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1.
Elife ; 102021 10 04.
Article in English | MEDLINE | ID: mdl-34605404

ABSTRACT

Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type-specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.


Subject(s)
Brain/metabolism , Enhancer Elements, Genetic , Animals , Brain/growth & development , High-Throughput Nucleotide Sequencing , Mice
2.
Genome Med ; 13(1): 69, 2021 04 26.
Article in English | MEDLINE | ID: mdl-33910599

ABSTRACT

BACKGROUND: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified. METHODS: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior. RESULTS: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits. CONCLUSIONS: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.


Subject(s)
Epilepsy/genetics , Gene Expression Regulation , NAV1.1 Voltage-Gated Sodium Channel/genetics , Sequence Deletion/genetics , Animals , Attention , Base Sequence , Brain/metabolism , Brain/pathology , Chromatin/metabolism , Conserved Sequence/genetics , Disease Models, Animal , Electroencephalography , Epilepsy/diagnostic imaging , Evolution, Molecular , Female , HEK293 Cells , Heterozygote , Homozygote , Humans , Male , Maze Learning , Memory Disorders/genetics , Mice, Inbred C57BL , Neurons/metabolism , Open Field Test , Phenotype , Protein Binding , Regulatory Sequences, Nucleic Acid/genetics , Survival Analysis , Temperature , Trans-Activators/metabolism
3.
Front Endocrinol (Lausanne) ; 11: 580879, 2020.
Article in English | MEDLINE | ID: mdl-33240218

ABSTRACT

The brain is responsible for maintaining whole-body energy homeostasis by changing energy input and availability. The hypothalamus and dorsal vagal complex (DVC) are the primary sites of metabolic control, able to sense both hormones and nutrients and adapt metabolism accordingly. The mitochondria respond to the level of nutrient availability by fusion or fission to maintain energy homeostasis; however, these processes can be disrupted by metabolic diseases including obesity and type II diabetes (T2D). Mitochondrial dynamics are crucial in the development and maintenance of obesity and T2D, playing a role in the control of glucose homeostasis and whole-body metabolism across neurons and glia in the hypothalamus and DVC.


Subject(s)
Brain/physiology , Eating , Glucose/metabolism , Homeostasis , Metabolic Diseases/physiopathology , Mitochondrial Dynamics , Animals , Energy Metabolism , Humans
4.
Chem Commun (Camb) ; 56(45): 6098-6101, 2020 Jun 07.
Article in English | MEDLINE | ID: mdl-32355935

ABSTRACT

Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by 'click' chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using 'click' chemistry.


Subject(s)
Azides/administration & dosage , Cadmium Compounds/administration & dosage , Cholera Toxin/administration & dosage , Motor Neurons/metabolism , Nanoparticles/administration & dosage , Selenium Compounds/administration & dosage , Sulfides/administration & dosage , Zinc Compounds/administration & dosage , Animals , Azides/chemistry , Brain Stem/metabolism , Cadmium Compounds/chemistry , Cholera Toxin/chemistry , Mice , Nanoparticles/chemistry , Selenium Compounds/chemistry , Sulfides/chemistry , Zinc Compounds/chemistry
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