chromatin

Do Zebrafish Hold the Key to Heart Regeneration? 

Posted on by Shannon Sindermann
Heart regeneration after heart attacks can stop poor health outcomes, read for more research

The human body has an incredible capacity for self-repair. Our skin can regenerate after a small cut, bones can heal after fractures and even the liver can regrow to its original size after 70% is lost or removed (3). However, when it comes to the heart, the story is very different. As Miley Cyrus once sang, “nothing breaks like a heart” – and science agrees. Unlike other organs, the heart has almost no ability to regenerate itself after injuries. In instances like myocardial infarctions, more commonly known as heart attacks, large amounts of cardiomyocytes (CMs)—the cells responsible for heart muscle contraction—are lost and cannot be regenerated, causing the formation of non-regenerative fibrotic scar tissue and, ultimately, decline in heart function (1).  

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Designing BET(ter) Inhibitors to Guide Therapy for Cancer and Inflammatory Diseases

Posted on by Ken Doyle
bet proteins brd nanoluc

Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.

One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).

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Protein:DNA Interactions—High-Throughput Analysis

Posted on by Gary Kobs
Copyright: molekuul / 123RF Stock Photo
Copyright: molekuul / 123RF Stock Photo

Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.

The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Coupled Reticulocyte Lysate). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.

Literature Cited

  1. Ren, B. et al. (2007) Genome-wide mapping of in vivo protein-DNA binding proteins. Science 316, 1497–502.
  2. Garner, M.M. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nuc. Acids. Res. 9, 3047-60.
  3. Glick,Y et al. (2016) Integrated microfluidic approach for quantitative high throughput measurements of transcription factor binding affinities. Nuc. Acid Res. 44, e51.