Project description:Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.
Project description:To better understand the molecular bases of resin production, a major source of terpenes for industry, the transcriptome of adult Pinus elliottii var. elliottii (slash pine) trees under field commercial resinosis was obtained.
Project description:Histone modifications represent one of the key factors contributing to proper genome regulation. One of the histone modifications involved in gene silencing is H3K9 methylation, which is found in the chromosomes across different eukaryotes and controlled by SU(VAR)3-9 and its orthologs. Although SU(VAR)3-9 was discovered over two decades ago, little is known about the details of its chromosomal distribution pattern. To fill in this gap, we used DamID-seq approach and obtained high-resolution genome-wide profiles for SU(VAR)3-9 in two somatic and two germline tissues of fruitfly.
Project description:Ananas comosus var. bracteatus has high ornamental value and widespread application because of its chimeric leaves. However, little is known about the molecular mechanism regulating this characteristic. Here, comparative transcriptomic and proteomic analyses of the white parts (Whs) and green parts (Grs) of the chimeric leaves were performed to identify differentially expressed genes (DEGs) and differentially expressed proteins (DEPs). In total, 1,685 DEGs, including 712 up- and 973 down-regulated ones, and 5,428 DEPs, including 1,018 up- and 795 down-regulated ones, were identified between the Whs and Grs. Comparisons with the GO and KEGG annotations revealed that the DEGs were involved mostly in carbon fixation, porphyrin and chlorophyll metabolism and oxidative phosphorylation. The DEPs were mainly involved in ribosomes, photosynthesis, photosynthesis antennas, and porphyrin and chlorophyll metabolism. Combined analysis showed that nine proteins related to chlorophyll biosynthesis, photosynthetic pigments, and photosynthesis were unchanged at mRNA level but suppressed at protein level. These results indicated that the albino phenotype of the Whs was caused by the proteomic-level suppression of key enzymes involved in the chlorophyll biosynthesis pathway and that translational and post-translational regulation may play important roles in both the biosynthesis of photosynthetic pigments and photosynthesis. Biological significance: Leaves of Ananas comosus var. bracteatus serve as the best materials for the study of albino mechanism. Because the chemic trait of A. comosus var. bracteatus is unstable and the molecular mechanism of the albino cells was poorly understood, we performed comparative analyses both at the transcriptome and proteome levels. This work revealed suppressed proteomic-level and translational and post-translational regulation contribute to the albino phenotype formation. Our results provide better information concerning the molecular mechanism within the chimeric leaves of A. comosus var. bracteatus.
Project description:The P. falciparum genome is equipped with several subtelomeric gene families that are implicated in parasite virulence and immune evasion. The members of these gene families are uniformly positioned within heterochromatic domains of the genome and are thus subject to variegated expression. The best-studied example is that of the var gene family encoding the major parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). Transcriptional regulation of other subtelomeric gene families and their role in parasite biology is much less understood. Here, we investigated the mode of transcriptional control of var, rif, stevor, phist and pfmc-2tm families by comparative genome-wide transcriptional profiling of transgenic parasite lines. Our results establish a clear functional distinction between var and non-var transcriptional control mechanisms. Unlike var promoters, we find that promoters of non-var families are not silenced by default. Moreover, we show that mutually exclusive transcription is unique to the var gene family.
Project description:modENCODE_submission_5128 This submission comes from a modENCODE project of Gary Karpen. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We aim to determine the locations of 125 chromosomal proteins across the Drosophila melanogaster genome. The proteins under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) using the Illumina NGS platform. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); Developmental Stage: 3rd Instar Larvae; Genotype: wild type; EXPERIMENTAL FACTORS: Strain Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); Antibody Su(var)3-9-Q2598 (target is Su(var)3-9); Developmental Stage 3rd Instar Larvae
Project description:We have investigated the effect of RRP6 depletion on the transcriptome of S2 cells using Affymetrix whole-genome tiling arrays. We have also carried out Illumina ChIP-seq analysis of RRP6 genome occupancy in control S2 cells (GFP-KD) and in cells depleted of SU(VAR)3-9.
Project description:modENCODE_submission_3888 This submission comes from a modENCODE project of Gary Karpen. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We aim to determine the locations of the major histone modifications across the Drosophila melanogaster genome. The modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) using genomic tiling arrays. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: Su(var) 3-9 06 17 mutant(official name : Su(var)3-9^06/Su(var)3-9^17 genotype : Su(var)3-9^06/Su(var)3-9^17 outcross : n/a transgene : n/a tags : X-ray tag : GFP description : Stocks with the Su(var)3-9^06 and Su(var)3-9^17 alleles were maintained as heterozygous stocks over a third chromosome balancer. To generate the transheterozygotes description : individuals from the heterozygous stocks were crossed together. The F1 individuals lacking the balancer chromosome were used for these experiments. made_by : Stocks were obtained from Gary Karpen ); Developmental Stage: 3rd Instar Larvae; Genotype: Su(var)3-9^06/Su(var)3-9^17; Transgene: n/a; NUMBER OF REPLICATES: 4; EXPERIMENTAL FACTORS: Strain Su(var) 3-9 06 17 mutant(official name : Su(var)3-9^06/Su(var)3-9^17 genotype : Su(var)3-9^06/Su(var)3-9^17 outcross : n/a transgene : n/a tags : X-ray tag : GFP description : Stocks with the Su(var)3-9^06 and Su(var)3-9^17 alleles were maintained as heterozygous stocks over a third chromosome balancer. To generate the transheterozygotes description : individuals from the heterozygous stocks were crossed together. The F1 individuals lacking the balancer chromosome were used for these experiments. made_by : Stocks were obtained from Gary Karpen ); Antibody H3K36me3 abcam (target is H3K36me3); Developmental Stage 3rd Instar Larvae