Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a, Left: PCR amplicons on agarose gel around exon 5 of Phf14 (wild-type: 654 bp, KO: around 460 bp). Right: Phf14 mRNA-levels (library size normalized counts mapping to the gene) in Phf14 KO and WT ESCs. b , Approximate stoichiometry of the Phf14 complex from pulldowns with Phf14 antibody from ESCs and NPCs. Calculated as 10^(top3-value)*signal_sum(given TMT lane)/signal_sum(all TMT lines). n = 3 (ES cells) and n = 2 (NPCs) c , mRNA levels (library size normalized counts mapping to the gene) of Tcf20 and Rai1 in control cells during differentiation (D0 – ESCs, D4 – embryoid bodies, D8 – NPCs, D12 – neurons). d, Hmg20a mRNA-levels (library size normalized counts mapping to the gene) in Phf14 KO and WT ESCs. e , Widefield microscope images of control and Phf14 KO neurons. Scale bar = 100 μm. f , GO enrichment analysis for genes differentially expressed in Phf14 KOs compared to controls in ESCs. g Cdkn2 and Cdkn2b levels from mRNA-sequencing (library size normalized counts mapping to the gene) and RT-qPCR at day 4 (embryoid body stage).

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Agarose Gel Electrophoresis, Control, Microscopy, Sequencing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Phf14 interacting proteins in ESCs and NPCs identified by quantitative tandem mass tag (TMT) spectrometry. Immunoprecipitation samples from control cells are compared to Phf14 KO. Red - significantly enriched proteins with adj. p-value < 0.05, grey – not significant. P-values and fold changes were obtained from limma statistical analysis. n = 2. b , Tcf20 and Rai1 normalized to Phf14 signal (with the same TMT-label) in ESCs and NPCs, related to ( a ). c , Western blot of nuclear lysate of control and Phf14 KO cells. The experiment was repeated three times with similar results. Histone H3 serves as a nuclear loading control. d , Control-normalized values of number of NPCs derived from the same amount of ESCs at the start of differentiation. e , Trypan-blue based live-cell percentage of NPCs. n = 3, two-sided unpaired Student’s t-test. f , Principal component analysis plot of Phf14 KO and control samples including all timepoints. (day 0 – ESCs, day 4 – embryoid bodies (EBs), day 8 – NPCs, day 12 – neurons). g , GO enrichment analysis for genes differentially expressed in Phf14 KOs compared to controls on day 4. h , Representative cell cycle distribution plots for monolayer-differentiated NPCs (day 4). i , Quantified data related to ( h ). Two-sided unpaired student’s t-test was used for calculating p-values.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Immunoprecipitation, Control, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a, Western blot showing HA-tagged Rai1 at the expected size (slightly below 250 kDa) in single-cell selected clonal lines. b , Co-immunoprecipitation and inputs of RAI1 ePHD and PHF14 constructs detected by Western blot in HEK293T cells. Constructs: HA-FLAG-RAI1(1569-1906), myc-PHF14(1-888), myc-PHF14(1-719), myc-PHF14(1-502) and myc-PHF14(1-312). c , MODified histone peptide array of purified ePHD domain of Rai1. Dnmt3a PWWP and ADD domains were used as a positive control. Representative images from 2 replicates. d , Rai1 mRNA normalized counts from mRNA-seq data and IGV browser views of the reads mapped to Rai1 gene with the deleted exon 4 highlighted. e , Volcano plot from mRNA-seq analysis with DESeq2 showing all differentially expressed genes in neurons with adj. p-value < 0.05. (3 independent replicates in both groups) (Full list of identified DEGs provided in Source data.) f , Comet tail moments after etoposide treatment, quantified using CometScore 2.0 software.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Western Blot, Immunoprecipitation, Construct, Modification, Peptide Microarray, Purification, Positive Control, Software

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , CldU/IdU track ratios from DNA fiber spreading using Phf14 KO and control ESCs and monolayer-differentiated NPCs. Over 100 tracks were quantified per condition. Two-sided Wilcoxon rank-sum test was used for calculating p-values. b , Widefield microscope images of an alkaline comet assay of control and Phf14 KO neurons. Cell were collected either untreated or 1 h after 5 μM etoposide treatment. Scale bar = 100 μm. c , Comet tail moments after etoposide treatment, quantified using CometScore 2.0 software. Vast majority of untreated comets had a tail moment of 0. d , Principle of hprtDRGFP reporter lines. The GFP sequence contains a stop codon that disrupts the expression of GFP near an I-SceI restriction site. The incomplete iGFP can be used as a repair template in HDR to restore a functional GFP. e , Homology-directed repair rates in Phf14 KO and control hprtDRGFP ESCs and monolayer-differentiated NPCs after double strand break induction using lentivirus-transduced I-SceI-T2A-BFP enzyme. GFP-positive cells were counted using flow cytometry and the HDR rate was calculated as the ratio of GFP-positive cells among cells gated for BFP for successful transduction. f , Amount of BFP-positive cells (cells that have I-SceI expression) in the measured population in control and Phf14 KO cells. Same amount of cells was plated the day before transduction with the same amount of lentivirus. g , Heatmap showing the z-scores from bait ratios (identified protein signal to Phf14 signal in the sample) of proteins that were significantly enriched in both untreated and etoposide treated IPs over KO controls. Phf14 interacting proteins were identified in ESCs without and after 10 μM etoposide treatment for 1 h identified by quantitative tandem mass tag (TMT) spectrometry. n = 3 controls, 2 KOs.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Control, Microscopy, Alkaline Single Cell Gel Electrophoresis, Software, Sequencing, Expressing, Functional Assay, Flow Cytometry, Transduction

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Immunofluorescence staining in monolayer-differentiated NPCs after laser microirradiation. Anti-phospho-histone H2A.X (Ser139) signal serves as a DNA damage marker. Cells were fixed 5-15 minutes after damage generation. b , Live-cell imaging of fluorescently tagged PHF14 complex proteins and PARP1 and ZMYND8 as positive controls (transient transfection into U2OS cells). DNA damage was induced with 355 nm laser at the indicated location. c , Quantified recruitment kinetics from live-cell laser microirradiation experiments for the PHF14 complex and controls (in U2OS for RAI1, PARP1 and ZMYND8, other proteins from HEK293T cells, no difference was observed between different cell lines in terms of recruitment kinetics). Time is set to 0 at the time of DNA damage induction and intensities are normalized such that the maximum reached is 1. A first-order exponential equation f(t) = 1-exp(−t / τ) was fitted to all data per protein. The time required for the normalized fluorescence intensity to reach 50% of its maximal value is represented as t50. The last panel includes fitted curves from the other panels only. n = 8 (PHF14), 14 (HMG20A), 8 (TCF20), 8 (RAI1), 8 (PARP1), 6 (ZMYND8). d , Laser microirradiation in transiently transfected U2OS cells after treatment with 1 μM olaparib for 1 h or equivalent amount of DMSO. All scale bars = 10 μm.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Immunofluorescence, Staining, Marker, Live Cell Imaging, Transfection, Fluorescence

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Immunofluorescence staining in monolayer-differentiated NPCs after laser microirradiation. Anti-phospho-histone H2A.X (Ser139) signal serves as a DNA damage marker. Cells were fixed 1 h after DNA damage was generated. Laser microirradiation in transiently transfected U2OS cells, 1 hour after DNA damage induction. b , Live-cell imaging of mEGFP-HMG20A and mRuby-PHF14 transiently transfected into U2OS cells. 1 hour after damage induction. c , Live-cell imaging of mEGFP and TCF20-mCherry (transient co-transfection into U2OS cells). DNA damage was induced with 355 nm laser at the indicated location. d , Immunofluorescence staining in control and Phf14 KO ESCs after laser microirradiation. e , Live-cell imaging of mEGFP-HMG20A, transiently transfected into control and Phf14 KO ESCs. DNA damage was induced with 355 nm laser at the indicated location. f , Quantified recruitment kinetics for mEGFP-HMG20A in control and Phf14 KO ESCs. g , Immunofluorescence staining in U2OS cells after laser microirradiation. DNA damage was induced after treatment with 1 μM olaparib for 1 h or equivalent amount of DMSO. All scale bars = 10 μm.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Immunofluorescence, Staining, Marker, Generated, Transfection, Live Cell Imaging, Cotransfection, Control

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Protein gel staining of gel filtration of the purified proteins from co-lysis of PHF14 and HMG20A expressing insect cells. Collected fractions are indicated with the square bracket. b , SDS-PAGE input PHF14 and HMG20A, and after cross-linking with DSS. c , Protein gel staining (left) and graph of gel filtration of the purified complex with PHF14, HMG20A, TCF20 and RAI1. Final eluted fractions were confirmed by SDS-PAGE for expression of the PHF14 complex. Collected fractions are indicated with the square bracket. d , Purified proteins from ( c ) were tested for optimal cross-linker concentration, DSS. SDS-PAGE of cross-linked and input complex. e , Intra-protein interactions of XL-MS result visualized by xiNET. Related to . f , Schematic diagram of PHT multigene expression cassette and protein gel staining from the final purified proteins from gel filtration. g , XL-MS results showing the interactions of PHT complex.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Staining, Filtration, Purification, Lysis, Expressing, SDS Page, Concentration Assay, Structural Proteomics

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Visualization of XL-MS results using a cross-link viewer (xiNET) of PHF14 and HMG20A (PH). The green lines are indication of interactions. b , Visualization of XL-MS results using a cross-link viewer (xiNET) of PHF14, HMG20A, TCF20 and RAI1 and a schematic diagram of multigene expression vector used for expressing the proteins used in XL-MS. c , Relative abundances of the PHF14 complex proteins from iBAQ. Protein lysates are related to ( b ), n = 3. d , Non-denaturing electrophoresis gel of PHF14, HMG20A and TCF20 (PHT) and iBAQ results from the indicated native gel bands (n=2). e , Negative-stain EM micrographs of PH and PHT. Scale bar = 50 nm. Blue boxes and red boxes indicate PH and PHT, respectively. Three representative images of each proteins complex are shown below. Scale bar = 10 nm.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Structural Proteomics, Expressing, Plasmid Preparation, Electrophoresis, Staining

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Visualization of DNA shapes used in EMSA. The nucleotide sequences were adopted from Bianchi et al., 1989. The identical sequences among the different DNAs are in the same color (green and red). The arrows indicate the direction of DNAs from 5’ to 3’. b , EMSA with HMG20A proteins on a non-denaturing polyacrylamide gel. HMG20A proteins were incubated with different shapes of DNA indicated above of each panel and stained for both DNA and proteins. Arrows indicate positions of DNA bands bound by HMG20A protein showing shift in mobility. c , EMSA with PHF14 and HMG20A proteins, incubated together with different shapes of DNA, on a non-denaturing polyacrylamide gel. Arrows indicate the position of PHF14 protein. Asterisk indicates non-specific bands. d , Purified proteins from incubated with DNA of different conformations. Arrows indicate the locations of the band with just PHF14 and HMG20A (PH) and all three proteins (PHF14, HMG20A and TCF20; PHT). In all experiments, DNA concentration was 1 μM and protein concentrations were 1 μM of each protein for ( b ) and ( c ). Oligonucleotide sequences are listed in Supplementary Table 2.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Incubation, Staining, Purification, Concentration Assay

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Purified HMG20A protein incubated with the DNA indicated each lane. Protein concentrations are as indicated. b , Individual proteins of PHF14 and HMG20A purified separately and analyzed by non-denaturing gel electrophoresis separately and mixed together.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Purification, Incubation, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Predictions of IDRs from amino acid sequences of PHF14 complex. Each upper-most panel indicates both predicted protein domains and IDRs from Simple Modular Architecture Research Tool (SMART), then Predictor of Natural Disordered Regions (PONDR) and Prion-Like Amino Acid Composition (PLAAC). Scores greater than 0.5 are indicative of disorder propensity. Values on the x-axis correspond to amino acid residue number of the corresponding protein. b , Graphical summary of the phase separation hypothesis. PHF14 and HMG20A form a binary complex that is recruited to DNA damage sites as a first step at the same speed as PARP1. PARP1 PARylates (shown in turquoise) both of them and its other targets at DNA damage sites. TCF20 and RAI1 are recruited later, PARylation-dependently and a tertiary or quaternary protein complex is assembled at the damage sites. The local increase in protein concentration triggers the formation of biomolecular condensates that separate the damage sites from the surrounding nucleoplasm and facilitate other repair factor recruitment. c , Confocal microscopy images of FRAP in U2OS cells transfected with mEGFP-HMG20A or TCF20-mEGFP. FRAP areas were bleached with 488 nm laser at the indicated locations. DNA damage was induced with 355 nm laser and then proteins were allowed to reach maximal accumulation before FRAP, where applicable. Scale bars = 10 μm. d, Comparison of turbidity changes of the PHF14 complex after phase separation (PH – PHF14 and HMG20A, PHT – PHF14, HMG20A and TCF20). Quantification was done by measuring OD 600 nm. e , Phase diagrams of proteins indicated in each diagram, with changes in protein concentration on the x-axis and NaCl concentration on the y-axis. The experiment was repeated three times and representative results are shown. Dark blue = clear droplets, light blue = some droplets, white = no droplets. f , Confocal images of individual proteins and the three-protein complex (PHT) after undergoing phase separation with the addition of Cy5-labeled 4WJ DNA. Scale bars = 20 μm.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Residue, Protein Concentration, Confocal Microscopy, Transfection, Comparison, Concentration Assay, Labeling

Journal: bioRxiv

Article Title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

doi: 10.1101/2021.10.12.462922

Figure Lengend Snippet: a , Charge, hydrophobicity and pi-pi contact predictions. For pi-pi contacts, greater than 4 is indicative of phase separation. Values on the x-axis correspond to amino acid residue number of the corresponding protein. b , Quantified recovery kinetics from FRAP experiments in . c , Phase contrast images of condensates that are formed with indicated proteins and 10% of PEG. Images were taken with a widefield Nikon Ti-E microscope d , Glass surface wetting assay of PHT proteins with Cy5-labeled DNA from confocal microscope. Scale bar = 20 μm. e , Phase diagram of PHF14 and HMG20A proteins with different shapes of DNA (200 nM) (y-axis) and increasing protein concentration (x-axis) at physiological salt concentration (150 mM NaCl). Dark blue = clear droplets, light blue = some droplets, white = no droplets.

Article Snippet: Phf14 antibody (proteintech 24787-1-AP) was coupled to M-280 Sheep Anti-Rabbit IgG Dynabeads (Invitrogen 11203D) according to manufacturer’s instructions overnight (7 μL antibody, 50 μL beads per IP).

Techniques: Residue, Microscopy, Labeling, Protein Concentration, Concentration Assay

Journal: Nature Communications

Article Title: BMP9 stimulates joint regeneration at digit amputation wounds in mice

doi: 10.1038/s41467-018-08278-4

Figure Lengend Snippet: Bone morphogenetic protein 9 (BMP9) stimulates regeneration of joint structures. a Bmp9 transcripts are localized to digit joints undergoing cavitation (arrowhead) in E18.5 embryos. b P2 level amputation of neonatal digits is used to test for induced regeneration. Following amputation (red line) at postnatal day 3 (PN3), epidermal closure is completed 4 days later (PN7), and an agarose microcarrier bead (blue dot) is implanted between the wound epidermis and the stump. c Microcomputed tomography (µCT) rendering of a BMP9-treated digit showing regeneration of a skeletal element articulating with the stump after 4 weeks (PN35). d Mallory trichrome staining of BMP9-treated digit 9 weeks after BMP9 treatment (PN70). Regenerated skeletal element (arrow) forms a joint-like structure with the digit stump. Articular cartilage of the P1/P2 joint is shown at higher magnification in the inset. e Higher magnification of the inset in ( d ) shows a cavity (c) separating a layer of articular cartilage (arrowhead) lining the regenerated skeletal element (n) and the stump bone (s). Small patches of articular cartilage (star) form on the stump bone. f – j In situ hybridization of cavity (arrow)-forming BMP9-treated digits showing localization of transcripts to the regenerated chondrogenic nodule but not the digit stump (s) 72 h after bead (*) implantation. f Col2a1 . g Sox9 . h Acan . i Fmod . j Ucma . k , l Immunofluorescence staining of Doublecortin (Dcx) expression. k Immunostaining shows Dcx+ cells (arrowhead) lining the synovial cavity of an uninjured joint at PN10. l Isolated superficial cells of the BMP9-induced nodule express Dcx (arrowhead) 72 h after treatment (PN10). The cavity is outlined with dashed white lines. Dcx+ cells are also observed internal to the superficial layer. m – q In situ hybridization of bovine serum albumin (BSA)-treated control digits 72 h after bead implantation showing no expression associated with the amputation wound distal to the stump. m Col2a1 . n Sox9 . o Acan . p Fmod . q Ucma . r – v In situ hybridization of non-cavity-forming BMP9-treated digits showing localization of transcripts to the distal stump 72 h after bead implantation. r Col2a1 . s Sox9 . t Acan . u Fmod . v Ucma . Right is distal, top is dorsal. Scale bars: a , f – j , m – v = 100 µm; d = 250 µm; e , k = 50 µm; l = 25 µm

Article Snippet: Briefly, a single agarose microcarrier bead (Affi-Gel Blue Gel beads, Bio-Rad, Hercules, CA) was used to deliver BMP9, BMP2 or BSA to the amputation wound.

Techniques: Tomography, Staining, In Situ Hybridization, Immunofluorescence, Expressing, Immunostaining, Isolation, Control