Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A ) PHF6 ChIP-seq cross-correlation analysis was conducted using cross-correlation metrics as described in Landt et al, (Landt et al, ). ( B ) Example ChIP-seq tracks for PHF6 pull-down and IgG control. (CA)n repeats are demarcated with red boxes, while the blue boxes represent the identified PHF6 peak.

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: ChIP-sequencing, Control

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A ) The numbers of PHF6 sites that overlap different genomic regions are shown in the pie chart. The right pie chart shows a breakdown of sites that overlap exonic regions. The colour gradient, shown on the right, represents the logarithm of enrichment of PHF6 sites in each region relative to random expectation. Only PHF6 sites with P < 10 –5 are included in the charts. ( B ) The heatmap on the left shows the distribution of PHF6 sites relative to TSS’. The peaks are sorted by ascending order of their P -values (shown in the middle) from the top to the bottom. The colour gradient depicts the frequency of PHF6 sites relative to the position of the nearest TSS, also shown for top-ranking PHF6 sites using the histogram on the right. ( C – F ) PHF6 binds to (CA) n -microsatellite repeats. ( C ) The sequence logo depicts the top motif identified by MEME-ChIP [PMID: 21486936]. ( D ) The distribution of the (CA) n motif relative to the peak summits is shown, as revealed by CentriMo [PMID: 22610855]. ( E ) Dot plot representation of the GO terms that are enriched near PHF6 sites. Only the top 15 terms with the most significant p -values are shown. The x -axis shows the fold-enrichment of the term, while the dot size and colour represent the number of PHF6 targets that overlap the GO term and the hypergeometric p-value, respectively. ( F ) Each dot in the scatterplot represents a GO term that is significantly enriched in both the GREAT analysis of (CA) n simple repeats and the GREAT analysis of PHF6 sites. The x - and y -axes reflect the logarithm of the hypergeometric fold-enrichment of the terms. The GO terms with the largest enrichment are highlighted. n = 6 mouse cortices were pooled for each PHF6 ChIP and IgG control ChIP, where n represents an independent biological sample.

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Sequencing, Control

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) Phf6 KD and control cortical progenitors were subjected to mRNA-seq analysis ( n = 3). Plots represent differentially regulated candidate target genes ( A ), and functional annotation of downregulated versus upregulated genes ( B ). GO term enrichment analysis was performed using CPDB (Kamburov et al, ). ( C , D ) PHF6 peak-gene associations within +/− 2Kb of TSS and the effect of Phf6 KD ( n = 3) on expression is presented. ( E ) PolII signal near the TSS of the PHF6-bound genes is shown using the colour gradient in the heatmap. The rows represent the genes, sorted based on the position of the PHF6 site. The PHF6 binding sites are depicted in blue. The vertical dotted lines delineate the +/−300 bp region around the TSS’. The horizontal dotted lines delineate the genes with a PHF6 site within this +/−300 bp region. ( F ) The expression changes in Phf6 KD cells as a function of the binding position of PHF6. Each data point shows the average for 50 genes that have PHF6 binding, with the binding site location relative to the TSS shown on the x -axis. Data information: Error bars represent ± SEM. mRNA-seq raw reads were mapped to mm10 genome using HISAT2 (Kim et al, ), followed by duplicate read removal using samtools. Gene-level read counts were obtained by HTSeq (Anders et al, ), using gene annotations from GENCODE (release M9). Genes with a minimum of 150 reads in at least one sample were retained. Gene set analysis was performed using ConsensusPathDB (Kamburov et al, ). n represents an independent biological sample.

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Control, Functional Assay, Expressing, Binding Assay

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – C ) N2A cells were transfected with Phf6 ( PHF6-GFP) or GFP-expressing control (GFP) constructs. ( A ) Gene expression was assessed by RT-qPCR ( n = 3). ( B ) Samples were subjected to KI67 staining for assessment of proliferation ( n > 3, representative image shown). Scale bar represents 20 µm. ( C ) Quantification of percent KI67 positive cells are shown ( n > 3). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired student t -test). n represents an independent biological sample.

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Transfection, Expressing, Control, Construct, Gene Expression, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – C ) eNSC were isolated and cultured from WT mice at E14 and Phf6 KD was induced using an siRNA approach. Samples were analyzed using a limiting dilution assay (LDA) ( A , B ) and immunoblotting ( C ) using antibodies indicated on the blot. ( D – J ) eNSCs were cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - mouse brains at ~E15 and were subjected to immunoblotting analysis ( D ), ELDA ( E ) ( p = 0.00686), LDA ( F ), sphere diameter ( G , H ) ( p < 0.0001), RT-qPCR analysis using Nestin and Sox2 primers ( I ), and 5-ethynyl-2’-deoxyuridine (EdU) analysis ( J ). ( K , L ) eNSCs were cultured from C99F ( K ), R342X ( L ) and corresponding wild-type control mice. mRNA expression of Nestin and Sox2 were analyzed by RT-qPCR. ( M – P ) eNSC were cultured from R342X mice and wild-type control mice and were subjected to ELDA ( M , N ) ( p = 0.0211), LDA ( O ), and alamarBlue analysis ( P ) 7 days post-plating. Scale bar represents 100 µm. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired student t -test). Representative plots of n > 3 independent replicates are shown in ( A , C – E , G , J , M , N ), data in panels ( B , F , H , I , K , L , O , P ) are plotted with n > 3 mean +/− SEM. n represents an independent biological sample. .

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Isolation, Cell Culture, Limiting Dilution Assay, Western Blot, Control, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) Immunofluorescence (IF) staining of coronal sections from P0 ( A ) and E13.5 ( B ) for Phf6 -/Y / Nestin-Cre + and Phf6 loxp/Y / Nestin-Cre - male mice using a PHF6 antibody (green) in the cerebral cortex. Nuclei were counterstained by Hoechst. Scale bars represent 50 µm. ( C ) Phf6 -/Y / Nestin-Cre + and Phf6 loxp/Y / Nestin-Cre − male mice were collected at P0 and subjected to Nissl staining with sagittal sections shown. Scale bars represent 500 µm in lower magnification and 250 µm in higher magnification photomicrographs. ( D ) IF staining of coronal sections from ~E15 male mice using a SOX2 antibody is shown. Scale bar represents 100 µm at lower magnification and 10 µm at higher magnification. ( E ) IF staining of coronal sections from P0 using cortical layer markers: SATB2 (green, layer II-V), TBR1 (red, layer VI), and CTIP2 (grey, layer V). Nuclei were counterstained by Hoechst. The cortical wall spanning from the basal of ventricle zone to the pial surface was equally divided into ten bins, the bin 1 covers the most superficial layer and bin 10 covers the deepest layer. ( F ) Comparative analysis of SATB2+ neurons in each segment of P0 male mice ( n = 3). ( G ) Comparative analysis of Hoechst+ nuclei in each segment of P0 male mice ( n =3). Scale bars represent 50 µm. ( H , I ) mRNA and protein of E14 R342X and wild-type control mice were subjected to RT-qPCR for Hopx expression ( n > 3) ( H ) ( p = 0.0021), and immunoblotting analysis of cell type-specific markers ( I ) ( n = 3, representative blots shown). ( J ) R342X and WT mice were collected at P0 and subjected to Nissl staining ( n = 2, representative image shown). Coronal sections are shown. Scale bars represent 500 µm. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. two-tailed unpaired student t -test ( H ). two-way ANOVA with multiple comparisons ( F , G ). n represents an independent biological sample. .

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Expressing, Western Blot, Two Tailed Test

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – F ) Low-dimensional representation of single cells from mouse cerebral cortex, based on UMAP embedding of single-cell RNA-seq data [Data ref: (Di Bella et al, )] are shown. Cells are coloured based on animal age ( A ), or the expression of Phf6 ( B ), EphA4 ( C ), EphA7 ( D ), EphB1 ( E ), or EphB2 ( F ). ( G ) Heatmap representation of the Pearson correlation coefficients between Phf6 and EphR across various cell types are shown. Correlation values were calculated using imputed gene expression profiles after applying MAGIC (Van Dijk et al, ). ( H ) UMAP embedding of cells are coloured by cell type. UMAP coordinates and cell type annotations are from [Data ref: (Di Bella et al, ) (GEO GSE153164)].

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: RNA Sequencing, Expressing, Gene Expression

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A – E ) Dot plots showing expression of Phf6 ( A ), EphA4 ( B ), EphA7 ( C ), EphB1 ( D ), and EphB2 ( E ) in the mouse cerebral cortex during development where the colour of each dot represents the mean normalized expression values per cell type for a given timepoint. The size of the circle represents the percentage of cells expressing each gene. Single cell mouse RNA-seq data was obtained from GEO GSE153164 [Data ref: (Di Bella et al, )]. ( F – J ) Analysis of PHF6 and EPHR expression in the human cortex. Average reads per kilobase million (RPKM) values over human developmental time (post-conceptual weeks; pcw) for gene analysis of PHF6 ( F ), EPHB1 ( G ), EPHA4 ( H ), EPHA7 ( I ), and EPHB2 ( J ) are shown. Gene analysis was taken from publicly available RNA-seq data taken from the human ventral frontal cortex (VFC) of the Allen Brain Atlas BrainSpan dataset [Data ref: (BrainSpan Atlas of the Developing Human Brain, )].

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Expressing, RNA Sequencing

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) eNSCs were cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - at ~E15 and mRNA and protein expression of EphR were analyzed by RT-qPCR ( A ) and immunoblotting ( B ). ( C , D ) mRNA and protein of brain tissue obtained from E14 R342X and wild-type control mice were analyzed as described in ( A , B ). ( E , F ) Cerebral cortical tissues were isolated from WT and R342X mice at E14 ( E ) or at P0 ( F ). Samples were subjected to ChIP-qPCR using a PHF6 antibody. Zfp735 loci was used as negative control for the PCR. ( G ) Dual luciferase reporter assay was performed in WT or R342X eNSC cultures 48 h following electroporation with pGL4.23- EphA4 , pGL4.23- EphA7 , pGL4.23- EphB1 or pGL4.23-basic reporter plasmids. RLU Relative luminescence units. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-tailed unpaired student t -test ( A , C , G ), one-way ANOVA ( E , F ). Representative data of n > 3 independent replicates are shown in panels ( B , D ). Data in panels ( A , C , E – G ) are plotted with n > 3 mean ± SEM. n represents an independent biological sample. .

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Cell Culture, Control, Expressing, Quantitative RT-PCR, Western Blot, Isolation, ChIP-qPCR, Negative Control, Luciferase, Reporter Assay, Electroporation, Two Tailed Test

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) mRNA and protein of E14 C99F-m and wild-type control mice were subjected to RT-qPCR and immunoblotting analysis ( n ≥ 3). ( C ) GFP or PHF6-GFP expressing N2A cells were subjected to ChIP using an antibody to PHF6 or IgG control followed by PCR analysis using primers to EphA4 , EphA7 and EphB1 . Zfp locus was used as control ( n = 3). ( D ) GFP or PHF6-GFP- expressing cells were electroplated with a luciferase reporter plasmid driven by a promoter containing 583 bp of the EphA4 gene (pGL4.23- EphA4 ), 550 bp of the EphA7 gene (pGL4.23- EphA7 ) or 709 bp of the EphB1 gene (pGL4.23- EphB1 ). The pGL4.23-basic reporter plasmid (pGL4.23) was used as a control. Renilla expression plasmid was used as an internal control for all samples. RLU Relative luminescence unit. Dual luciferase reporter assay was performed 48 h following electroporation ( n = 3). ( E ) N2A cells were electroporated with siRNA against Phf6 (si Phf6 ) or control siRNA (siCtl) followed by dual luciferase reporter assay at 48 h ( n = 3). ( F ) EPHA4, EPHA7 and PHF6 levels were analyzed by immunoblotting in PHF6-GFP- expressing N2A cells. TUBULIN was used as a loading control. ( G ) Densitometric quantification of PHF6, EPHA4 and EPHA7 protein level normalized to TUBULIN is shown ( n = 3). ( H ) E14-Cerebral cortical tissues from WT and C99F-m mice were subjected to ChIP-PCR analysis, as described in panel ( C ). ( I ) eNSCs cultured from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - ~E15 mouse brains were subjected to immunoprecipitation (IP) using PHF6 antibody or IgG as control followed by immunoblotting analysis using a PHF6 antibody. ( J ) eNSCs from Phf6 -/Y / Nestin-CreERT2 + and control Phf6 loxP/Y / Nestin-CreERT2 - mouse brains at ~E15, were subjected to ChIP-PCR using a PHF6 antibody. Zfp735 loci was used as control for the PCR ( n = 2). ( K , L ) Protein expression of EPHB1 ( K ), EPHB2 ( L ), SOX2 and NESTIN were analyzed by immunoblotting in EphB1 and EphB2 knockdown (KD) cells. Loading controls of ß-ACTIN and GAPDH were used ( n = 2). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. [( C , H ) one-way ANOVA, ( A , D , E , G ) two-tailed unpaired student t -test]. n represents an independent biological sample.

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Control, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Plasmid Preparation, Reporter Assay, Electroporation, Cell Culture, Immunoprecipitation, Knockdown, Two Tailed Test

Journal: EMBO Reports

Article Title: PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

doi: 10.1038/s44319-024-00082-0

Figure Lengend Snippet: ( A , B ) Protein expression of PHF6, SOX2, and NESTIN in C99F-m ( A ) or R342X ( B ) E14 brains were analyzed with immunoblotting. GAPDH or TUBULIN were used as loading controls. ( C – F ) E14 brains were sectioned at a thickness of 8 μm and were subjected to staining using SOX2 and TBR2 antibodies. DAPI was used as a nuclei marker. Percentage of SOX2+ ( C ) ( p = 0.0084), TBR2+ ( D ) ( p = 0.001), and SOX2+/TBR2+ merged ( E ) cells were quantified using FIJI software. Representative images are shown ( F ). Scale bar represents 100 µm. Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 for panels ( C – E ), two-tailed unpaired student t -test ( n > 3 independent replicates). Data in ( A , B ) represents 3 biological replicates ( n = 3 mice). .

Article Snippet: 80 μg of total cell extracts from Phf6 loxP/Y / Nestin-CreERT2 - or Phf6 -/Y / Nestin-CreERT2 + eNSCs were employed for immunoprecipitation (IP), using either 1 μg of IgG or PHF6 antibody (NOVUS, NB100-68262, 1:1000).

Techniques: Expressing, Western Blot, Staining, Marker, Software, Two Tailed Test

Journal: Genes & Development

Article Title: A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth

doi: 10.1101/gad.254151.114

Figure Lengend Snippet: Phf6 is a developmentally specified, in vivo-specific regulator of tumor cell growth. ( A ) Scatter plot showing the behavior of single shRNAs in in vitro and in vivo validation assays. All hairpins target genes found within common amplicons in human ALL. P -values were calculated using a Student’s t -test. ( B ) A scatter plot showing the effect of hairpin-mediated Phf6 suppression on leukemia cell representation in vitro and in vivo. ( C ) A graph showing suppression of the in vivo effect of a Phf6 shRNA in a population of leukemia cells via expression of a nontargetable Phf6 cDNA. ( D ) Scatter plots showing the effect of Phf6 suppression in leukemia cells harvested from the spleen and bone marrow of tumor-bearing animals. ( E ) Peripheral leukemia cell counts 9 d following tumor cell transplantation. ( F ) Longitudinal monitoring of the percentage of vector control or shPhf6 -infected leukemia cells in partially transduced tumor cell populations. ( G ) A scatter plot showing the effect of Phf6 suppression in distinct Eµ-myc transplanted B-cell lymphomas. ( H ) A graph showing the relative impact of Phf6 suppression in a transplanted AML. The Phf6 and control shRNAs were induced with doxycycline at day 14, and the relative percentage of infected cells over time is shown. ( I ) Scatter plots showing the impact of Phf6 suppression and overexpression in a transplanted T-cell lymphoma.

Article Snippet: Proteins were detected with the following antibodies: Phf6 (1:1000 in TBS-T with 5% BSA; Novus Biologicals, NB100-68262), Runx1 (1:1000 in TBS-T with 5% BSA; Abcam, ab23980), and HSP60 (1:1000 in TBS-T with 5% BSA; Santa Cruz Biotechnology, SC1722).

Techniques: In Vivo, In Vitro, Biomarker Discovery, shRNA, Expressing, Transplantation Assay, Plasmid Preparation, Control, Infection, Over Expression

Journal: Genes & Development

Article Title: A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth

doi: 10.1101/gad.254151.114

Figure Lengend Snippet: ( A ) A gene track showing the binding of Phf6, Rnap2, and histone modifications (H3K4me3, H3K79me2, H3K27ac, and H3K27me3) in a T-ALL cell line (Jurkat) at the MLL1 locus. The X -axis indicates the linear sequence of genomic DNA, and the Y -axis represents the total number of mapped reads. The black horizontal bar indicates the genomic scale in kilobases. ( B ) A pie chart showing Phf6 binding to genes in the genome classified as active (green), initiated (blue), bivalent (orange), polycomb silent (gray), and silent (red) in T-ALL (Jurkat). The proportion of genes in each class bound by Phf6 is shown by black radial lines. P -values indicate significance of the difference within each class. ( C – F ) Phf6 occupies the promoters of genes/pathways that are frequently altered in hematopoietic malignancies. Gene tracks represent binding of Phf6, Rnap2, and histone modifications (H3K4me3, H3K79me2, H3K27ac, and H3K27me3) in the Jurkat T-ALL cell line at the NOTCH1 ( C ), JAG1 ( D ), RUNX1 ( E ), and DMNT3A ( F ) loci. The X -axis indicates the linear sequence of genomic DNA, and the Y -axis shows the total number of mapped reads.

Article Snippet: Proteins were detected with the following antibodies: Phf6 (1:1000 in TBS-T with 5% BSA; Novus Biologicals, NB100-68262), Runx1 (1:1000 in TBS-T with 5% BSA; Abcam, ab23980), and HSP60 (1:1000 in TBS-T with 5% BSA; Santa Cruz Biotechnology, SC1722).

Techniques: Binding Assay, Sequencing

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Phf6 loss decreases the leukemogenic potential of cells in vivo and triggers a change in disease presentation. ( A ) PHF6 is a lineage-specific regulator of tumor growth in B-ALL and T-cell acute lymphoblastic leukemia (T-ALL). ( B ). Kaplan-Meier survival analysis of mice injected with either 10 3 (dotted) or 10 6 (solid) Phf6 WT (blue) and Phf6 KO (red) B-ALL cells. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6 WT cells. P- values are shown for the comparisons. ( C ) Representative hematoxylin and eosin (H&E) ( top ) and immunohistochemistry ( bottom ) staining of serial sections from lymph nodes (LNs) and lymphoma (mass) of recipient mice injected with Phf6 WT , shPhf6, and Phf6 KO cells. mCherry immunochemistry demarcates tumor cells. Bars, 600 µm. ( D , top ) Size comparison of representative LNs from Phf6 WT ( left ) and Phf6 KO ( right ) recipient mice. ( Bottom ) Quantification of combined LN weight of Phf6 WT (blue; n = 5) and Phf6 KO (red; n = 5) recipients. ( E ) Tumor burden in the blood of Phf6 WT (blue; n = 7) and Phf6 KO (red; n = 8) recipient mice. mCherry demarcates tumor cells. ( F ) Bar graphs showing the percentage of the CD4 + fraction among mCherry + cells isolated from Phf6 WT (blue; n = 9) and Phf6 KO (red; n = 5) tumors in bone marrow ( left ) and LNs ( right ). Data represent the mean ± standard deviation (SD) in D – F . Statistics were calculated with two-sided Student's t -test. (***) P < 0.001; (****) P < 0.0001.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: In Vivo, Injection, Immunohistochemistry, Staining, Isolation, Standard Deviation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: The absence of Phf6 promotes an altered gene expression program in B-cell leukemia. ( A ) Heat map showing differentially expressed genes (fold change >4, false discovery rate [FDR] <0.05) in pairwise comparisons between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Each column represents a replicate sample. The scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. ( B ) The top gene ontology (GO) and PANTHER terms found to be enriched in Phf6 KO cells. The P -value for each term is plotted as −log 10 ( P -value). ( C ) GSEA plot depicting significant ( P < 0.001) changes in pre-B lymphocyte signature genes upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( D ) Quantitative PCR (qPCR) analysis of Phf6 WT (blue) and Phf6 KO (red) cells transduced with empty vector (EV; solid) or a vector expressing Phf6 cDNA (cDNA; dotted). Relative mRNA levels for B-cell-associated genes are shown: Phf6 , Cd22 , Cd74 , Il4ra , Lyn , Ly86 , and Blk . ( E ) Schematic representation of ICA used to identify differential expression signatures (independent components [ICs]) in the integrated RNA-seq data set comprised of Phf6 WT , shPhf6, and Phf6 KO cells. Hinton diagram representation of ICA-derived signatures. Columns denote signatures, and rows denote samples. Colors denote relative directionality of gene expression ([red] up-regulation; [green] down-regulation), and the size of each square represents the magnitude of the contribution of each sample to the respective IC. Each signature is two-sided. Vertical boxes denote statistically significant ( P = 0.01, Mann-Whitney test) independent components. IC2 identified a Phf6 KO -specific gene signature. ( F ) GSEA plot depicting ( P = 0.08) enrichment in T-cell signal transduction signature upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. Data represent the mean ± SD. Statistics for these data were calculated with two-sided Student's t -test. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001; (n.s.) not significant.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: Expressing, RNA Sequencing Assay, Transformation Assay, Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Derivative Assay, MANN-WHITNEY

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: PHF6 exerts transcriptional regulation by interacting with histones rather than binding sequence-specific DNA sites. ( A ) Pie chart showing the distribution of 77,749 PHF6-binding sites across genomic regions in B-ALL cells. (TTS) Transcription termination site; (UTR) untranslated region. ( B ) De novo DNA sequence motifs identified in PHF6-bound regions at promoters of differentially expressed genes with their associated P -values. Shown are sequence logos of de novo position-weight matrices found by the MEME motif discovery tool ( left ) or those of known transcription factors whose motifs are found to be most similar to the de novo motif discovery results by Tomtom software ( right ). ( C ) Endogenous coimmunoprecipitation (co-IP) assay of TCF12, NF-κB, and PHF6 in the absence ( top ) or presence ( bottom ) of ethidium bromide (EtBr). Input is 7% of immunoprecipitation lysate. ( D , left ) Metagene tracks of PHF6 ChIP-seq signal averaged over all promoter–TSS tracks grouped by relative expression levels. (Red) High; (green) genomic; (purple) low. ( Right ) Metagene track of H3K27ac ChIP-seq signal averaged over all promoter–TSS regions. Shaded regions around average tracks denote estimates of 95% confidence interval (CI) of the metagene average signals based on resampling. ( E ) Metagene tracks of PHF6 ChIP-seq signal and correlation with histone marks: H3K27ac (blue), H3K4me3 (yellow; GSE66234), H3K27me3 (green). Pearson correlation of PHF6 and histone ChIP-seq signals across 10-kb regions spanning the TSS. Differentially expressed genes (solid line) and genome-wide genes (dotted line) are shown. ( F ) Endogenous co-IP assay of histone H3 and PHF6 in the absence ( top ) or presence ( bottom ) of EtBr. Input is 7% of immunoprecipitation lysate.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: Binding Assay, Sequencing, Software, Co-Immunoprecipitation Assay, Immunoprecipitation, ChIP-sequencing, Expressing, Genome Wide

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: PHF6 has distinct binding patterns within lineage-specific genes that result in drastic changes in nucleosome occupancy upon genetic deletion. ( A ) Heat maps comparing gene expression of curated CD19 + B-cell ( left ) and CD4 + T-cell ( right ) gene sets between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. Each column represents a replicate sample. ( B ) Motif enrichment analysis for DNA regions that undergo significant changes in chromatin accessibility in Phf6 KO cells. ( Top ) DNA motifs with decreased chromatin accessibility upon loss of Phf6 . ( Bottom ) DNA motifs with increased chromatin accessibility upon loss of Phf6 . ( C ) Metagene analysis of nucleosome positions ( top ) and fold enrichment of PHF6 binding ( bottom ) plotted for global analysis ( left ), the CD19 + B-cell gene set ( middle ), and the CD4 + T-cell gene set ( right ) assessed at TSSs ±1-kb genomic regions. ( Top ) Nucleosome positions are shown for Phf6 WT (solid line) and Phf6 KO (dotted line) cells, called by the NucleoATAC algorithm and normalized for batch effects using the chromVAR package. ( Bottom ) Metagene tracks of PHF6 ChIP-seq signal averaged over the TSSs ±1 kb in Phf6 WT cells. (−1/+1) +1/−1 nucleosomes flanking the TSS; (NFR) nucleosome-free region. Gray bars indicate major changes in nucleosome positioning that correspond to enriched PHF6 binding. Shaded regions around average tracks denote estimates of 95% CI of the metagene average signals based on resampling.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Transformation Assay, ChIP-sequencing

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Chromatin instability allows for aberrant T-cell transcription factor signaling. ( A ) Cell proliferation assay comparing Phf6 WT (blue; up arrow), Phf6 KO (red; up arrow), Phf6 WT + control vector (blue; circle-dotted), Phf6 KO + control vector (red; circle-dotted), Phf6 WT + NICD cDNA (blue; down triangle), and Phf6 KO + NICD cDNA (red; down triangle) cells. n = 3. ( B ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes infected with control vector or activated NICD vector. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6 WT + control vector cells. P -values are shown for the comparisons. ( C ) Quantification of the combined thymus weight of Phf6 WT + control vector (blue; n = 4), Phf6 KO + control vector (red unfilled; n = 6), and Phf6 KO + NICD cDNA (red patterned; n = 7) recipients. ( D ) Bar graphs showing the percentage of the CD4 + fraction among mCherry + cells isolated from Phf6 WT + control vector (blue; n = 5), Phf6 KO + control vector (red unfilled; n = 4), and Phf6 KO + NICD cDNA vector (red patterned; n = 6) tumors from LNs. Data represent the mean ± SD in C and D . Statistics were calculated with two-sided Student's t -test. (*) P < 0.05; (**) P < 0.01; (****) P < 0.0001.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: Proliferation Assay, Plasmid Preparation, Injection, Infection, Isolation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Loss of Phf6 results in decreased dependence on the driving oncogene BCR–ABL1 in vivo. ( A ) GSEA plots depicting significant changes in the targets of the BCR–ABL1 fusion signature ( P = 0.0129) and dasatinib resistance signature ( P = 0.0056) upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( B ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes with subsequent mock or ponatinib treatment (30 mg/kg daily for four consecutive days). The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P -value is shown for the comparison. ( C ) Kaplan-Meier survival analysis of mice injected with 10 6 B-ALL cells of the indicated genotypes with mock or 10 mg/kg doxorubicin (single immunoprecipitation dose) treatment. The number ( n ) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P -value is shown for the comparison.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: In Vivo, Injection, Immunoprecipitation

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: Model of PHF6 as a chromatin state regulator, permitting transcription factor binding through chromatin accessibility. ( A ) In wild-type cells, PHF6 binds to the ±1 nucleosome flanking the open TSSs of genes, allowing B-cell-specific transcription factors to bind, drive gene expression, and maintain B-cell identity. Conversely, PHF6 binds nucleosomes surrounding the TSSs of T-cell-specific genes, coordinating chromatin compaction and thus blocking the binding of T-cell-specific transcription factors. ( B ) In the absence of PHF6, chromatin is no longer maintained in an open state, B-cell transcription factors cannot bind, and expression of B-cell identity genes is down-regulated. However, T-cell identity genes are no longer inaccessible, allowing T-cell-specific transcription factors to bind and activate aberrant transcriptional programs.

Article Snippet: Antibodies used for immunoprecipitation were anti-PHF6 (Novus, 68262), anti-TCF12 (Cell Signaling Technology, 11825), anti-NF-kB (p65) (Santa Cruz Biotechnology, sc-372), anti-Histone H3 (Bethyl Laboratories, A300-823A), and normal rabbit IgG (Santa Cruz Biotechnology, sc-2027).

Techniques: Binding Assay, Expressing, Blocking Assay