Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell-specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein-protein relationships and protein functions at the chromatin template.Chemical Immunolog

Recommendations for intervertebral disc notochordal cell investigation: from isolation to characterization

Background Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called “the notochord”. However, changes in the cells derived from the notochord which form the NP (i.e., notochordal cells [NCs]), in terms of their phenotype and functional identity from early developmental stages to skeletal maturation are less understood. These key issues require further investigation to better comprehend the role of NCs in homeostasis and degeneration as well as their potential for regeneration. Progress in utilizing NCs is currently hampered due to poor consistency and lack of consensus methodology for in vitro NC extraction, manipulation, and characterization. Methods Here, an international group has come together to provide key recommendations and methodologies for NC isolation within key species, numeration, in vitro manipulation and culture, and characterization. Results Recommeded protocols are provided for isolation and culture of NCs. Experimental testing provided recommended methodology for numeration of NCs. The issues of cryopreservation are demonstrated, and a pannel of immunohistochemical markers are provided to inform NC characterization. Conclusions Together we hope this article provides a road map for in vitro studies of NCs to support advances in research into NC physiology and their potential in regenerative therapies

Expanding the Epi-discovery tool box: Systematic and direct screening for chromatin regulators and chromatin binders using DNA barcoding

Eukaryotic genomes are packaged in chromatin, which consists of DNA, histones, and associated proteins. Chapter 1 describes the challenges this brings in understanding general principles of chromatin such as regulation of the gene expression, and the maintenance of chromatin states during transcription, DNA repair and DNA replication. To understand the dynamics of chromatin at a detailed level, this thesis has focused on dissecting locus-specific chromatin regulatory pathways and protein binding events. Chapter 2 and Chapter 3 describes Epi-ID, a screening strategy in yeast that enables the direct assessment of locus specific chromatin status in thousands of gene mutants in parallel. Epi-ID takes advantage of short DNA sequences called DNA barcodes that are introduced into an array of yeast knock-out mutants, at a common chromosomal location in the genome. Chromatin immunoprecipitation on pools of barcoded mutant strains followed by barcode counting by high throughput sequencing reports on the abundance of the chromatin mark of interest in each mutant strain. Further, transcription, replication, and repair processes all involve interactions of many different proteins and how these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown. To acquire this type of direct evidence Chapter 4 and Chapter 5 describes the development and application of Epi-Decoder, a Tag-ChIP-Barcode-Seq technology in yeast. Epi-Decoder is orthogonal to proteomics approaches and does not rely on MS but instead takes advantage of DNA sequencing. Chapter 6 further summarizes the findings and focusses on their limitations and horizons

NuRD-interacting protein ZFP296 regulates genome-wide NuRD localization and differentiation of mouse embryonic stem cells

Contains fulltext : 197898.pdf (publisher's version ) (Open Access)11 p

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism

Chemical Immunolog