Journal: Nucleic Acids Research

Article Title: The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex

doi: 10.1093/nar/gku274

Figure Lengend Snippet: Identification of FOXK2 binding proteins by RIME. ( A ) Schematic illustration of the RIME protocol used to identify FOXK2 associated factors in the context of chromatin association. ( B ) Summary of FOXK2 interaction proteins belong to the SIN3A core complex and the PR-DUB complex. HCFC1 is shown in brackets as it is unclear whether this is part of the core PR-DUB complex. ( C ) Visualization using STRING of a sub-network of interactions between FOXK2 binding proteins. ( D ) Validation of FOXK2 interaction with endogenous components of SIN3A and PR-DUB core complex proteins by co-immunoprecipitation (IP) experiments using anti-Flag (FOXK2) antibody in U2OS–FOXK2–HF cells. Precipitated proteins were detected by immunoblotting (IB) using the antibodies as indicated. Arrows represent the bands corresponding to each of the full-length proteins. Note that the ASXL2 blot is from a different IP experiment. ( E ) PLA analysis of interaction between Flag-tagged FOXK2 and endogenous HCFC1 in U2OS–FOXK2–HF (top and bottom left panels) or U2OS (bottom right) cells. The combinations of antibodies used are shown above and below each panel (IgG represents a non-specific antibody). DNA is stained using DAPI (blue) and the PLA signal is red. Quantitative analysis of PLA signals in the nucleus is shown below. The level of signals/nuclei in the control PLA sample (Flag and non-specific IgG antibodies) was set as 1.

Article Snippet: Raw intensity CEL files were normalized as before using RMA, and differential expression between the six shBAP1 arrays and three shControl arrays estimated using the limma package in R. For ChIP-seq analysis, we used our Illumina ChIP-seq read libraries for FOXK2-bound compared to input DNA in U2OS cells (ArrayExpress: E-MTAB-2204), together with externally published ChIP-seq read libraries for DNA bound by HCFC1, compared to input, in HeLa cells (GEO: GSE31417) ( ).

Techniques: Binding Assay, Biomarker Discovery, Immunoprecipitation, Western Blot, Staining, Control

Journal: Nucleic Acids Research

Article Title: The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex

doi: 10.1093/nar/gku274

Figure Lengend Snippet: Interactions between endogenous FOXK2 and BAP1. Reciprocal co-immunoprecipitation experiments using either FOXK2 ( A ) or BAP1 ( B ) antibodies for immunoprecipitation (IP) from U2OS cells. Co-precipitated endogenous BAP1 and FOXK2 were detected by immunoblotting (IB). Arrows represent the bands corresponding to each of the full-length proteins. ( C ) Immunofluorescence detection of endogenous FOXK2 (green) and BAP1 (red) and their co-localization in the nucleus [indicated by DAPI staining of DNA (blue)]. ( D ) Imaging and quantification of PLA signals generated by the indicated combinations of antibodies (IgG represents a non-specific antibody). DNA is stained using DAPI (blue) and the PLA signal is red. Quantitative analysis of PLA signals in the nucleus is shown on the right. The level of signals/nuclei in the control PLA sample (BAP1 and non-specific IgG antibodies) was set as 1.

Article Snippet: Raw intensity CEL files were normalized as before using RMA, and differential expression between the six shBAP1 arrays and three shControl arrays estimated using the limma package in R. For ChIP-seq analysis, we used our Illumina ChIP-seq read libraries for FOXK2-bound compared to input DNA in U2OS cells (ArrayExpress: E-MTAB-2204), together with externally published ChIP-seq read libraries for DNA bound by HCFC1, compared to input, in HeLa cells (GEO: GSE31417) ( ).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Imaging, Generated, Control

Journal: Nucleic Acids Research

Article Title: The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex

doi: 10.1093/nar/gku274

Figure Lengend Snippet: Mapping the FOXK2–BAP1 interaction domains. ( A ) Schematic representation of full-length FOXK2 and the GST fusions to the C-terminal (amino acids 1–218) and N-terminal (amino acids 189–660) regions of FOXK2. The positions of the forkhead associated (FHA) and forkhead domains (FOX) are shaded in grey. ( B ) GST pulldown assays using bacterially expressed GST-tagged FOXK2(1–218) and FOXK2(189–660) and in vitro translated BAP1. Arrows mark the positions of full-length GST–FOXK2 fusion proteins. A total of 10% input is shown. ( C ) Co-immunoprecipitation experiments to analyse interactions between FOXK2(1–218) and BAP1. HEK293T cells were transfected with the indicated plasmids encoding FOXK2(1–218) fused with the Gal4 DNA binding domain and Flag-tagged BAP1, followed by immunoprecipitation (IP) with anti-Flag antibody and immunoblotting (IB) with either an anti-Flag or an anti-Gal4 antibody. ERK2 is a loading control. The asterisk marks a non-specific signal. ( D and E ) GST pulldown assay using either GST, or wild-type (WT) or FHA mutant (R58A) versions of the GST–FOXK2(1–218) fusion protein and total cell extracts from U2OS cells. Interacting BAP1, HCFC1, SIN3A and input GST fusion proteins were revealed by IB. Arrows mark the positions of full-length GST–FOXK2 fusion proteins. A total of 3% cell lysate input is shown. Ethidium bromide was added to the GST pulldown reactions where indicated. ( F ) Co-immunoprecipitation experiments using FOXK2 antibodies for IP from U2OS cells. Co-precipitated endogenous BAP1 was detected by IB. Where indicated, the final co-IP was treated with λ phosphatase. ( G ) Schematic representation of full-length BAP1 and the indicated N- and C-terminal truncation mutants. The locations of the ubiquitin carboxyl-terminal hydrolase (UCH) domain and the C-terminal domain (CTD) are shown. ( H ) Co-immunoprecipitation analysis of FOXK2 interactions with BAP1 deletion mutants. HEK293T cells were transfected with the indicated plasmids encoding FOXK2(1–218) fused to the Gal4 DNA binding domain and Flag-tagged full-length or truncated mutants of BAP1, followed by IP with anti-Flag antibody and IB with the anti-Gal4, anti-HCFC1 and anti-Flag antibodies. The asterisk marks a non-specific signal.

Article Snippet: Raw intensity CEL files were normalized as before using RMA, and differential expression between the six shBAP1 arrays and three shControl arrays estimated using the limma package in R. For ChIP-seq analysis, we used our Illumina ChIP-seq read libraries for FOXK2-bound compared to input DNA in U2OS cells (ArrayExpress: E-MTAB-2204), together with externally published ChIP-seq read libraries for DNA bound by HCFC1, compared to input, in HeLa cells (GEO: GSE31417) ( ).

Techniques: In Vitro, Immunoprecipitation, Transfection, Binding Assay, Western Blot, Control, GST Pulldown Assay, Mutagenesis, Co-Immunoprecipitation Assay, Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex

doi: 10.1093/nar/gku274

Figure Lengend Snippet: FOXK2 and the PR-DUB bind to the same chromatin regions. ChIP-qPCR analysis of endogenous BAP1 ( A ) and the other PR-DUB components HCFC1 and ASXL2 ( B ) binding to the indicated FOXK2 genomic binding regions. Blacks bars in (B) represent binding to FOXK2 binding regions associated with the MCM3 and KDM3A loci; white bars are negative control region which does not bind to FOXK2 ( MCM3-int9 ). Data are normalized against input DNA and shown relative to binding to non-specific IgG (taken as 1). Data are the average of three independent experiments. ( C ) Genome wide correlation between FOXK2 and HCFC1 binding regions. Heatmap of HCFC1 and FOXK2 read densities mapped onto FOXK2 peak summits and ranked according to FOXK2 signal. ( D ) UCSC genome browser view of FOXK2 and HCFC1 binding profiles associated with the DDX19A and VPS51 loci.

Article Snippet: Raw intensity CEL files were normalized as before using RMA, and differential expression between the six shBAP1 arrays and three shControl arrays estimated using the limma package in R. For ChIP-seq analysis, we used our Illumina ChIP-seq read libraries for FOXK2-bound compared to input DNA in U2OS cells (ArrayExpress: E-MTAB-2204), together with externally published ChIP-seq read libraries for DNA bound by HCFC1, compared to input, in HeLa cells (GEO: GSE31417) ( ).

Techniques: ChIP-qPCR, Binding Assay, Negative Control, Genome Wide

Journal: Nucleic Acids Research

Article Title: The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex

doi: 10.1093/nar/gku274

Figure Lengend Snippet: FOXK2 acts as a chromatin targeting factor for the BAP1-containing deubiquitinase complex. ChIP analysis of endogenous FOXK2 and BAP1 binding to the indicated genomic loci in U2OS cells treated with non-targeting control siRNAs (siNT, black bars) or siRNA against FOXK2 (siFOXK2, white bars) ( A ) or siRNA against BAP1 (siBAP1, grey bars) ( B ). ChIP was performed with nonspecific IgG or anti-FOXK2 and BAP1 antibodies; the data are shown relative to the input DNA and are the averages of two (B) or three (A) experiments. ( C ) ChIP analysis of H2A and H2AK119 ubiquitin levels at the indicated genomic loci in U2OS cells treated with non-targetting control siRNAs (siNT; light grey bars), siRNA against BAP1 (siBAP1; dark grey bars), siRNA against FOXK2 (siFOXK2; white bars) or both BAP1 and FOXK2 (black bars). ChIP was performed with non-specific IgG, anti-H2A and ubiquitinated K119 H2A antibodies (H2Aub); the data are the averages of three experiments. Statistically significant differences between siNT and siBAP1/siFOXK2 treated samples are shown; P -value <0.05 (*) and <0.01(**). ( D ) Model depicting the role of FOXK2 in nucleating the recruitment of chromatin remodelling complexes to chromatin. FOXK2 recruits both the PR-DUB and the SIN3A complex, potentially through direct or indirect interactions with the shared subunit HCFC1. Question marks denote uncertainty about which interactions are direct. BAP1 can then cause local histone deubiquitination, and histone deacetylation can potentially be achieved through the HDAC1 component of the SIN3A complex.

Article Snippet: Raw intensity CEL files were normalized as before using RMA, and differential expression between the six shBAP1 arrays and three shControl arrays estimated using the limma package in R. For ChIP-seq analysis, we used our Illumina ChIP-seq read libraries for FOXK2-bound compared to input DNA in U2OS cells (ArrayExpress: E-MTAB-2204), together with externally published ChIP-seq read libraries for DNA bound by HCFC1, compared to input, in HeLa cells (GEO: GSE31417) ( ).

Techniques: Binding Assay, Control, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Methyl-lysine readers PHF20 and PHF20L1 define two distinct gene expression–regulating NSL complexes

doi: 10.1016/j.jbc.2022.101588

Figure Lengend Snippet: PHF20 and PHF20L1 bind to the promoters of highly expressed genes. A , Venn diagram indicating the numbers and the overlap of 3xHA PHF20 and 3xHA PHF20L1 ChIP-Seq peaks. B , pie chart of the genomic distribution of 3xHA PHF20-binding ( left ) and 3xHA PHF20L1-binding sites. C , average normalized input and HA ChIP-Seq profiles of 3xHA PHF20 ( left ) and 3xHA PHF200L1 ( right ) across transcription start sites (TSSs). D , Venn diagram indicating the numbers and overlap of 3xHA PHF20, 3xHA PHF20L1, and KANSL3 ChIP-Seq peaks. E , boxplot showing the expression of genes that do not have PHF20 or PHF20L1 binding to their promoters (nonbound), genes that have both PHF20 and PHF20L1 binding to their promoters (dual targets), or genes that have only PHF20 binding to their promoters (PHF20_only targets) in U2OS cells. ∗∗∗∗ p (by two-sided t test) <0.0001. ChIP-Seq, chromatin immunoprecipitation sequencing; HA, hemagglutinin; PHF20, plant homeodomain finger protein 20; PHF20L1, PHF20-like protein 1.

Article Snippet: HA-ChIPs and library preparation for sequencing PHF20-bound and PHF20L-bound DNA (ChIP-Seq) were performed at MD Anderson Epigenomics Profiling Core as described earlier ( ).

Techniques: ChIP-sequencing, Binding Assay, Expressing