A multi-parametric flow cytometric assay to analyze DNA–protein interactions

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA–protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein–DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro

The Redshift Evolution of the Mean Temperature, Pressure, and Entropy Profiles in 80 SPT-Selected Galaxy Clusters

We present the results of an X-ray analysis of 80 galaxy clusters selected in the 2500 deg2 South Pole Telescope survey and observed with the Chandra X-ray Observatory. We divide the full sample into subsamples of ∼20 clusters based on redshift and central density, performing a joint X-ray spectral fit to all clusters in a subsample simultaneously, assuming self-similarity of the temperature profile. This approach allows us to constrain the shape of the temperature profile over 0 R500) regions than their low-z (0.3 < z < 0.6) counterparts. Combining the average temperature profile with measured gas density profiles from our earlier work, we infer the average pressure and entropy profiles for each subsample. Confirming earlier results from this data set, we find an absence of strong cool cores at high z, manifested in this analysis as a significantly lower observed pressure in the central 0.1R500 of the high-z cool-core subset of clusters compared to the low-z cool-core subset. Overall, our observed pressure profiles agree well with earlier lower-redshift measurements, suggesting minimal redshift evolution in the pressure profile outside of the core. We find no measurable redshift evolution in the entropy profile at r . 0.7R500 – this may reflect a long-standing balance between cooling and feedback over long timescales and large physical scales. We observe a slight flattening of the entropy profile at r & R500 in our high-z subsample. This flattening is consistent with a temperature bias due to the enhanced (∼3×) rate at which group-mass (∼2 keV) halos, which would go undetected at our survey depth, are accreting onto the cluster at z ∼ 1. This work demonstrates a powerful method for inferring spatially-resolved cluster properties in the case where individual cluster signal-to-noise is low, but the number of observed clusters is high.Physic

MicroRNAs are required for the feature maintenance and differentiation of brown adipocytes

Brown adipose tissue (BAT) is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs (miRNAs) as essential regulators of brown adipocyte differentiation, but whether miRNAs are required for the feature maintenance of mature brown adipocytes remains unknown. To address this question, we ablated Dgcr8, a key regulator of the miRNA biogenesis pathway, in mature brown as well as in white adipocytes. Adipose tissue–specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat and were intolerant to cold exposure. Primary brown adipocyte cultures in vitro confirmed that miRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that miRNAs are essential for the browning of subcutaneous white adipocytes in vitro and in vivo. Using this animal model, we performed miRNA expression profiling analysis and identified a set of BAT-specific miRNAs that are upregulated during brown adipocyte differentiation and enriched in brown fat compared with other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of miRNAs in the maintenance as well as in the differentiation of brown adipocytes

A multi-parametric flow cytometric assay to analyze DNA–protein interactions

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA–protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein–DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro