Determinants of the DNA binding and gene regulatory specificity for type II nuclear receptor signaling

The type II nuclear receptors (NRs) function as heterodimeric transcription factors with the retinoid X receptor (RXR) to regulate diverse biological processes in response to endogenous ligands and therapeutic drugs. Due to their importance as therapeutic targets, the NRs have been extensively studied; however, the rules dictating NR transcriptional specificity remain unclear. Type II NRs regulate both distinct and overlapping gene programs. DNA-binding specificity has been proposed as a primary mechanism dictating how individual NRs distinguish their genomic targets. However, many in vivo targets of NRs identified in ChIP-seq data lack an identifiable binding site, suggesting that current models of DNA binding specificity of the type II NRs are incomplete. A more thorough characterization of the DNA binding and gene regulatory specificity of the type II NRs will be informative for understanding the role of DNA binding in achieving NR transcriptional specificity. NRs function can be altered by ligand binding, post-translational modifications (PTMs), and interactions with other proteins. Therefore, to understand how cellular context may alter NR regulatory specificity, analysis of NR DNA binding in the presence of different ligands or cell-specific coregulator proteins will be informative. I used protein binding microarrays (PBMs) to characterize the DNA binding preferences of twelve NR:RXRα heterodimers. I find more promiscuous NR DNA binding than has been reported, challenging the view that NR binding is defined by half-site spacing. I show that NRs bind DNA using two distinct modes, explaining widespread half-site binding in vivo. Finally, we show that current models of NR DNA binding preferences better reflect binding-site activity rather than binding-site affinity. Examining how DNA binding is altered in a cellular context, I find that NR-DNA binding is significantly altered in the presence of soluble nuclear components, such as other transcription factors and transcriptional coregulators. In the presence of other nuclear proteins NR DNA binding is less promiscuous, suggesting interactions with nuclear proteins can modulate NR DNA binding specificity. Our rich PBM dataset and revised NR binding models provide a framework for understanding NR specificity and will facilitate more accurate analyses of genomic datasets

Motif grammar: The basis of the language of gene expression

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Sca-1 is involved in the adhesion of myosphere cells to αVβ3 integrin

Summary A myosphere cell is a unique type of muscle stem cell that is able to maintain its pre-myogenic state in culture over time. These cells are propagated in culture as free-floating, non-adherent spheres. We believe that the 3-dimensional adhesive cell-cell interactions involved in maintaining the sphere-like myosphere structures are also involved in maintaining their longevity in culture. We found that Sca-1, which is highly expressed by myosphere cells, plays a role in the growth and the formation of the myospheres. In comparing adhesion molecules expressed by 3-dimensionally grown myosphere cells to those expressed by 2-dimensionally grown primary myoblasts, we found that there was a distinct difference in the expression of β3 integrin. Upon further investigation we discovered that there is an adhesive interaction between Sca-1+ cells and αVβ3 integrin. Here we show that Sca-1+ cells (myosphere cells and NIH3T3 cells) adhere to αVβ3 integrin and that Sca-1− cells (primary myoblasts) do not adhere. The interaction between Sca-1 and αVβ3 integrin was confirmed using antibody blocking, shRNA knockdown of Sca-1 in Sca-1+ cells, and by expressing Sca-1 cDNA in Sca-1− cells, which demonstrated that the level of adhesion of these cells to αVβ3 integrin was dependent on the presence of Sca-1. Additionally, we found that the co-expression of Sca-1 and β3 resulted in significantly greater adhesion of Sca-1+ cells to αVβ3 integrin. In conclusion, our data indicate that Sca-1 is involved in maintaining the 3-dimensional myosphere cell-cell contacts and that Sca-1 is involved in the binding of cells to αVβ3 integrin

Restoration Efforts on Lost Creek, Upper Clark Fork Basin

Lost Creek is a third order stream entering the upper Clark Fork River near Warm Springs, Montana. Lost Creek’s water quality and habitat issues include nutrient loading, excessive sedimentation, habitat and flow alterations, channelization, loss of woody riparian vegetation, and fish passage barriers. Among all tributaries, Lost Creek is the largest contributor of nitrogen to the upper Clark Fork River above Deer Lodge. The Tri-State Council’s Clark Fork River voluntary nutrient reduction program lists Lost Creek as the number one priority among non-point nutrient sources in the upper Clark Fork River basin. Montana Fish, Wildlife and Parks is undertaking a major watershed restoration effort to reduce excessive nutrient and sediment discharge and improve the degraded channel condition of the lower creek. The Lost Creek watershed project involves the coordination of riparian and upland restoration activities across six ranches and 27 stream miles stretching from the community of Lost Creek downstream to the Clark Fork River. Overall,the project includes installation of two fish passage structures, corral relocation, habitat enhancement, riparian revegetation, bank stabilization, historic channel reactivation, bridge replacement, riparian fencing, and grazing management. In addition, this project will evaluate the effectiveness of various in-stream restoration techniques for improving habitat and reducing bank erosion in Lost Creek. These techniques include the use of root wads, coir logs, straw bales, tree revetments, bank reshaping, channel relocation, willow transplanting, and willow sprigging

Restoration Efforts on Lost Creek, Upper Clark Fork Basin

Lost Creek is a third order stream entering the upper Clark Fork River near Warm Springs, Montana. Lost Creek’s water quality and habitat issues include nutrient loading, excessive sedimentation, habitat and flow alterations, channelization, loss of woody riparian vegetation, and fish passage barriers. Among all tributaries, Lost Creek is the largest contributor of nitrogen to the upper Clark Fork River above Deer Lodge. The Tri-State Council’s Clark Fork River voluntary nutrient reduction program lists Lost Creek as the number one priority among non-point nutrient sources in the upper Clark Fork River basin. Montana Fish, Wildlife and Parks is undertaking a major watershed restoration effort to reduce excessive nutrient and sediment discharge and improve the degraded channel condition of the lower creek. The Lost Creek watershed project involves the coordination of riparian and upland restoration activities across six ranches and 27 stream miles stretching from the community of Lost Creek downstream to the Clark Fork River. Overall,the project includes installation of two fish passage structures, corral relocation, habitat enhancement, riparian revegetation, bank stabilization, historic channel reactivation, bridge replacement, riparian fencing, and grazing management. In addition, this project will evaluate the effectiveness of various in-stream restoration techniques for improving habitat and reducing bank erosion in Lost Creek. These techniques include the use of root wads, coir logs, straw bales, tree revetments, bank reshaping, channel relocation, willow transplanting, and willow sprigging

CASCADE: high-throughput characterization of regulatory complex binding altered by non-coding variants

Summary: Non-coding DNA variants (NCVs) impact gene expression by altering binding sites for regulatory complexes. New high-throughput methods are needed to characterize the impact of NCVs on regulatory complexes. We developed CASCADE (Customizable Approach to Survey Complex Assembly at DNA Elements), an array-based high-throughput method to profile cofactor (COF) recruitment. CASCADE identifies DNA-bound transcription factor-cofactor (TF-COF) complexes in nuclear extracts and quantifies the impact of NCVs on their binding. We demonstrate CASCADE sensitivity in characterizing condition-specific recruitment of COFs p300 and RBBP5 (MLL subunit) to the CXCL10 promoter in lipopolysaccharide (LPS)-stimulated human macrophages and quantify the impact of all possible NCVs. To demonstrate applicability to NCV screens, we profile TF-COF binding to ∼1,700 single-nucleotide polymorphism quantitative trait loci (SNP-QTLs) in human macrophages and identify perturbed ETS domain-containing complexes. CASCADE will facilitate high-throughput testing of molecular mechanisms of NCVs for diverse biological applications