ABSTRACT: Genome resolved taxonomic and functional profiling during periods of high performance and system upset in a full-scale mixed microalgal wastewater resource recovery facility
Project description:Gene expression profiling of mixed cell lines, used to evaluate the performance of deconvolution methods. This study was part of Danaher & Kim et al., "Advances in mixed cell deconvolution enable quantification of cell types in spatially resolved gene expression data"
Project description:Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells which are mutant for upSET. We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone post-translational modification quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. We show that this result applies to heterochromatin genes generally using nascent-RNA sequencing in the upSET-mutant S2 lines. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.
Project description:Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domaincontaining protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin. For determining DamID based UpSET chromatin profile, three biological replicates were used. For evaluating chromatin accessibility, two biological replicates were performed.
Project description:Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domaincontaining protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.
Project description:Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domaincontaining protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.
Project description:Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domaincontaining protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.
Project description:An experiment was designed to assess the reproducibility of SWATH-MS measurements collected from different mass spectrometers in a single facility over a period spanning approximately four months. Data were acquired with 90-minute gradient lengths at the Australian Cancer Research Foundation International Centre for the Proteome of Human Cancer (ProCan) on six SCIEX TripleTOF 6600 QTOF mass spectrometers. Multiple replicate aliquots were prepared for eight samples. These comprised a dilution series of ovarian cancer tissue (0%, 3.125%, 6.25%, 12.5%, 25% and 50%) offset by yeast and a fixed proportion (50%) of prostate cancer tissue (Samples 1-6), a 1:1 mix of ovarian cancer tissue and yeast cells (Sample 7), and a human cell line (HEK293T; Sample 8). On each mass spectrometer, sets of 20 replicate aliquots (three aliquots of Samples 2-5, and two aliquots of Samples 1, 6-8) were run during each of thirteen 48-hour periods. Experimental data were acquired in 48-hour time periods on each instrument continuously for eight days (with sets of 20 replicates commencing on days 1, 3, 5 and 7), once per week for the remainder of the month (commencing on days 14, 21 and 28), and then once per month for the remainder of the first three months (commencing on days 56 and 84). After each instrument underwent a major clean, the sets of 20 replicates were again run continuously for a further eight days (commencing on days 101, 103, 105 and 107). Data were therefore acquired during a total of thirteen 48-hour periods over approximately four months, during which time the mass spectrometry facility was fully operational. Mass spectrometer maintenance schedules varied according to each individual instrument's performance, and each instrument commenced data acquisition asynchronously within 28 days from the experiment start.
Project description:Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domaincontaining protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin. Total mRNA from wildtype and epic mutant ovaries was extracted, labeled and hyb on a custom tiling arrays. Two biological replicates were performed.