Project description:Heritable heterochromatin domain formation is initiated by dynamic changes in the proteome on specialized loci, leading to chromatin modifier recruitment, heterochromatin assembly and propagation. Here we characterize the proteomes associated with a defined heterochromatin nucleation region in three distinct chromatin states: before de novo H3K9 methylation nucleation, following heterochromatin assembly and upon heterochromatin disassembly. Our analyses separate sequence-dependent from sequence-independent processes, providing a systematic examination of the template-driven nucleation and epigenetic wiring underlying heterochromatin transmission. We identify functional interactions that reveal an integral role for the conserved nucleolytic Grc3 complex, and its associated network, as pioneer factors in heterochromatin assembly. More broadly, we demonstrate the power of quantitative proteomics-based strategies for the unbiased scrutiny of the single regions assembled in distinct chromatin states.
Project description:We have completed the high quality reference genome for domestic sheep (Oar v3.1). Early-stage Illumina GA sequence platform sequenced less reads in high GC content regions than in other regions. To read through higher GC content regions, we generated 2 Gb MeDIP-seq data for filling gaps in sheep reference genome assembly.
Project description:We compiled the transcriptome by extracting mRNA, reverse transcription and Illumina sequencing, followed by assembly and annotation by comparison with public databases (including; Nr, SwissProt and COG). Quantitative data on the transcription abundance of each putative protein sequence is provided as assesed by the SOAPdenovo_trans assembly tool.
Project description:C2H2 zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting 3 (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion, providing one explanation for their remarkable evolutionary success. Given a set of modules with defined 3-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences. However, a recent survey of a large number of three-finger ZFAs engineered by modular assembly reported high failure rates (~70%), casting doubt on the generality of modular assembly. Here, we used protein-binding microarrays to analyze 28 ZFAs that failed in the aforementioned study. Most (17) preferred specific sequences, which in all but one case resembled the intended target sequence. Like natural ZFAs, the engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences. Thus, the failure of these proteins in previous assays is not due to lack of sequence-specific DNA-binding activity. Our findings underscore the relevance of individual C2H2-ZF sequence specificities within tandem arrays, and support the general ability of modular assembly to produce ZFAs with sequence-specific DNA-binding activity. Protein binding microarray (PBM) experiments were performed for a set of 20 artificial zinc finger arrays (ZFAs). Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006.
Project description:we mapped the locations of DNA segments occupied by GATA1 using chromatin immunoprecipitation (ChIP). We have produced genome-wide GATA1 ChIP datasets after restoration and activation in G1E-ER4 cells. we employed the sequence census methodology of ChIP-seq , using Illumina GA2 technology to produce 23 million reads (36 nucleotides long) uniquely mapped to the mouse genome (mm8 assembly) for the GATA1 ChIP DNA and 15 million mapped reads for the input DNA Examination of transcription factor GATA1 occupancy