Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

<p>Abstract</p> <p>Background</p> <p>Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i) a source for purified cells (or nuclei) of distinct types, and (ii) a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models) or to amalgamations of diverse cell types (tissue chunks, whole organisms).</p> <p>Results</p> <p>Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine <it>in vivo </it>expression of a bacterial DNA adenine methyltransferase (DAM) with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture) towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of <it>C. elegans </it>strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis), our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the <it>C. elegans </it>(diploid) genome.</p> <p>Conclusions</p> <p>Validating the DALEC mapping results, we observe a strong association between observed coverage by nucleosomes and low DAM accessibility. Strikingly, we observed no extended regions of inaccessible chromatin for any of the tissues examined. These results are consistent with "local choreography" models in which differential gene expression is driven by intricate local rearrangements of chromatin structure rather than gross impenetrability of large chromosomal regions.</p

Shaping Skeletal Growth by Modular Regulatory Elements in the Bmp5 Gene

Cartilage and bone are formed into a remarkable range of shapes and sizes that underlie many anatomical adaptations to different lifestyles in vertebrates. Although the morphological blueprints for individual cartilage and bony structures must somehow be encoded in the genome, we currently know little about the detailed genomic mechanisms that direct precise growth patterns for particular bones. We have carried out large-scale enhancer surveys to identify the regulatory architecture controlling developmental expression of the mouse Bmp5 gene, which encodes a secreted signaling molecule required for normal morphology of specific skeletal features. Although Bmp5 is expressed in many skeletal precursors, different enhancers control expression in individual bones. Remarkably, we show here that different enhancers also exist for highly restricted spatial subdomains along the surface of individual skeletal structures, including ribs and nasal cartilages. Transgenic, null, and regulatory mutations confirm that these anatomy-specific sequences are sufficient to trigger local changes in skeletal morphology and are required for establishing normal growth rates on separate bone surfaces. Our findings suggest that individual bones are composite structures whose detailed growth patterns are built from many smaller lineage and gene expression domains. Individual enhancers in BMP genes provide a genomic mechanism for controlling precise growth domains in particular cartilages and bones, making it possible to separately regulate skeletal anatomy at highly specific locations in the body

A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage.

Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation