Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

Remodeling of Zn2+ homeostasis upon differentiation of mammary epithelial cells.

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother's milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.

ß-Propeller protein-associated neurodegeneration: a new X-linked dominant disorder with brain iron accumulation.

Neurodegenerative disorders with high iron in the basal ganglia encompass an expanding collection of single gene disorders collectively known as neurodegeneration with brain iron accumulation. These disorders can largely be distinguished from one another by their associated clinical and neuroimaging features. The aim of this study was to define the phenotype that is associated with mutations in WDR45, a new causative gene for neurodegeneration with brain iron accumulation located on the X chromosome. The study subjects consisted of WDR45 mutation-positive individuals identified after screening a large international cohort of patients with idiopathic neurodegeneration with brain iron accumulation. Their records were reviewed, including longitudinal clinical, laboratory and imaging data. Twenty-three mutation-positive subjects were identified (20 females). The natural history of their disease was remarkably uniform: global developmental delay in childhood and further regression in early adulthood with progressive dystonia, parkinsonism and dementia. Common early comorbidities included seizures, spasticity and disordered sleep. The symptoms of parkinsonism improved with l-DOPA; however, nearly all patients experienced early motor fluctuations that quickly progressed to disabling dyskinesias, warranting discontinuation of l-DOPA. Brain magnetic resonance imaging showed iron in the substantia nigra and globus pallidus, with a 'halo' of T1 hyperintense signal in the substantia nigra. All patients harboured de novo mutations in WDR45, encoding a beta-propeller protein postulated to play a role in autophagy. Beta-propeller protein-associated neurodegeneration, the only X-linked disorder of neurodegeneration with brain iron accumulation, is associated with de novo mutations in WDR45 and is recognizable by a unique combination of clinical, natural history and neuroimaging features.

Analysis of ATP13A2 in large neurodegeneration with brain iron accumulation (NBIA) and dystonia-parkinsonism cohorts.

Several causative genes have been identified for both dystonia-parkinsonism and neurodegeneration with brain iron accumulation (NBIA), yet many patients do not have mutations in any of the known genes. Mutations in the ATP13A2 lead to Kufor Rakeb disease, a form of autosomal recessive juvenile parkinsonism that also features oromandibular dystonia. More recently, evidence of iron deposition in the caudate and putamen have been reported in patients with ATP13A2 mutations. We set out to determine the frequency of ATP13A2 mutations in cohorts of idiopathic NBIA and dystonia-parkinsonism. We screened for large deletions using whole genome arrays, and sequenced the entire coding region in 92 cases of NBIA and 76 cases of dystonia-parkinsonism. A number of coding and non-coding sequence variants were identified in a heterozygous state, but none were predicted to be pathogenic based on in silico analyses. Our results indicate that ATP13A2 mutations are a rare cause of both NBIA and dystonia-parkinsonism.