MCF-7 Luciferase, PARP-1, PARG, SIRT1, and macroH2A Knockdown
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE12971 | GEO | 2010/01/31
SECONDARY ACCESSION(S): PRJNA112579
REPOSITORIES: GEO
Similar Datasets
Project description:Affymetrix expression arrays were used to compare expression patterns upon knockdown of PARP-1, PARG, SIRT1, or macroH2A in comparison to Luciferase control. Total RNA isolated from three independently generated control-matched MCF-7 knockdown cell lines targeting Luciferase (control), PARP-1, PARG, SIRT1, and macroH2A was used to probe Affymetrix Human U133A 2.0 Expression Microarrays. The raw array data was processed by Affymetrix GeneChip Operating Software (GCOS) to obtain detection calls (ABS_CALL) and signal values (VALUE_2). The signals were then normalized by scaling to a target value of 500 using GCOS. To adjust for batch effects due to day-to-day differences in RNA isolations, the empirical Bayes method was applied to the data set. After adjusting any values less than 0.01 to 0.01, the data was log2 transformed, median centered for each array, and median centered for each individual probe set (VALUE). Filters were then applied to obtain final gene lists.
2010-02-21 | E-GEOD-12971 | biostudies-arrayexpress
Project description:Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins in the nucleus by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs (shRNAs) to stably knockdown PARP-1 or PARG in MCF-7 cells, followed by expression microarray analyses.
2009-10-23 | GSE12952 | GEO
Project description:SIRT1 is a nuclear NAD+-dependent protein deacetylase. Expression microarray analysis was used to study the effect of SIRT1 knockdown on gene expression in MCF-7 breast cancer cells. Experiment Overall Design: SIRT1 stable knockdown was achieved using two retroviral shRNA constructs. An shRNA directed against Luciferase was used to generate the Luc control cells. Three independent biological replicates with matching Luc controls were analyzed using Affymetrix U133A 2.0 microarrays. Experiment Overall Design: These 6 samples were part of a 15-sample microarray analysis (GSE12971) examining expression regulation by SIRT1, PARP-1, PARG and macroH2A. All 15 samples were included for the following data normalization steps: data sets obtained using the GCOS software were grouped based on date of experiment and adjusted for batch effect using the parametric empirical Bayes method (Combat R); all values < 0.01 were adjusted to 0.01; the data were log2 transformed, median centered for each array, and median centered for each gene.
2009-06-08 | E-GEOD-13459 | biostudies-arrayexpress
Project description:Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins in the nucleus by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs (shRNAs) to stably knockdown PARP-1 or PARG in MCF-7 cells, followed by expression microarray analyses. Experiment Overall Design: Two shRNAs targeting Luciferase (Luc, control), PARP-1, or PARG were cloned into the pSUPER.retro vector (puro or neo resistant) and sequentially introduced into parental MCF-7 cells using a standard retroviral infection technique. Luc, PARP-1, and PARG control-matched cell lines were generated three independent times. Experiment Overall Design: Total RNA was isolated from stable Luc, PARP-1, and PARG knockdown cells was analyzed for global patterns of gene expression. Briefly, 7 μg of total RNA was labeled using Affymetrix's standard one-cycle amplification and labeling protocol. The labeled cRNA was then hybridized to Affymetrix Human U133A 2.0 GeneChips, which were scanned using a GeneChip Scanner 3000. Each biological replicate (1, 2 or 3) represents a single RNA isolation from each set of cell lines. Experiment Overall Design: These 9 samples were samples were part of a 15-sample microarray analysis (GSE12971) examining expression regulation by SIRT1, PARP-1, PARG and macroH2A. All 15 samples were included for the data normalization steps. Experiment Overall Design: The raw array data was processed by Affymetrix GeneChip Operating Software (GCOS) to obtain detection calls (ABS_CALL) and signal values (VALUE_2). The signals were then normalized by scaling to a target value of 500 using GCOS. To adjust for batch effects due to day-to-day differences in RNA isolations, the empirical Bayes method was applied to the data set. After adjusting any values less than 0.01 to 0.01, the data was log2 transformed, median centered for each array, and median centered for each individual probe set (VALUE). Filters were then applied to obtain final gene lists. Experiment Overall Design: Specifically, the criteria for a gene to be considered regulated by PARP-1 or PARG was: (1) detection call flagged as present or marginal in 2 out of 3 array replicates for both control and factor knockdown cell lines and (2) significance of values between control and knockdown cell lines for any given gene had a two-tailed Studentâ??s t-test with a p-value < 0.05. To determine the genes most robustly regulated by PARP-1 or PARG, a fold change criteria was applied, namely log2 fold change > 0.5 or < -0.5 when compared to the Luc control knockdown cells.
2009-10-30 | E-GEOD-12952 | biostudies-arrayexpress
Project description:MCF-7 Luciferase, PARP-1, PARG, SIRT1, and macroH2A Knockdown
| PRJNA112579 | ENA
Project description:MacroH2As core histone variants have a unique structure that includes C-terminal nonhistone domain. MacroH2As are highly conserved in vertebrates, and are thought to regulate gene expression. However the nature of genes regulated by macroH2As and the biological significance of macroH2As for the organism remain unclear. Our gene expression studies indicate that macroH2A.1 and macroH2A.2 work together to regulate specific genes. In these studies we examine the distributions of macroH2A.1 and macroH2A.2 nucleosomes to determine if they are localized to the genes that show altered expression in macroH2A knockout mouse liver. MacroH2A.1 and macroH2A.2 nucleosomes prepared from ~ 50 fetal mouse livers were purified by thio-affinity chromatography. Five samples were sequenced: Thiopropyl Sepharose, Normal Liver - contains mononucleosomal DNA from macroH2A.1-containing nucleosomes; Activate Thiol Sepharose, Normal Liver - contains mononucleosomal DNA primarily from macroH2A.2-containing nucleosomes. Starting Material, Normal Liver - this is a reference samplefor the first two samples. It contains mononucleosomal DNA from bulk fetal liver chromatin. Activated Thiol Sepharose, Knockout Livers - this is a control sample that contains mononucleosomal DNA from non-macroH2A nucleosomes that contaminate the macroH2A.2 nucleosomes. This fraction was prepared from macroH2A1/2 double knockout fetal livers; Starting Material, Knockout Liver - this is a reference sample for the fourth sample. It contains mononucleosomal DNA from bulk chromatin prepared from macroH2A1/2 double knockout fetal livers.
2014-10-15 | E-GEOD-57796 | biostudies-arrayexpress
Project description:MacroH2As core histone variants have a unique structure that includes C-terminal nonhistone domain. MacroH2As are highly conserved in vertebrates, and are thought to regulate gene expression. However the nature of genes regulated by macroH2As and the biological significance of macroH2As for the organism remain unclear. Our gene expression studies indicate that macroH2A.1 and macroH2A.2 work together to regulate specific genes. In these studies we examine the distributions of macroH2A.1 and macroH2A.2 nucleosomes to determine if they are localized to the genes that show altered expression in macroH2A knockout mouse liver.
2014-10-15 | GSE57796 | GEO
Project description:SIRT1 is a nuclear NAD+-dependent protein deacetylase. Expression microarray analysis was used to study the effect of SIRT1 knockdown on gene expression in MCF-7 breast cancer cells.
2009-05-28 | GSE13459 | GEO
Project description:Interaction proteomics in cell extracts on tagged macroH2A, either expressed in cells or using recombinant dimers with H2B.
2023-10-24 | PXD045696 | Pride
Project description:Genetic loss-of-function studies in development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by H3K9me3. Loss of macroH2A leads to major defects in nuclear organization including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by repeat sequences when depleted of macroH2A are disorganized, expanded and fragmented and mildly re-expressed. On the molecular level we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein Lamin B1. Taken together our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher order chromatin architecture.
2017-03-21 | GSE58175 | GEO