlies the gene gating of MYC in human colon cancer cells, and could potentially be targeted for diagnostic or therapeutic uses. In summary, this thesis explores the dynamics of the stochastic interactomes impinging on the MYC oncogene, and provides new insights on the role of 3D chromatin orchestration in the transcriptional regulation of MYC. Our analyses uncovered the molecular factors involved in the gene gating of MYC, and thus increase our understanding of tumour development. These findings could potentially be beneficial for future diagnostic approaches, or for targeted therapeutic strategies in the treatment of cancer

Characterisation of a gain-of-function mutant of CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) in Arabidopsis thaliana

Receptor-like kinases (RLKs) comprise a large superfamily of proteins in plant genomes, and play essential roles in plant growth, development and response to biotic and abiotic stresses. The CYSTEINE-RICH RECEPTOR-LIKE KINASES (CRKs) comprise one of the largest subfamilies of RLKs with over 40 members in Arabidopsis thaliana, and although a few members of the family have been initially characterised, their precise biological functions remain largely unknown. This thesis reports the characterisation of a novel gain-of-function allele of CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) in A. thaliana which was isolated from a chemical mutagenesis screen. This mutation causes the substitution of alanine 397 with a threonine residue in subdomain III / αC-helix of the kinase domain of CRK10, and this novel allele has been accordingly registered as crk10-A397T with the Arabidopsis community database. The crk10- A397T mutant is a dwarf, and anatomical characterisation unveiled severely collapsed xylem vessels in the root and hypocotyl of the plant. Reporter lines suggested CRK10 is expressed in close association to vascular tissues, and a translational fusion with the fluorescent protein mCherry indicates that CRK10 is a plasma membrane-bound protein. Analysis of the recombinant WT and crk10- A397T versions of the cytoplasmic kinase domain of CRK10 demonstrated their auto-phosphorylation activity, and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis concluded that Thr397 acts as an additional auto-phosphorylation site in situ. Furthermore, an RNA-seq experiment revealed the constitutive induction of defence-related genes in the transcriptome of crk10- A397T mutant hypocotyls, including genes involved in the signalling pathways of the stress hormones salicylic acid (SA) and abscisic acid (ABA). Analysis of the composition of cell walls in the crk10-A397T mutant hypocotyls revealed extensive differences compared to the WT, an indication of cell wall remodelling mechanisms that are likely associated with the collapse of xylem vessels in this organ. Bioassays with the soil-borne vascular pathogen Fusarium oxysporum revealed that crk10-A397T mutant has a greater probability of survival to infection compared to WT plants. Analysis of genetic crosses demonstrated that key components of SA signalling pathways are required for the disease resistance phenotype of the crk10-A397T mutant