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, chromosomes & cancer

Article Title: Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

doi: 10.1002/gcc.22983

Figure Lengend Snippet: Frequent mutations of SMARCAD1 in humans and zebrafish suggest it is a tumor suppressor. (A) Heat map of human chromosome 4. SAMRCAD1 is located on this chromosome. The locus of SMARCAD1 (green line) was found to be underrepresented in ~26% (16 out of 61) human MPNSTs. (B) Zebrafish smarcad1a is located on chromosome 8, which is underrepresented in about 70% (103 out of 147) of zebrafish MPNSTs. (C) Zebrafish smarcad1b is located on chromosome 10, which is overrepresented in about 60% (88 out of 147) of zebrafish MPNSTs. Samples are sorted top-to-bottom by decreasing deletion amplitude at the respective SAMRCAD1/smarcad1 locus, indicated by a green line. Blue and red bars on the right side of each panel indicate samples with SAMRCAD1/smarcad1 losses (blue) or gains (red). Color densities are corresponding to the degree of loss and gain as previously described. (D) SMARCAD1 mutation frequency of human cancers in TCGA database. (E) S MARCAD1 mutation types and positions found in human cancers in TCGA database. (F) SMARCAD1 expression level of 6 human MPNST cell lines (all NF1 mutant except STS26T) and HEI-193, immortalized human schwannoma

Article Snippet: For Western blot, a commercial anti-SMARCAD1 antibody (Bethyl, A301–593A 1:2000 dilution) was chosen.

Techniques: Mutagenesis, Expressing

Journal: Genes, chromosomes & cancer

Article Title: Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

doi: 10.1002/gcc.22983

Figure Lengend Snippet: Zebrafish have two smarcad1 genes. (A) Extended majority-rule consensus tree for the Bayesian phylogenetic analysis of SMARCAD1 proteins. Numbers at each node indicate posterior probability (pp) values based on 20 million runs. Branch lengths are proportional to the means of the pp densities for their expected replacements per site. The ME and ML phylogenetic trees ( and ) were generally in agreement with the BP phylogeny: most of the metazoan species have one SMARCAD1 gene, while there are usually two smarcad1 genes in teleost genomes. The two smarcad1s most likely resulted from teleost-specific whole-genome duplications, as each formed a distinct clade. The tree was rooted with yeast. (B) Syntenic relationship between human and zebrafish chromosomes. Zebrafish chromosome 8 (Dre8) that contains smarcad1a and three other genes ( FAM190A , GRID2 , and ATOH1 , bottom row) are orthologous to, and in the same order as, genes in the portion of human chromosome 4 (Hsa4) that contains SMARCAD1 (middle row). A portion of Dre10 contains smarcad1b , but not the other three directly linked genes. However, this part of Dre10 is co-orthologous to the portion of Hsa4 that contains SMARCAD1 , as they share other syntenies (top row). Orthologous genes are indicated with colored lines

Article Snippet: For Western blot, a commercial anti-SMARCAD1 antibody (Bethyl, A301–593A 1:2000 dilution) was chosen.

Techniques:

Journal: Genes, chromosomes & cancer

Article Title: Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

doi: 10.1002/gcc.22983

Figure Lengend Snippet: The two smarcad1 zebrafish genes are expressed in both overlapped and distinct regions during embryonic development. Whole-mount in situ hybridization of zebrafish embryos at stages 12S (A, E), 20S (B, F), 24 hpf (C, D, G, H), 48 hpf (I–P), and 72 hpf (Q–X). Anterior is to the left in all whole-mount images, and dorsal is to the top in all transverse sections. Gene names are labeled at the upright corner in the panels of whole-mount images. (A–D, I–L, and Q–T): gene expression of smarcad1a . (E–H, M–P, and U–X): gene expression of smarcad1b . (A and C) Lateral view of the expression of smarcad1a at 12S, 20S, and 24dpf, respectively. (D) Transverse section through the trunk region of the embryos in panel C. smarcad1a is mainly expressed in the neural tissues, endoderm, and intermediate cell mass at these stages. (E and G) Lateral view of the expression of smarcad1b at 12S, 20S, and 24dpf, respectively. (H) Transverse section through the trunk region of the embryos in panel G. The expression patterns of smarcad1b are very similar to smarcad1a at these early stages. (I, J) Lateral and dorsal view of the expression of smarcad1a at 48 hpf. (K and L) Transverse section through the head and pectoral fin regions. (M and N) Lateral and dorsal view of the expression of smarcad1b at 48 hpf. (O and P) Transverse section through the head and pectoral fin regions. (Q and R) Lateral and dorsal view of the expression of smarcad1a at 72 hpf. (S and T) Transverse section through the head and pectoral fin regions. (U and V) Lateral and dorsal view of the expression of smarcad1b at 72 hpf. (W and X) Transverse section through the head and pectoral fin regions. The dashed lines indicate the positions of sections. The letters below the dashed lines correspond to the panels. de , diencephalon; dm , dermomyotome; end , endoderm; fb , forebrain; hb , hindbrain; icm , intermediate cell mass; ir , iris; le , lens; mand , mandibular cartilage; mb , middle brain; mhb , midbrain-hindbrain boundary; mz , ciliary marginal zone; n , notochord; nt , neural tube; op , optical cup; pf , pectoral fin; pa , pharyngeal; quad , quadrate cartilage; ret , retina; tb , tail bud

Article Snippet: For Western blot, a commercial anti-SMARCAD1 antibody (Bethyl, A301–593A 1:2000 dilution) was chosen.

Techniques: In Situ Hybridization, Labeling, Gene Expression, Expressing

Journal: Genes, chromosomes & cancer

Article Title: Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

doi: 10.1002/gcc.22983

Figure Lengend Snippet: Human cells show a defect in double-strand DNA damage repair. (A, B) Double-strand DNA damage repair was measured by Western blots with anti-histone H2AX S139ph (phospho Ser139) in SMARCAD1 knockdown schwannoma cell line HEI-193 after X-ray irradiations (10 and 20 Gy, respectively). ULTRA-3351712 knockdown cell was used for this experiment. Doxycycline (final concentration: 0.1 μg/ml) was added 48 h ahead of the experiments. (C, D) Double-strand DNA damage repair in SMARCAD1 overexpression MPNST cells (STS26T) by H2AX S139ph. Doxycycline (final concentration: 1 μg/ml) was added 24 h ahead of the experiments. Both cell lines were harvested at 1, 5, and 10 h post-irradiation along with untreated samples. un, untreated sample. Beta-actin (ACTB) was used as a loading control. The densitometry ratios of H2A X S139ph over ACTB was listed underneath the blots

Article Snippet: For Western blot, a commercial anti-SMARCAD1 antibody (Bethyl, A301–593A 1:2000 dilution) was chosen.

Techniques: Western Blot, Knockdown, Concentration Assay, Over Expression, Irradiation, Control

Journal: Genes, chromosomes & cancer

Article Title: Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

doi: 10.1002/gcc.22983

Figure Lengend Snippet: Cell proliferation and independent growth were inhibited in both SMARCAD1 knockdown and overexpression. Proliferation was measured by MTT assay in SMARCAD1 knockdown HEI193 cells (A) and overexpression STS26T cells (B). Growth differences were evident from day 3 in both cases. Anchorage-independent growth was examined by soft-agar assay in SMARCAD1 knockdown HEI193 cells (C) and overexpression STS26T cells (D). All the assays were done with three biological replicates. Asterisk (*) indicates statistical significance, p < 0.05

Article Snippet: For Western blot, a commercial anti-SMARCAD1 antibody (Bethyl, A301–593A 1:2000 dilution) was chosen.

Techniques: Knockdown, Over Expression, MTT Assay, Soft Agar Assay

Journal: Genes, chromosomes & cancer

Article Title: Loss of smarcad1a accelerates tumorigenesis of malignant peripheral nerve sheath tumors in zebrafish

doi: 10.1002/gcc.22983

Figure Lengend Snippet: Model of SMARCAD1 tumor suppressor mechanisms. Three mechanisms (DNA damage response, heterochromatin maintaining, and transcription regulation) were proposed based on current knowledge. , , , , , , , The bidirectional arrows indicate protein–protein interactions. The arrows imply cellular consequences. The dashed arrows show a possible result. The involved genes and pathways are solely based on loss-of-function studies due to the lack of overexpression reports

Article Snippet: For Western blot, a commercial anti-SMARCAD1 antibody (Bethyl, A301–593A 1:2000 dilution) was chosen.

Techniques: Protein-Protein interactions, Over Expression