Journal: bioRxiv

Article Title: 53BP1 deficiency leads to hyperrecombination using break-induced replication (BIR)

doi: 10.1101/2024.09.11.612483

Figure Lengend Snippet: (a). Recruitment of RAD51 (left and middle) and 53BP1 (left and right) to γH2AX sites was analyzed by PLA in U2OS cells treated with IR (4 Gy, 2h after for PLA) or HU (1 mM, 24h, 30 min after release for PLA). Left: representative PLA images. Right: quantification of PLA foci per nucleus. Also see Supplementary Figure 9a. Scale bar =2 μm. (n=300 cells) (b). Recruitment of 53BP1 (left), PCNA (middle) and PIF1 (right) to γH2AX sites was analyzed by PLA in U2OS cells expressing SMARCAD1 shRNAs with vector as a control after HU treatment (1 mM, 24h). Quantification of PLA foci per nucleus is displayed. Also see Supplementary Figure 9b. (n=300 cells) (c). Recruitment of PCNA (left) and PIF1 (right) to γH2AX sites was analyzed by PLA in U2OS WT and 53BP1 -KO cells after HU treatment (1 mM, 24h). Quantification of PLA foci per nucleus is displayed. Also see Supplementary Figure 9c. (n=300 cells) (d and e). Recruitment of 53BP1 (d), PCNA (e, left) and PIF1 (e, right) to γH2AX sites was analyzed by PLA in U2OS cells expressing Flag-SMACRAD1-WT or Flag-NΔ-SMACRAD1 with endogenous SMACRAD1 depleted by shRNA after HU treatment (1 mM, 24h). Quantification of PLA foci per nucleus is displayed. Also see Supplementary Figure 9d for d and Supplementary Figure 9e for e. (n=300 cells) a to e: Three experiments were performed with ∼100 nuclei analyzed in each experiment. Quantification of PLA foci per nucleus from a total of ∼300 nuclei are displayed. (f). HR frequency was determined in U2OS (EGFP-HR/STGC) cells expressing SMACRAD1 shRNA with a vector control (left) or in cells expressing Flag-SMACRAD1-WT or Flag-NΔ-SMACRAD1 with endogenous SMACRAD1 depleted by shRNAs, 5 days after I-Sce1 lentiviral infection. Also see Supplementary Figure 9d. (n=5 replicates) (g). U2OS (EGFP-BIR/LTGC-Flex1) cells expressing Flag-SMACRAD1-WT or Flag-NΔ-SMACRAD1 with endogenous SMACRAD1 depleted by shRNA were further infected with lentiviruses encoding FANCM shRNA (left) or synchronized to S-phase using double thymidine block followed by HU treatment (1 mM, 24h, right). BIR frequency was assessed by FACS 6 days after. Also see S9f. (n=5 replicates)

Article Snippet: Antibodies used were: 53BP1 (NB100-305, Novus Biologicals), FLAG (F1804, Sigma-Aldrich), PCNA (SC-56, Santa Cruz), HA (E10176EF, Covance), PRIM1 (10773-1-AP, Proteintech), RPA2 (NA19L, Calbiochem), SMARCAD1 (A5850, ABclonal), and KU70 (E-5, SC-17789, Santa Cruz Biotechnology).

Techniques: Expressing, Plasmid Preparation, Control, shRNA, Infection, Blocking Assay

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 1. Data mining of FLAG-SMARCAD1 affinity purification from human cells reveals co- purification of architectural proteins. (a) Flow chart of the experimental approach used in [57]. IgG and FLAG pulldowns were performed, and peptides were quantified using label-free mass spectrom- etry. Identified peptides for the proteins we validated here are shown in Supplementary Figure S1. (b) The six polypeptides of the hTFIIIC complex and their known interactions according to the STRING physical network. TFIIIC complex division into two sub-complexes is indicated; modules A and B bind A and B box DNA loci, respectively [60]. Shown in bold are the five subunits that were identified by mass spectrometry as associated with tagged, overexpressed SMARCAD1 by [57].

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Mass Spectrometry

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 2. Validation of architectural proteins as bona fide SMARCAD1-associated proteins by reciprocal co-immunoprecipitation of endogenous proteins. (a) Flow chart of the experimental approach. (b) Endogenous co-immunoprecipitation experiments (n = 2–4) from nuclear extracts from HeLa cells with anti-SMARCAD1 or anti-TFIIIC antibodies (subunits 63 and 90) analyzed by Western blot with the indicated antibodies. Inputs, 9% of the nuclear extract, are from the same blot as the IP. IgG, immunoglobin G. KAP1, a known binding partner of SMARCAD1, was analyzed in parallel (positive control).

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

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

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 3. hTFIIIC102 binds directly to hSMARCAD1. (a) Schematic representation of the GST fusion proteins tested for interaction with His-SMARCAD1. Apart from KAP1 (Materials and Methods), all proteins were full-length. Amino acid numbers are given. (b) GST-pulldown assay. Top panel: Bound proteins detected by Western blot with an anti-SMARCAD1 antibody. His-SMARCAD1 expressed in insect cells (Input, lane 2, 20%), GST (lane 3), GST-KAP1 ∆PB (lane 4), and GST-TFIIIC102 (lane 5). Molecular weight markers are indicated (kDa). Bottom panel: Instant-blue-stained PAGE gels of purified GST proteins.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: GST Pulldown Assay, Western Blot, Molecular Weight, Staining

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 4. Architectural proteins associate with SMARCAD1 in mouse pluripotent ESCs. (a) Experi- mental set-up. (b) Western blot analysis of endogenous immunoprecipitates (IPs) from 46C nuclear extracts; 6% of nuclear extract was used as input. The IPs were performed in the presence of ethidium bromide and benzonase to exclude associations mediated by nucleic acids. SMARCAD1 runs as a doublet in these cells. The lower band in the TFIIIC63 panel corresponds to the heavy chain of the antibody used in the IP.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Western Blot

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 5. Total (T), soluble (S for supernatant), and chromatin (P for pellet) fractions were prepared from control (Ctrl) and stable Smarcad1 knockdown (KD) ESCs (PGK12.1) and analyzed by Western blot with indicated antibodies. GAPDH and histone H3 were used as control for cytoplasm- and chromatin-bound fractions, respectively. Molecular weight markers are indicated in kDa. For TFIIIC90, bands of different mobility were detected, likely corresponding to differently modified versions of the protein.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Control, Knockdown, Western Blot, Molecular Weight

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 6. Gene expression analysis after depletion of Smarcad1 in mESCs. (a) Experimental outline for testing the ability of SMARCAD1 to affect the transcription of TFIIIC- and TFIIIC-regulated loci. Two ESC lines were investigated, namely PGK12.1 and E14. SU, subunits. (b,c) RT-qPCR-based characterization of the relative expression of (b) TFIIIC subunits and (c) selected loci corresponding to different TFIIIC binding sites in control or Smarcad1 knockdown ESCs. Gene expression is normalized to the average of two housekeeping genes and is presented as mean ± S.E. (error bar) of three technical triplicates. Biological replicates of datasets for b and c are shown in Supplementary Figure S2.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Binding Assay, Control, Knockdown

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

doi: 10.3390/genes14091793

Figure Lengend Snippet: Figure 7. In mESCs, there is no enrichment of SMARCAD1 at TFIIIC sites. (a) Genome-wide, there is no significant overlap between an unfiltered ChIP-seq dataset of SMARCAD1 [51] and common peaks of TFIIIC110 and TFIIIC 220 [21] that mark active Pol III genes and extra-TFIIIC (ETC) sites. (b) Venn diagram showing the overlap of TFIIIC-bound tRNA genes with unfiltered SMARCAD1 ChIP-seq peaks in ESCs. (c,d) SMARCAD1 and TFIIIC enrichment (% of input) at tRNA genes in either undifferentiated (PGK12.1, Panel (c)) or 5-day-differentiated mESCs (E14, Panel (d)). The specificity of SMARCAD1 ChIP was evaluated using as a positive control intracisternal particle A (IAPez) and as a negative control an intergenic region. Percentage of input values are the mean ± S.E. of three technical replicates.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Genome Wide, ChIP-sequencing, Positive Control, Negative Control

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: Data mining of FLAG-SMARCAD1 affinity purification from human cells reveals co-purification of architectural proteins. ( a ) Flow chart of the experimental approach used in . IgG and FLAG pulldowns were performed, and peptides were quantified using label-free mass spectrometry. Identified peptides for the proteins we validated here are shown in . ( b ) The six polypeptides of the hTFIIIC complex and their known interactions according to the STRING physical network. TFIIIC complex division into two sub-complexes is indicated; modules A and B bind A and B box DNA loci, respectively . Shown in bold are the five subunits that were identified by mass spectrometry as associated with tagged, overexpressed SMARCAD1 by .

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Affinity Purification, Copurification, Mass Spectrometry

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: Validation of architectural proteins as bona fide SMARCAD1-associated proteins by reciprocal co-immunoprecipitation of endogenous proteins. ( a ) Flow chart of the experimental approach. ( b ) Endogenous co-immunoprecipitation experiments (n = 2–4) from nuclear extracts from HeLa cells with anti-SMARCAD1 or anti-TFIIIC antibodies (subunits 63 and 90) analyzed by Western blot with the indicated antibodies. Inputs, 9% of the nuclear extract, are from the same blot as the IP. IgG, immunoglobin G. KAP1, a known binding partner of SMARCAD1, was analyzed in parallel (positive control).

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

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

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: hTFIIIC102 binds directly to hSMARCAD1. ( a ) Schematic representation of the GST fusion proteins tested for interaction with His-SMARCAD1. Apart from KAP1 (Materials and Methods), all proteins were full-length. Amino acid numbers are given. ( b ) GST-pulldown assay. Top panel: Bound proteins detected by Western blot with an anti-SMARCAD1 antibody. His-SMARCAD1 expressed in insect cells (Input, lane 2, 20%), GST (lane 3), GST-KAP1 ΔPB (lane 4), and GST-TFIIIC102 (lane 5). Molecular weight markers are indicated (kDa). Bottom panel: Instant-blue-stained PAGE gels of purified GST proteins.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: GST Pulldown Assay, Western Blot, Molecular Weight, Staining, Purification

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: Architectural proteins associate with SMARCAD1 in mouse pluripotent ESCs. ( a ) Experimental set-up. ( b ) Western blot analysis of endogenous immunoprecipitates (IPs) from 46C nuclear extracts; 6% of nuclear extract was used as input. The IPs were performed in the presence of ethidium bromide and benzonase to exclude associations mediated by nucleic acids. SMARCAD1 runs as a doublet in these cells. The lower band in the TFIIIC63 panel corresponds to the heavy chain of the antibody used in the IP.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Western Blot

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: Total (T), soluble (S for supernatant), and chromatin (P for pellet) fractions were prepared from control (Ctrl) and stable Smarcad1 knockdown (KD) ESCs (PGK12.1) and analyzed by Western blot with indicated antibodies. GAPDH and histone H3 were used as control for cytoplasm- and chromatin-bound fractions, respectively. Molecular weight markers are indicated in kDa. For TFIIIC90, bands of different mobility were detected, likely corresponding to differently modified versions of the protein.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Control, Knockdown, Western Blot, Molecular Weight, Modification

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: Gene expression analysis after depletion of Smarcad1 in mESCs. ( a ) Experimental outline for testing the ability of SMARCAD1 to affect the transcription of TFIIIC- and TFIIIC-regulated loci. Two ESC lines were investigated, namely PGK12.1 and E14. SU, subunits. ( b , c ) RT-qPCR-based characterization of the relative expression of ( b ) TFIIIC subunits and ( c ) selected loci corresponding to different TFIIIC binding sites in control or Smarcad1 knockdown ESCs. Gene expression is normalized to the average of two housekeeping genes and is presented as mean ± S.E. (error bar) of three technical triplicates. Biological replicates of datasets for b and c are shown in .

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Binding Assay, Control, Knockdown

Journal: Genes

Article Title: The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse

doi: 10.3390/genes14091793

Figure Lengend Snippet: In mESCs, there is no enrichment of SMARCAD1 at TFIIIC sites. ( a ) Genome-wide, there is no significant overlap between an unfiltered ChIP-seq dataset of SMARCAD1 and common peaks of TFIIIC110 and TFIIIC 220 that mark active Pol III genes and extra-TFIIIC (ETC) sites. ( b ) Venn diagram showing the overlap of TFIIIC-bound tRNA genes with unfiltered SMARCAD1 ChIP-seq peaks in ESCs. ( c , d ) SMARCAD1 and TFIIIC enrichment (% of input) at tRNA genes in either undifferentiated (PGK12.1, Panel ( c )) or 5-day-differentiated mESCs (E14, Panel ( d )). The specificity of SMARCAD1 ChIP was evaluated using as a positive control intracisternal particle A (IAPez) and as a negative control an intergenic region. Percentage of input values are the mean ± S.E. of three technical replicates.

Article Snippet: Antibodies used were IgG rabbit (NA934V; GE Healthcare, Buckinghamshire, UK); SMARCAD1 (A301-592A; Bethyl, Biomol, Hamburg, Germany); TFIIIC63 (A302-242A; Bethyl, Biomol, Hamburg, Germany); TFIIIC90 (PA5-31288; ThermoFisher Scientific, TFS, Waltham, MA, USA).

Techniques: Genome Wide, ChIP-sequencing, Positive Control, Negative Control