atf4 (sc-200) antibody  (Santa Cruz Biotechnology)

Journal: PLOS Genetics

Article Title: Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs

doi: 10.1371/journal.pgen.1011014

Figure Lengend Snippet: (A) Number of peaks called in ATF4 ChIP-Seq experiments in human cell types. Data from published experiments was reprocessed from raw reads using a uniform pipeline. To obtain a common peak set across all cell types, overlapping peak regions were merged. (B) Peak length distribution for the merged set of ATF4 peak regions. (C) Fraction of ATF4 ChIP-Seq peaks called in more than one cell type. (D) Genomic context distribution of ATF4 peaks. (E) Functional state of chromatin in ATF4 ChIP-Seq peak regions and genome-wide. Epigenomic maps were obtained from the EpiMap project , which annotated chromatin into 18 states based on combinations of histone modifications. For HepG2, K562, HAP1 and MSC, basal (non-stress condition) data was available in EpiMap (accessions BSS00558, BSS00762, BSS00491 and BSS01260, respectively). For erythroblast, HUDEP2 and DLD1, a similar EpiMap cell type was used as a proxy (erythroid differentiation of CD34+ cells for the former two, and immortalized colonic epithelial cells for the latter; EpiMap accessions BSS00545 and BSS00223, respectively). (F) Percentage of peak regions containing the C/EBP-ATF motif, a known ATF4 binding sequence, compared to matched background regions (as selected by HOMER). The peak summit coordinate +/- 100 bp was used as the search region. The HOMER motif database ATF4 motif (based on GSE35681) was used as a representative C/EBP-ATF motif. Each dot represents a ChIP-Seq library. (G) ChIP-Seq binding profile similarity between ATF4 and 207 other chromatin-binding proteins in HepG2 cells. The peak sets besides ATF4 were obtained from (GEO accession: GSE104247). For ATF4, peaks from all HepG2 ChIP-Seq samples were merged. All peaks were resized to equal length (+/- 100 bp around the center) and the bedtools Jaccard similarity statistic was calculated between the ATF4 binding regions and every other set of ChIP-Seq peaks.

Article Snippet: The blots were incubated with rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution; Cell Signaling Technology #7074).

Techniques: ChIP-sequencing, Functional Assay, Genome Wide, Binding Assay, Sequencing

Journal: PLOS Genetics

Article Title: Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs

doi: 10.1371/journal.pgen.1011014

Figure Lengend Snippet: (A) Gene ontology biological process enrichment analysis for genes with a TSS-proximal ATF4 peak called in ≥4 cell types (out of the 7 analyzed). One ChIP-Seq library was used to represent each cell type (the library with the most peaks called). (B) Genes most frequently binding ATF4 at the TSS/promoter. All genes with binding in 6 or 7 cell types are listed. (C) Number of ATF4-sensitive genes from RNA profiling studies of ATF4 pathway modulation. (D) Gene ontology biological process enrichment analysis for genes with ATF4-sensitive expression in ≥4 studies (out of the 7 analyzed). (E) Most frequently ATF-sensitive genes across the RNA profiling studies. (F) Intersection of genes with ATF4 binding at the TSS/promoter region in ≥4 ChIP-Seq studies and ATF4-sensitive expression in ≥4 RNA profiling studies.

Article Snippet: The blots were incubated with rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution; Cell Signaling Technology #7074).

Techniques: ChIP-sequencing, Binding Assay, Expressing

Journal: PLOS Genetics

Article Title: Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs

doi: 10.1371/journal.pgen.1011014

Figure Lengend Snippet: (A) Overview of SNPs selected for validation of ATF4-dependent allelic effect. Genetic association data was queried from OpenTargets. (B) Luciferase reporter activity for genomic regions containing SNP alleles (ref, light blue; alt, dark blue) in HepG2 cells without treatment (Ctrl), with tunicamycin treatment (Tun) and with ATF4 overexpression (ATF4). The mean ± SD is shown from 4–5 experiments performed on separate days. (C) Electrophoretic mobility shift assay (EMSA) with biotin-labeled oligonucleotides corresponding to either the ref or the alt allele of the SNP. Nuclear protein extract from HepG2 cells overexpressing ATF4 was used, and, in lanes labeled α-ATF4, antibody targeting ATF4 was included in the reaction. * p < 0.05, ** p < 0.005, *** p < 0.0005 by two-tailed t test with Bonferroni-Holm correction.

Article Snippet: The blots were incubated with rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution; Cell Signaling Technology #7074).

Techniques: Biomarker Discovery, Luciferase, Activity Assay, Over Expression, Electrophoretic Mobility Shift Assay, Labeling, Two Tailed Test

Journal: PLOS Genetics

Article Title: Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs

doi: 10.1371/journal.pgen.1011014

Figure Lengend Snippet: (A) LocusZoom plot of the mean platelet volume GWAS study conducted by Chen et al . The trait association p values for all variants studied in this region are shown and the orientation of the arrowheads indicates the effect direction (GWAS Catalog accession: GCST90002346). Linkage disequilibrium (LD) in the European population is shown relative to the lead SNP (rs532446), provided by LocusZoom . (B) Chromatin accessibility and ATF4 ChIP-Seq coverage near rs532446. The SNP location is indicated with a vertical dashed line, and the locations of CRISPR gRNAs are shown in red. Chromatin accessibility tracks were obtained from the human single-nucleus ATAC-Seq atlas , except K562, which was obtained from ENCODE accession ENCFF972GVB . (C) CRISPR gRNAs designed to introduce short indels within the C/EBP-ATF motif overlapping rs532446. The gRNA sequences targeting the reference (ref) and alternative (alt) alleles are shown, and the locations of the C/EBP-ATF motif (green), the rs532446 SNP (red), the gRNA PAM motif (blue), and the Cas9 cut site (arrowheads) are indicated. (D) Quantification of indels introduced into the rs532446-overlapping C/EBP-ATF motif by the CRISPR-Cas9 editing using gRNA 1+2 (described in panel C). (E) PCR-based validation of CRISPR-Cas9 mediated deletion of the rs532446 region using gRNA 3+4. (F) RT-qPCR quantification of GADD45A mRNA expression in untreated and arsenite-treated cells transfected with control gRNA (ctrl gRNA) or gRNAs designed to disrupt the regulatory region overlapping rs532446 (gRNA 1+2 or gRNA 3+4). Data are presented relative to the mRNA expression level in untreated cells transfected with ctrl gRNA. (G) Luciferase reporter activity for genomic regions containing SNP rs532446 (C/T) in untreated and arsenite-treated K562 cells. (H) EMSA analysis for SNP rs532446 allele-specific binding of ATF4. Biotin-labeled probes containing rs532446 C or T alleles were incubated with nuclear extracts from untreated and arsenite-treated K562 cells. In lanes labeled α-ATF4, antibody targeting ATF4 was included in the reaction. In panels F and G the mean ± SD is shown from 3–4 transfection experiments performed on separate days. ** p < 0.005, *** p < 0.0005 by two-tailed t test with Bonferroni-Holm correction. The coordinates shown are for GRCh38.

Article Snippet: The blots were incubated with rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution; Cell Signaling Technology #7074).

Techniques: ChIP-sequencing, CRISPR, Introduce, Biomarker Discovery, Quantitative RT-PCR, Expressing, Transfection, Control, Luciferase, Activity Assay, Binding Assay, Labeling, Incubation, Two Tailed Test

Journal: PLOS Genetics

Article Title: Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs

doi: 10.1371/journal.pgen.1011014

Figure Lengend Snippet: (A) LocusZoom plot of the myocardial infarction GWAS study conducted by Hartiala et al . The trait association p values for all variants studied in this region are shown and the orientation of the arrowheads indicates the effect direction (GWAS Catalog accession: GCST011364). Linkage disequilibrium (LD) in the European population is shown relative to the lead SNP (rs7011846), provided by LocusZoom . (B) Chromatin accessibility and ATF4 ChIP-Seq coverage near rs7011846. The SNP location is indicated with a vertical dashed line, and the locations of CRISPR gRNAs are shown in red. Chromatin accessibility tracks were obtained from the human single-nucleus ATAC-Seq atlas , except THP-1, which was obtained from GEO accession GSE96800 . (C) PCR-based validation of CRISPR-Cas9 mediated deletion of the rs7011846 region using gRNA 5+7 or gRNA 6+7. (D) RT-qPCR quantification of LPL mRNA expression in untreated and thapsigargin-treated cells transfected with control gRNA (ctrl gRNA) or gRNAs designed to disrupt the regulatory region overlapping rs7011846 (gRNA 5+7 or gRNA 6+7). Data are presented relative to the mRNA expression level in untreated cells transfected with ctrl gRNA. (E) EMSA analysis for SNP rs7011846 allele-specific binding of ATF4. Biotin-labeled probes containing rs7011846 G or A alleles were incubated with nuclear extracts from untreated and thapsigargin-treated THP-1 cells. In lanes labeled α-ATF4, antibody targeting ATF4 was included in the reaction. Where indicated, unlabeled competitor probe was added to the reaction in 5-fold excess. (F) Luciferase reporter activity for genomic regions containing SNP rs7011846 (G/A) in untreated and thapsigargin-treated THP-1 cells. In panels D and F the mean ± SD is shown from 4–6 transfection experiments performed on separate days. * p < 0.05, ** p < 0.005, *** p < 0.0005 by two-tailed t test with Bonferroni-Holm correction. The coordinates shown are for GRCh38.

Article Snippet: The blots were incubated with rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:3000 dilution; Cell Signaling Technology #7074).

Techniques: ChIP-sequencing, CRISPR, Biomarker Discovery, Quantitative RT-PCR, Expressing, Transfection, Control, Binding Assay, Labeling, Incubation, Luciferase, Activity Assay, Two Tailed Test

Journal: Lipids in Health and Disease

Article Title: Atorvastatin helps preserve pancreatic β cell function in obese C57BL/6 J mice and the effect is related to increased pancreas proliferation and amelioration of endoplasmic-reticulum stress

doi: 10.1186/1476-511X-13-98

Figure Lengend Snippet: Atorvastatin upregulates PDX-1 and LXR- β expression and downregulates the protein expressions of ER stress markers. Total RNA was extracted from the pancreas of C57 mice and analyzed by quantitative real-time PCR. A comparative threshold cycle (CT) method was used for relative quantification of gene expression using beta-actin for normalization. Measurements were carried out in triplicate for each sample. (A) Relative mRNA levels of PDX-1 and LXR-β in pancreatic cells. Western blot analysis of pancreatic (B) PDX-1 (C) phosphorylated eIF2α (D) ATF4 (E) CHOP in C57 mice compared in three groups. Beta actin served as loading control. Data represented the mean of at least three independent experiments ± S.E.M. **p < 0.01, ***p < 0.001 vs. control.

Article Snippet: The following antibodies were used (1:1000 dilution unless otherwise indicated): total eIF2α (sc-11386, 1:500), ATF4 (sc-200), PDX-1 (sc-25403), and CHOP (sc-575), Bcl-2 (sc-7382) (all from Santa Cruz Biotechnology), phospho-EIF2α (Ser51, 9721) (Cell Signaling Technology, Danvers, MA) and β-actin (Abmart, 1:2000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1)

doi: 10.1016/j.jbc.2022.102277

Figure Lengend Snippet: ChIP-seq analysis of ATF4 in response to GCN2 activation. A , heatmaps of ATF4 ChIP-seq read densities in a window of ± 2.5kb from peak summits centered at 0 for WT and GCN2 KO MEFs exposed to control (+Leu) or leucine deficient (-Leu) medium for 24 h. Each row represents the ChIP-seq read density around a peak summit for each identified peak per condition. Read densities are proportional to color intensities across groups. ChIP-seq data represent a single sequencing experiment on a ChIP conducted using chromatin pooled from two independent experiments each performed with at least five replicates. In WT MEFs, leucine deprivation reprogrammed ATF4 binding and augmented the number of binding sites, an effect that is lost in GCN2 KO MEFs. B , average ATF4 ChIP-seq signal intensities from peaks identified in ( A ) normalized per reads for WT and GCN2 KO MEFs ± leucine (Leu) for 24 h. C , Venn diagrams illustrating the effect of leucine (Leu) deprivation on ATF4 ChIP-seq target gene identification in WT and GCN2 KO MEFs. The analysis was restricted to genes harboring peaks identified within ±5 kb of gene TSSs. D , enriched (adjusted p -value < 0.05) MSigDB Hallmark gene signatures in ATF4 ChIP-seq target gene sets with binding peaks found within ±5 kb of gene TSSs. E and F functional enrichment analysis of an ATF4-targeted 145-gene set with binding peaks present within ±5 kb of gene TSSs in WT MEFs ± leucine. Using a redundancy reduction of significant terms, the top 10 significantly enriched (Benjamin-Hochberg (BH)-corrected FDR < 0.05) GO biological processes determined by WebGestalt are shown with the associated genes. Node size and color are proportional to the number of genes found in a biological category. An enrichment ratio >1 denotes that the number of overlapping genes with a functional term is greater than by chance with a random set of genes. ChIP, chromatin-immunoprecipitation; FDR, false discovery rate; GO, Gene Ontology; MEF, mouse embryonic fibroblast; TSS, transcription start site.

Article Snippet: Antibodies used were ATF4 (sc-200) (for ChIP-seq), ATF-4 antibody (D4B8) from Cell Signaling (For ChIP-qPCR), ATF4/CREB-2 (sc-390063, Santa Cruz) (for WB analysis presented in D ), ATF5 (Santa Cruz, sc-377168), LARP1 (sc-515873, Santa Cruz or ab86359, Abcam), p-eIF2α (ab32157, Abcam), Actin (ab179467, Abcam), GCN1 (ab8613, Abcam), GCN2 (3302S, Cell Signaling), anti-FLAG (F3165, Sigma), antimouse IgG, horeseradish peroxidase conjugated (W402B, Promega), anti-rabbit IgG, horeseradish peroxidase conjugated (W401B, Promega), and G3BP (catalog no.: #611126, BD Transduction Laboratories).

Techniques: ChIP-sequencing, Activation Assay, Control, Sequencing, Binding Assay, Functional Assay, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1)

doi: 10.1016/j.jbc.2022.102277

Figure Lengend Snippet: GCN2-dependent regulation of the LARP1 via ATF4. A , UCSC Genome browser views for ATF4 ChIP-seq binding events in WT and GCN2 KO MEFs that have been exposed to control (+Leu) or leucine-deficient (-Leu) medium for 24 h for a subset of genes associated with mTOR signaling. ATF4 ChIP-seq binding profiles in WT and ATF4 KO MEFs treated with tunicamycin (Tm) ( GSE35681 ) are also shown below each panel validating the specificity of the ATF4 targets found. ChIP-seq data represent a single sequencing experiment on a ChIP conducted using chromatin pooled from two independent experiments, each performed with at least five replicates. B , a heatmap shows MACS2 fold enrichment for a selected ATF4 ChIP-seq target genes associated with mTOR signaling, the integrated stress response and amino acid metabolism with binding peaks found within ±5 kb of gene TSSs. ChIP-seq data represent a single sequencing experiment on a ChIP conducted using chromatin pooled from two independent experiments each performed with at least five replicates. C and E , ATF4 ChIP-qPCR analysis of Larp1 and Ddit3 promoters in WT and GCN2 KO MEFs ( C ) or WT and ATF4 KO MEFs ( E ) exposed to control (+Leu) or leucine-deficient (-Leu) medium for 6 h and 24 h. D and F , RT-qPCR analysis of Larp1 and Ddit3 in WT and GCN2 KO MEFs ( D ) or WT and ATF4 KO MEFs ( F ) exposed to control (+Leu) or leucine-deficient (-Leu) medium for 6 h and 24 h. Data in ( C – F ) are presented as means ± SD (n = 3). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; Two-way ANOVA followed by Bonferroni post hoc test. ChIP, chromatin-immunoprecipitation; MEF, mouse embryonic fibroblast.

Article Snippet: Antibodies used were ATF4 (sc-200) (for ChIP-seq), ATF-4 antibody (D4B8) from Cell Signaling (For ChIP-qPCR), ATF4/CREB-2 (sc-390063, Santa Cruz) (for WB analysis presented in D ), ATF5 (Santa Cruz, sc-377168), LARP1 (sc-515873, Santa Cruz or ab86359, Abcam), p-eIF2α (ab32157, Abcam), Actin (ab179467, Abcam), GCN1 (ab8613, Abcam), GCN2 (3302S, Cell Signaling), anti-FLAG (F3165, Sigma), antimouse IgG, horeseradish peroxidase conjugated (W402B, Promega), anti-rabbit IgG, horeseradish peroxidase conjugated (W401B, Promega), and G3BP (catalog no.: #611126, BD Transduction Laboratories).

Techniques: ChIP-sequencing, Binding Assay, Control, Sequencing, ChIP-qPCR, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1)

doi: 10.1016/j.jbc.2022.102277

Figure Lengend Snippet: GCN2 suppresses TOP mRNA translation. A , analysis of ribosome profilings performed in a previous study (Darnell 2018) on WT and GCN2 KO HEK293T cells after 6 h of arginine or leucine starvation or nutrient replete conditions (n = 1). Scatter plots of footprints per million (FPM) for each transcript are plotted for WT and GCN2 KO cells ± arginine or leucine starvation. Yellow points indicate 5′TOP mRNAs (the list of 5′TOP mRNAs obtained from Table S2 of Yamashita et al . 2008) . B , violin plots of Log2 (GCN2 KO/WT) values for all transcripts in each nutrient condition described in ( A ). A two-sided Wilcoxon signed-rank test was performed to assess whether the Log2 (GCN2 KO/WT) values of 5′TOP mRNAs increased significantly compared to non-5′TOP mRNAs. Resulting p -values are shown for each condition. C , RT-qPCR analysis of LARP1 expression in WT and GCN2 KO HEK 293T cells or in HEK293T cells exposed to scrambled shRNA (Scr.) or shRNA against ATF4 (shATF4) in the presence or absence of leucine (Leu). Data are presented as means ± SD (n = 3). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; Two-way ANOVA followed by Bonferroni post hoc test. D , absorption profiles of ribosomes and RT-qPCR analysis of WT and GCN2 KO HEK 293T cells cultured in control medium (+Leu) for 6 h. 40S and 60S denote the corresponding ribosomal subunits and 80S refers to monosomes. E , absorption profiles of ribosomes and RT-qPCR analysis of WT and GCN2 KO HEK293T cells cultured in the absence of leucine (-Leu) for 6 h. 40S and 60S denote the corresponding ribosomal subunits and 80S refers to monosomes. RT-qPCR, reverse transcription quantitative PCR; TOP, terminal oligopyrimidine.

Article Snippet: Antibodies used were ATF4 (sc-200) (for ChIP-seq), ATF-4 antibody (D4B8) from Cell Signaling (For ChIP-qPCR), ATF4/CREB-2 (sc-390063, Santa Cruz) (for WB analysis presented in D ), ATF5 (Santa Cruz, sc-377168), LARP1 (sc-515873, Santa Cruz or ab86359, Abcam), p-eIF2α (ab32157, Abcam), Actin (ab179467, Abcam), GCN1 (ab8613, Abcam), GCN2 (3302S, Cell Signaling), anti-FLAG (F3165, Sigma), antimouse IgG, horeseradish peroxidase conjugated (W402B, Promega), anti-rabbit IgG, horeseradish peroxidase conjugated (W401B, Promega), and G3BP (catalog no.: #611126, BD Transduction Laboratories).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Cell Culture, Control, Reverse Transcription, Real-time Polymerase Chain Reaction