Project description:Transcriptional regulation is impacted by multiple layers of genome organization. Here, we identified and mapped out all the transcriptionally active chromosomal domains in the chicken immature erythrocyte genome, including the known β- and α-globin domains, by combining a powerful chromatin fractionation method with next generation DNA and RNA sequencing. To characterize transcriptionally active chromatin domains, we used two biological replicates. Each replicate set has one control SE fraction and one transcriptionally active chromatin fraction (F1)
Project description:Transcriptional regulation is impacted by multiple layers of genome organization. Here, we report that highly expressed genes were associated with H3K4me3 and H3K27ac.Our data provide a genome-wide profile of chromatin signatures in relation to expression levels in chicken immature erythrocytes. Examination of two different histone modifications in immature erythrocyte cells
Project description:Transcriptional regulation is impacted by multiple layers of genome organization. Here, we identified and mapped out all the transcriptionally active chromosomal domains in the chicken immature erythrocyte genome, including the known β- and α-globin domains, by combining a powerful chromatin fractionation method with next generation DNA and RNA sequencing. Two biological replicates of total RNA were sequenced to characterize genes in chicken erythrocyte cells
Project description:We employed high-throughput sequencing of both short (~18-24nt) and long (>200nt) RNAs in human erythrocytes. We obtained blood from five healthy individuals for the short (small) RNA-seq library preparations and blood from three individuals for the long RNA-seq library preparations. We identified an abundant, diverse population of RNAs. Both polyadenylated and nonpolyadenlated long RNAs were identified. Additionally, known and novel microRNAs were identified in the short RNA dataset using the probabalistic modeling algorithm miRDeep. These RNAs lend insight into erythrocyte biology and provide utility as potential biomarkers. To determine both shared and unique aspects of the erythrocyte long RNA transcriptome, we compared this transcriptome with that of the PBMC and CD34+ erythroid progenitor transcriptomes. Sequencing of RNAs from mature erythrocytes of healthy individuals
Project description:The nucleosome plays a central role in genome regulation. Traditional methods for mapping nucleosomes depend on the resistance of the nucleosome core to micrococcal nuclease (MNase). However, the lengths of the protected DNA fragments are heterogeneous, limiting the accuracy of nucleosome position information. To resolve this problem, we removed residual linker DNA by simultaneous digestion of yeast chromatin with MNase and exonuclease III (ExoIII). Paired-end sequencing of mono-nucleosomes revealed not only core particles (145-147 bp), but also intermediate particles in which ~8 bp project from one side (154 bp) or both sides (161 bp) of the nucleosome core. We term these particles "pseudo-chromatosomes" because they are present in yeast lacking linker histone. They are also observed after MNase-ExoIII digestion of chromatin reconstituted using recombinant core histones. We propose that the pseudo-chromatosome provides a DNA framework to facilitate H1 binding. Comparison of budding yeast nucleosome sequences obtained using micrococcal nuclease (MNase-seq) and MNase + exonuclease III (ExoIII) (MNase-ExoIII-seq): wild type cells and hho1-null cells. Nucleosome sequences from native chromatin and H1-depleted chromatin from mouse liver. Nucleosome sequences from a plasmid reconstituted into nucleosomes using recombinant yeast histones or native chicken erythrocyte histones.
Project description:In order to identify protein involved in sensing DNA double strand breaks and recruiting the repair machinry, we performed in vitro assembly of chromatin on small circular and linear DNA templates, respectively. Proteins involved in chromatin assembly and sensing of free DNA ends were isolated and subjected to proteomics analysis.
Project description:Organic matter recycling in marine systems is largely driven by microbial processes, particularly in the Arctic where primary production and inputs can be temporally offset from upper trophic level consumption. This study followed bacterial dynamics in the chlorophyll maximum of the Bering Strait and sediment-water interface of the Chukchi Sea using metaproteomic and 16S rRNA methods to measure cellular function and taxonomic composition under low and high marine-derived particulate organic matter (POM) treatments at 0°C. Parallel analysis of major organic components (lipids and amino acids) allowed a comparison of microbial-POM interactions. Over the 10 day experimental period, bacteria under both treatments showed rapid community responses and changes in proteomic expression, accompanied by small changes in the concentration and distributions of organic components. In the Bering Strait community, protein translation was an important immediate cellular response under both POM scenarios while specific metabolic processes were more distinct between treatments. For example, under both conditions, evidence for carboxylic acid metabolism increased at day 6 while carbohydrate utilization as an energy source showed unique patterns as the experiments progressed. With POM additions to the Bering Strait community, nitrogen transport and regulation went up, including nitrogen fixation and ammonia assimilation, by day 6. In addition, a number of vitamin enzymatic cofactors were enriched by this day, providing evidence for an increase in C1 metabolism at that time. Low POM conditions stimulated the cycling and synthesis of amino acids, which was not as pronounced under the high POM treatment. In the Chukchi Sea community, nitrate reduction and substrate-specific transporter activity was statistically higher than in the Bering Strait, especially under low POM conditions. Taxonomic inference revealed that a wide range of bacterial classes were associated with the shifting cellular functions, but that Alphaproteobacteria, Gammaproteobacteria and Flavobacteria controlled most of these protein abundances. Activities of all classes were highly variable, with less dominant bacterial groups exhibiting a particularly strong degree of niche separation.
Project description:We applied ChIP-seq to explore the effect of missense mutations in TFs on their genome wide binding profile. Using a retroviral expression system in chicken mesenchymal stem cells, we elucidated the mechanism underlying a novel missense mutation in HOXD13 (Q317K) associated with a complex hand and foot malformation phenotype. The glutamine at position 317 (position 50 of the homeodomain) is conserved in most homeodomains, a notable exception being bicoid-type homeodomains that have K at this position. Our results show that the mutation results in a shift in the binding profile of the mutant towards a bicoid/PITX1 motif. Gene expression analysis and functional assays using in vivo overexpression studies confirm that the mutation results in a partial conversion of HOXD13 into a TF with bicoid/PITX1 properties. A similar shift was not observed with the mutation Q317R, which is associated with brachysyndactyly, suggesting that the bicoid/PITX1-shift observed for Q317K might be related to the severe clinical phenotype. For each wt or mutant transcription factor two independent biological replicates were used for ChIP-seq together with corresponding input DNA as reference samples. For expression analysis one RNA sample for each wt and mutant transcription factor was used and compared to the expression level of cells infected with empty vector by RNA-seq.