Morphology of Purkinje Cells in Cerebella of Epileptic Mice

Calcium ion (Ca2+) homeostasis is a critical component normal neuronal development. Abnormal Ca2+ concentrations can disrupt neuron development and nervous system function and lead to various neurological disorders, such as epilepsy. Leaner and tottering mice, which are spontaneous animal models of human absence epilepsy, exhibit reduced Ca2+ current density in neurons known as Purkinje cells within their cerebellum. This is a result of unique mutations that each type of mutant mouse carry in the P/Q-type calcium channel 1A subunit gene, and highly expressed in Purkinje cells. Consequently, both mutant mice exhibit severe juvenile onset of ataxia, paroxysmal dyskinesia, and absence seizures. Previous studies have revealed significant loss of Purkinje cells in leaner mice. However, the specific structural effects of these mutations on existing cerebellar neurons remain unclear. We examined the neuronal morphology of Purkinje cells in adult (6-8 months) male and female mice of both mutant genotypes. We found significant reduction in dendritic growth and arborization in Purkinje cells of leaner mice and a trend towards reduction in dendritic growth and arborization in Purkinje cells of tottering mice when compared to wild type mice. No difference in somatic area was observed between any two of the genotypes. Hence, we conclude that there are significant morphological deficits present in Purkinje cells of leaner mice compared to wild type mice that could, in part, explain the symptoms seen in the mice, but the lack of structural differences in Purkinje cells of tottering mice indicate that there are likely to be underlying functional impairments present that require study beyond the structural level

Discovery of non-directional and directional pioneer transcription factors by modeling DNase profile magnitude and shape

Here we describe Protein Interaction Quantitation (PIQ), a computational method that models the magnitude and shape of genome-wide DNase profiles to facilitate the identification of transcription factor (TF) binding sites. Through the use of machine learning techniques, PIQ identified binding sites for >700 TFs from one DNase-seq experiment with accuracy comparable to ChIP-seq for motif-associated TFs (median AUC=0.93 across 303 TFs). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into pre-pancreatic and intestinal endoderm. We identified (n=120) and experimentally validated eight ‘pioneer’ TF families that dynamically open chromatin, enabling other TFs to bind to adjacent DNA. Four pioneer TF families only open chromatin in one direction from their motifs. Furthermore, we identified a class of ‘settler’ TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and non-directional pioneer activity shapes the chromatin landscape for population by settler TFs

Discovery of directional and nondirectional pioneer transcription factors by modeling DNase profile magnitude and shape

We describe protein interaction quantitation (PIQ), a computational method for modeling the magnitude and shape of genome-wide DNase I hypersensitivity profiles to identify transcription factor (TF) binding sites. Through the use of machine-learning techniques, PIQ identified binding sites for >700 TFs from one DNase I hypersensitivity analysis followed by sequencing (DNase-seq) experiment with accuracy comparable to that of chromatin immunoprecipitation followed by sequencing (ChIP-seq). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into prepancreatic and intestinal endoderm. We identified 120 and experimentally validated eight 'pioneer' TF families that dynamically open chromatin. Four pioneer TF families only opened chromatin in one direction from their motifs. Furthermore, we identified 'settler' TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and nondirectional pioneer activity shapes the chromatin landscape for population by settler TFs.National Institutes of Health (U.S.) (Common Fund 5UL1DE019581)National Institutes of Health (U.S.) (Common Fund RL1DE019021)National Institutes of Health (U.S.) (Common Fund 5TL1EB008540)National Institutes of Health (U.S.) (Grant 1U01HG007037)National Institutes of Health (U.S.) (Grant 5P01NS055923