Foxf2: A Novel Locus for Anterior Segment Dysgenesis Adjacent to the Foxc1 Gene

Anterior segment dysgenesis (ASD) is characterised by an abnormal migration of neural crest cells or an aberrant differentiation of the mesenchymal cells during the formation of the eye's anterior segment. These abnormalities result in multiple tissue defects affecting the iris, cornea and drainage structures of the iridocorneal angle including the ciliary body, trabecular meshwork and Schlemm's canal. In some cases, abnormal ASD development leads to glaucoma, which is usually associated with increased intraocular pressure. Haploinsufficiency through mutation or chromosomal deletion of the human FOXC1 transcription factor gene or duplications of the 6p25 region is associated with a spectrum of ocular abnormalities including ASD. However, mapping data and phenotype analysis of human deletions suggests that an additional locus for this condition may be present in the same chromosomal region as FOXC1. DHPLC screening of ENU mutagenised mouse archival tissue revealed five novel mouse Foxf2 mutations. Re-derivation of one of these (the Foxf2W174R mouse lineage) resulted in heterozygote mice that exhibited thinning of the iris stroma, hyperplasia of the trabecular meshwork, small or absent Schlemm's canal and a reduction in the iridocorneal angle. Homozygous E18.5 mice showed absence of ciliary body projections, demonstrating a critical role for Foxf2 in the developing eye. These data provide evidence that the Foxf2 gene, separated from Foxc1 by less than 70 kb of genomic sequence (250 kb in human DNA), may explain human abnormalities in some cases of ASD where FOXC1 has been excluded genetically

The Tumor Suppressor PRDM5 Regulates Wnt Signaling at Early Stages of Zebrafish Development

PRDM genes are a family of transcriptional regulators that modulate cellular processes such as differentiation, cell growth and apoptosis. Some family members are involved in tissue or organ maturation, and are differentially expressed in specific phases of embryonic development. PRDM5 is a recently identified family member that functions as a transcriptional repressor and behaves as a putative tumor suppressor in different types of cancer. Using gene expression profiling, we found that transcriptional targets of PRDM5 in human U2OS cells include critical genes involved in developmental processes, and specifically in regulating wnt signaling. We therefore assessed PRDM5 function in vivo by performing loss-of-function and gain-of-function experiments in zebrafish embryos. Depletion of prdm5 resulted in impairment of morphogenetic movements during gastrulation and increased the occurrence of the masterblind phenotype in axin+/− embryos, characterized by the loss of eyes and telencephalon. Overexpression of PRDM5 mRNA had opposite effects on the development of anterior neural structures, and resulted in embryos with a shorter body axis due to posterior truncation, a bigger head and abnormal somites. In situ hybridization experiments aimed at analyzing the integrity of wnt pathways during gastrulation at the level of the prechordal plate revealed inhibition of non canonical PCP wnt signaling in embryos overexpressing PRDM5, and over-activation of wnt/β-catenin signaling in embryos lacking Prdm5. Our data demonstrate that PRDM5 regulates the expression of components of both canonical and non canonical wnt pathways and negatively modulates wnt signaling in vivo

Myc-regulated microRNAs attenuate embryonic stem cell differentiation

Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c-Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer-extension PCR assay we identified miRNAs, including, miR-141, miR-200, and miR-429 whose expression is regulated by c-Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c-Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c-Myc. We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation

Regulation of metabolism and inflammation in liver and skeletal muscle

Type 2 diabetes (T2D) is a complex metabolic disorder characterised by hyperinsulinaemia, hyperglycaemia and dyslipidaemia. Obesity is the major risk factor for development of insulin resistance, a main predictor of T2D. Recent evidence indicates that nutrient excess and obesity lead to chronic low-grade inflammation in metabolic tissues, which further promotes insulin resistance. AMP-activated protein kinase (AMPK), a central regulator of energy homeostasis, increases insulin sensitivity in liver and skeletal muscle and lowers the plasma glucose level, thus reverting the major metabolic disturbances in T2D. Serine/threonine protein kinase 25 (STK25) was found to be differentially expressed in skeletal muscle, comparing AMPKγ3 (Prkag3-/-) knockout mice to wild-type littermates, indicating a potential role for STK25 in regulation of energy homeostasis in skeletal muscle. In Paper I, genes regulating the circadian rhythm (Cry2, Nr1d1 and Bhlhb2) were shown to be differentially expressed in skeletal muscle from wild-type mice treated with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), while they remained unaltered in AMPKγ3 knockout mice. Furthermore, the respiratory exchange ratio (RER) was elevated during the dark period of observation in wild-type mice reflecting a diurnal shift in substrate utilisation from lipid oxidation at daytime to carbohydrate utilisation during nighttime. However, no day/night shift in the RER profile was observed in Prkag3-/- littermates. Thus, this study suggests that APMK, as a central energy sensor, could be one important node linking energy metabolism to the circadian clock function. In Papers II and III, the AMPK agonists, AICAR and metformin, are shown to markedly decrease the expression of IL-6-induced serum amyloid A (SAA) cluster genes, haptoglobin and suppressor of cytokine signalling 3 (SOCS3) in the human hepatocyte cell line HepG2. By repressing AMPK activity with small interfering (si)RNA the inhibitory effect of AMPK on SAA expression by both AICAR and metformin was reversed (Paper II), indicating that the effect of the agonists is mediated by AMPK activation. Further, we show that AMPK interferes with IL-6 signalling by decreasing IL-6-induced phosphorylation of Janus kinase 1 (JAK1), src homology 2 domain containing protein tyrosine phosphatase 2 (SHP2) and signal transducer and activator of transcription 3 (STAT3) in HepG2 cells (Papers II and III). In addition, pharmacological activation of AMPK was shown to repress IL-6-induced inflammation in vivo by suppression of STAT3 activity in mouse liver (Paper III). This suggests that AMPK is an important intracellular link between metabolic and inflammatory pathways in liver. In Paper IV we show that partial reduction of STK25 by siRNA increases uncoupling protein 3 (UCP3), glucose transporter 1 (GLUT1), GLUT4 and hexokinase 2 (HK2) in the rodent myoblast cell line L6, both at mRNA and protein level. Correspondingly, the rates of palmitate oxidation and insulin-stimulated glucose uptake were elevated after partial depletion of STK25. In conclusion, our studies suggest a role of STK25 as a negative regulator of glucose and lipid metabolism in skeletal muscle. Impaired glucose uptake and fatty acid metabolism by skeletal muscle is a hallmark of insulin resistance, and therefore, STK25 could be an important new mediator to be evaluated for therapeutic intervention in T2D and related complications

3-D chromatin conformation, accessibility, and gene expression profiling of triple-negative breast cancer

This work was supported by the Instituto de la Salud Carlos III (ISCIII) Sara Borrell project (#CD22/00026), Miguel Servet Project (#CPII22/00004), and AES 2022 (#PI22/01496) and co-funded by European Union, the Institut d'Investigació Sanitària Illes Balears (FOLIUM program and IMPETUS Call IMP21/10), the Govern de les Illes Balears (Margalida Comas program), the Fundación Francisco Cobos, the Asociación Española Contra el Cancer (AECC), the department of European Funds, University, and Culture of the Government of the Balearic Islands and the "CONTIGO Contra el Cancer de Mujer" foundation (#MERIT project). The group acknowledges support from the EASI-Genomics project, which has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 824110. This project (EPIMETN) was supported by the National Genomics Infrastructure in Stockholm, funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. The funding body played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript.We want to acknowledge Llabata. P and NIMGenetics group for their technical support in RNA-seq.Objectives: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with limited treatment options. Unlike other breast cancer subtypes, the scarcity of specific therapies and greater frequencies of distant metastases contribute to its aggressiveness. We aimed to find epigenetic changes that aid in the understanding of the dissemination process of these cancers. Data description: Using CRISPR/Cas9, our experimental approach led us to identify and disrupt an insulator element, IE8, whose activity seemed relevant for cell invasion. The experiments were performed in two well-established TNBC cellular models, the MDA-MB-231 and the MDA-MB-436. To gain insights into the underlying molecular mechanisms of TNBC invasion ability, we generated and characterized high-resolution chromatin interaction (Hi-C) and chromatin accessibility (ATAC-seq) maps in both cell models and complemented these datasets with gene expression profiling (RNA-seq) in MDA-MB-231, the cell line that showed more significant changes in chromatin accessibility. Altogether, our data provide a comprehensive resource for understanding the spatial organization of the genome in TNBC cells, which may contribute to accelerating the discovery of TNBC-specific alterations triggering advances for this devastating disease