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Knockdown of PHF8 in HeLa S3 cells

ABSTRACT: PHF8 is an H3K9me2 demethylase, interacts with H3K4me3 and RNA Polymerase II, is enriched at thousands of transcription start sites and can act as a transcriptional co-activator. In the following experiment we wanted to analyze the impact of PHF8-knockdown on transcript levels in HeLa cells. Three independent replicate knockdown experiments with PHF8-shA and control NT-sh were performed (6 samples in total). The two samples of each replicate experiment were hybridized in two color dye swap experiments (6 arrays in total).

ORGANISM(S): Homo sapiens

SUBMITTER: Klaus Fortschegger

PROVIDER: E-GEOD-20563 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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PHF8 targets histone methylation and RNA polymerase II to activate transcription

Project description:Mutations in PHF8 are associated with X-linked mental retardationand cleft lip/cleft palate. PHF8 contains a plant homeodomain(PHD) in its N-terminus and is member of a family of JmjC-domaincontaining proteins. While PHDs can act as methyl lysine recognitionmotifs, JmjC-domains can catalyze lysine demethylation. Here,we show that PHF8 is a histone demethylase that removes repressivehistone H3 dimethyl lysine 9 marks. Our biochemical analysisrevealed specific association of the PHF8 PHD domain with histoneH3 trimethylated at lysine 4 (H3K4me3). Chromatin-immunoprecipitationfollowed by high throughput sequencing indicated that PHF8 isenriched at transcription start sites of many active or poisedgenes, mirroring the presence of RNA polymerase II (RNAPII)and of H3K4me3-bearing nucleosomes. We show that PHF8 can actas a transcriptional co-activator and its activation functionlargely depends on binding of the PHD to H3K4me3. Furthermore,we present evidence for direct interaction of PHF8 with theC-terminal domain of RNAPII. Importantly, a PHF8 disease mutantis defective in demethylation and in co-activation. This isthe first demonstration of a chromatin-modifying enzyme whichis globally recruited to promoters through its association withH3K4me3 and RNAPII. This SuperSeries is composed of the following subset Series: GSE20563: Knockdown of PHF8 in HeLa S3 cells GSE20725: ChIP-Seq of PHF8 and H3K4me3 Refer to individual Series

2010-04-26 | E-GEOD-20753 | biostudies-arrayexpress

The mental retardation gene PHF8 mediates histone H4K20/H3K9 demethylation and regulates zebrafish brain apoptosis and craniofacial development: expression analysis

Project description:PHF8 (PHD Finger 8) mutations have been found in patients with X-linked mental retardation (XLMR) and craniofacial deformities. Here we identify PHF8 as the first enzyme that mediates demethylation of mono-methylated histone H4 lysine (K) 20 (H4K20me1), with additional activities towards H3K9me2/1 and H3K27me2. Patient mutations significantly compromise the demethylase activity, indicating functional importance. ChIP-seq identified PHF8 near the transcription start sites (TSS) of over 7000 target genes as well as in gene bodies and intergenic regions. PHF8 depletion resulted in up-regulation of H4K20me1 and H3K9me1 at the TSS-associated sites, and H3K9me2 in the gene bodies and intergenic regions, respectively, demonstrating differential substrate specificities at different target genomic locations. PHF8 depletion at the TSS results in decreased target gene expression, which is coincident with increased occupancy of the protein L3MBTL1 previously shown to induce chromatin compaction via binding to lower methyl states including H4K20me1 and H3K9me1. PHF8 RNAi and control RNAi stable HeLa cell lines were established. RNA were isolated from triplicate cell cultures. RNAs were subject to microarray expression analysis. Hybridizations were performed in duplicate.

2010-07-14 | E-GEOD-21555 | biostudies-arrayexpress

The mental retardation gene PHF8 mediates histone H4K20/H3K9 demethylation and regulates zebrafish brain apoptosis and craniofacial development: ChIP-Seq analysis

Project description:PHF8 (PHD Finger 8) mutations have been found in patients with X-linked mental retardation (XLMR) and craniofacial deformities. Here we identify PHF8 as the first enzyme that mediates demethylation of mono-methylated histone H4 lysine (K) 20 (H4K20me1), with additional activities towards H3K9me2/1 and H3K27me2. Patient mutations significantly compromise the demethylase activity, indicating functional importance. ChIP-seq identified PHF8 near the transcription start sites (TSS) of over 7000 target genes as well as in gene bodies and intergenic regions. PHF8 depletion resulted in up-regulation of H4K20me1 and H3K9me1 at the TSS-associated sites, and H3K9me2 in the gene bodies and intergenic regions, respectively, demonstrating differential substrate specificities at different target genomic locations. PHF8 depletion at the TSS results in decreased target gene expression, which is coincident with increased occupancy of the protein L3MBTL1 previously shown to induce chromatin compaction via binding to lower methyl states including H4K20me1 and H3K9me1. HeLa cells stably expressing control and PHF8 shRNA were used to profile the histone modifications including H4K20me1, H3K9me1 and 2 by ChIP-seq. For PHF8 ChIP-seq, chromatin was obtained by formaldehyde cross-link and sonication. For chIP-seq of Histone modifcation, chromatin was prepared by MNase digestion to obtain mono-nucleosomes.

2010-07-14 | E-GEOD-21108 | biostudies-arrayexpress

Genome-wide mapping of H3K4me3 and of PHF8 and ZNF711 binding sites in SH-SY5Y cells

Project description:We analyzed the genome wide binding sites for the potential XLMR genes PHF8 and ZNF711 in the neuroblastoma cell line SH-SY5Y using antibodies specific for PHF8 and ZNF711 and correlated this with binding pattern of H3K4me3. The total number of peaks detected for PHF8 based on 9.8 million sequence reads, 9.6 million sequence reads for ZNF711 and 9.1 million sequence reads for H3K4me3 after IgG normalization were 17075, 1875 and 19534, respectively. Annotation of the peaks to genes using the hg18 genome database revealed 10039 genes positive for H3K4me3, 7682 were bound by PHF8 and 1103 genes were bound by ZNF711 within +/-1000bp from TSS. As former genome wide analysis of H3K4me3 revealed is this histone mark preferentially associated with transcription start sites. The analysis revealed that 82% of H3K4me3 positive genes are also positive for PHF8. In general, PHF8 either co-localizes or partly overlaps with regions positive for H3K4me3 and covers the putative TSS of the target genes. Examination of two different proteins and one histone modification in a human neuroblastoma cell line

2010-03-08 | E-GEOD-20673 | biostudies-arrayexpress

Knockdown of PHF8 in HeLa S3 cells

Project description:PHF8 is an H3K9me2 demethylase, interacts with H3K4me3 and RNA Polymerase II, is enriched at thousands of transcription start sites and can act as a transcriptional co-activator. In the following experiment we wanted to analyze the impact of PHF8-knockdown on transcript levels in HeLa cells.

2010-04-27 | GSE20563 | GEO

The histone demethylase PHF8 is essential for cytoskeleton dynamics

Project description:PHF8 is a histone demethylase associated with X-linked mental retardation (XLMR). It has been described as a transcriptional coactivator involved in cell cycle progression, but its physiological role is still poorly understood. Here we show that PHF8 controls the expression of genes involved in cell adhesion and cytoskeleton organization such as RhoA, Rac1 and GSK3M-NM-2. A lack of PHF8 not only results in a cell cycle delay but also in a disorganized actin cytoskeleton and impaired cell adhesion. Our data demonstrate that PHF8 directly regulates the expression of these genes by demethylating H4K20me1 at promoters. Moreover, c-Myc transcription factor interacts with PHF8 and binds to the analyzed promoters, suggesting that c-Myc is involved on PHF8 recruitment to these promoters. Further analysis in the neuroblastoma cell line SH-SY5Y and in cortical neurons shows that depletion of PHF8 results in deficient neurite elongation. Overall, our results suggest that the mental retardation phenotype associated with loss of function of PHF8 could be due to abnormal neuronal connections as a result of alterations in cytoskeleton function. HeLa cells were transfected with either control or PHF8 siRNAs. Experiments were performed in biological triplicates. RNA were extracted and sujected to microarray analysis.

2012-07-31 | E-GEOD-38175 | biostudies-arrayexpress

Transcritomic analysis of PHF8 mediated genes in MKN28, human gastric cancer cells

Project description:PHF8, an important epigenetic regulator of cell cycle progression, rRNA synthesis and chromatin organization, is often overexpressed in different types of cancer including gastric cancer. Notably, elevated expression of PHF8 is associated with a poor clinical outcome in patients with gastric cancer. A global transcriptomic analysis between control and PHF8-knockdown MKN28 cells reveals differentially expressed genes involved in cell mortility and cell migration, suggesting PHF8 acts as an important regulator of cell migration related genes.

2020-09-23 | GSE117980 | GEO

RNA-seq data of PHF8 wild-type and PHF8 knockout TRAMP mouse

Project description:RNA-seq analysis were applied to elucidate the transcriptional differences of PHF8 wild-type and PHF8 knockout TRAMP mouse. A total of 2,092 differentially expressed genes (Fold Change > 2, or Fold Change＜0.5; FDR < 0.05) with 623 down- and 1469 up-regulated genes were identifed in Phf8-KO TRAMP mice.

2020-09-26 | GSE157621 | GEO

APOC2 increase PHF8 binding on chromatin

Project description:We performed PHF8 ChIP-seq analysis of APOC2 overexpressed RKO cells and control cells to identify high-confident PHF8 regulated targets.

2021-05-07 | GSE114089 | GEO

RNA-seq data of 786-O cells transfected with control-sgRNA (Control) and PHF8-sgRNA (PHF8-KO)

Project description:RNA-seq analysis were applied to elucidate the transcriptional differences between 786-O cell lines transfected with control-sgRNA (Control) and PHF8-sgRNA (PHF8-KO). A total of 3575 differentially expressed genes (Fold Change > 2, or Fold Change＜0.5; FDR < 0.05) with 2097 down- and 1478 up-regulated genes were identified in PHF8-KO 786-O cells

2022-10-08 | GSE214183 | GEO

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