Functional anatomy of distant-acting mammalian enhancers.

Transcriptional enhancers are a major class of functional element embedded in the vast non-coding portion of the human genome. Acting over large genomic distances, enhancers play critical roles in the tissue and cell type-specific regulation of genes, and there is mounting evidence that they contribute to the aetiology of many human diseases. Methods for genome-wide mapping of enhancer regions are now available, but the functional architecture contained within human enhancer elements remains unclear. Here, we review recent approaches aimed at understanding the functional anatomy of individual enhancer elements, using systematic qualitative and quantitative assessments of mammalian enhancer variants in cultured cells and in vivo. These studies provide direct insight into common architectural characteristics of enhancers including the presence of multiple transcription factor-binding sites and the mixture of both transcriptionally activating and repressing domains within the same enhancer. Despite such progress in understanding the functional composition of enhancers, the inherent complexities of enhancer anatomy continue to limit our ability to predict the impact of sequence changes on in vivo enhancer function. While providing an initial glimpse into the mutability of mammalian enhancers, these observations highlight the continued need for experimental enhancer assessment as genome sequencing becomes routine in the clinic.

Transmembrane protein 50b (C21orf4), a candidate for Down syndrome neurophenotypes, encodes an intracellular membrane protein expressed in the rodent brain.

Transmembrane protein 50b, Tmem50b, previously referred to as C21orf4, encodes a predicted transmembrane protein and is one of few genes significantly over-expressed during cerebellar development in a Down syndrome mouse model, Ts1Cje. In order to assess potential mechanisms by which Tmem50b could contribute to Down syndrome-related phenotypes, we determined the expression patterns of Tmem50b mRNA, as well as Tmem50b protein distribution, expression and subcellular localization. In situ hybridization in mice at embryonic day 14.5 showed cortical plate and spinal cord mRNA expression. By postnatal day 7, strong mRNA expression was seen in the cerebellum, hippocampus and olfactory bulb, with diffuse cortical expression. Quantitative PCR of adult mouse tissue showed Tmem50b mRNA expression in the brain, heart and testis. A rabbit polyclonal antibody was generated against Tmem50b and rat and mouse tissue screening by Western blot, and immunohistochemistry showed that protein expression concurred with mRNA expression. Double immunofluorescence revealed that Tmem50b is highly expressed in rat and mouse glial fibrillary acidic protein-positive cells in vivo and in vitro, but less so in neuronal MAP2- or beta-tubulin II-positive cells in vitro. Tmem50b is invariably expressed in cultured mouse neural precursor cells. In adult mouse cerebellum sections, Tmem50b immunoreactivity was found in Purkinje and Golgi cell somata and in Bergmann glial processes. Electron microscopy confirmed that Tmem50b was present on endoplasmic reticulum (ER) and Golgi apparatus membranes. Results indicate that Tmem50b is a developmentally-regulated intracellular ER and Golgi apparatus membrane protein that may prove important for correct brain development through functions associated with precursor cells and glia.

Development of high-throughput tools to unravel the complexity of gene expression patterns in the mammalian brain.

Genomes of animals contain between 15000 (e.g. Drosophila) and 50000 (human, mouse) genes, many of which encode proteins involved in regulatory processes. The availability of sequence data for many of these genes opens up opportunities to study complex genetic and protein interactions that underlie biological regulation. Many examples demonstrate that an understanding of regulatory networks consisting of multiple components is significantly advanced by a detailed knowledge of the spatiotemporal expression pattern of each of the components. Gene expression patterns can readily be determined by RNA in situ hybridization. The unique challenge emerging from the knowledge of the sequence of entire genomes is that assignment of biological functions to genes needs to be carried out on an appropriately large scale. In terms of gene expression analysis by RNA in situ hybridization, efficient technologies need to be developed that permit determination and representation of expression patterns of thousands of genes within an acceptable time-scale. We set out to determine the spatial expression pattern of several thousand genes encoding putative regulatory proteins. To achieve this goal we have developed high-throughput technologies that allow the determination and visualization of gene expression patterns by RNA in situ hybridization on tissue sections at cellular resolution. In particular, we have invented instrumentation for robotic in situ hybridization capable of carrying out in a fully automated fashion, all steps required for detecting sites of gene expression in tissue sections. In addition, we have put together hardware and software for automated microscopic scanning of gene expression data that are produced by RNA in situ hybridization. The potential and limitations of these techniques and our efforts to build a Web-based database of gene expression patterns are discussed.