Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2
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{"title"=>"Chemical inhibition of histone deacetylases 1 and 2 induces fetal hemoglobin through activation of GATA2", "type"=>"journal", "authors"=>[{"first_name"=>"Jeffrey R.", "last_name"=>"Shearstone", "scopus_author_id"=>"6506831676"}, {"first_name"=>"Olga", "last_name"=>"Golonzhka", "scopus_author_id"=>"15755412900"}, {"first_name"=>"Apurva", "last_name"=>"Chonkar", "scopus_author_id"=>"57193451192"}, {"first_name"=>"David", "last_name"=>"Tamang", "scopus_author_id"=>"57191975312"}, {"first_name"=>"John H.", "last_name"=>"Van Duzer", "scopus_author_id"=>"6508306296"}, {"first_name"=>"Simon S.", "last_name"=>"Jones", "scopus_author_id"=>"56528700900"}, {"first_name"=>"Matthew B.", "last_name"=>"Jarpe", "scopus_author_id"=>"7003635628"}], "year"=>2016, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84963854386", "doi"=>"10.1371/journal.pone.0153767", "issn"=>"19326203", "pui"=>"609839774", "sgr"=>"84963854386", "isbn"=>"10.1371/journal.pone.0153767", "pmid"=>"27073918"}, "id"=>"e5fc4260-3329-33b8-b973-f918f75160ed", "abstract"=>"Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and β-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult β-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene. [ABSTRACT FROM AUTHOR]", "link"=>"http://www.mendeley.com/research/chemical-inhibition-histone-deacetylases-1-2-induces-fetal-hemoglobin-through-activation-gata2", "reader_count"=>11, "reader_count_by_academic_status"=>{"Researcher"=>3, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>3, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Medicine and Dentistry"=>5, "Agricultural and Biological Sciences"=>3, "Psychology"=>1, "Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"Finland"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/4945474"], "description"=>"<p>BM cells were cultured in CS1 expansion media, and then placed in CS1 differentiation media for 8 days with vehicle or 1 μM ACY-957. Erythroid maturation stage was determined by flow cytometry using the cell surface markers TFRC and GYPA. Representative of experiments performed with cells from four independent donors.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3175939, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Effect_of_HDAC_inhibition_on_erythroid_differentiation_/3175939", "title"=>"Effect of HDAC inhibition on erythroid differentiation.", "pos_in_sequence"=>4, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945528"], "description"=>"<p>(A) Erythroid maturation stage of cells used in gene expression profiling experiments. BM cells were cultured in CS1 expansion media and then shifted to CS1 differentiation media for 5 days with vehicle or 1 μM ACY-957. At the end of the culture period, erythroid maturation stage was determined by flow cytometry and total RNA was isolated for analysis using Affymetrix GeneChips. Experiments were performed using cells from three independent donors. (B) Differentially expressed genes resulting from ACY-957 treatment using a filter of absolute fold change greater than 1.5 and a P-value less than 0.025. (C) Gene set enrichment analysis demonstrates that genes up-regulated by HDAC2 knockdown (‘Up in <i>HDAC2</i> KD’ gene set) are significantly overrepresented at the top of a ranked list of gene expression changes resulting from ACY-957 treatment. Significant enrichment is illustrated by the positive running enrichment score (ES) marked by the green line, normalized enrichment score (NES) = 2.5, and false discovery rate (FDR) P-value < 0.001. (D) Enrichment scores of the ‘Up in <i>HDAC1</i> KD’, ‘Up in <i>HDAC2</i> KD’, and ‘Down in <i>HDAC2</i> KD’ gene sets relative to all gene sets (2777 total) in the Molecular Signatures Database collection of Chemical and Genetic Perturbations. (E) GeneChip derived gene expression ratios of candidate <i>HBG</i> modulators following ACY-957 treatment, <i>HDAC1</i> knockdown, or <i>HDAC2</i> knockdown. Ratios expressed as treatment versus control. NS, not significant. (F) Gene expression of candidate <i>HBG</i> modulators by QPCR. BM cells were cultured in CS1 expansion media, and then shifted to CS1 differentiation media for 4 days in presence of vehicle or 1 μM ACY-957. Gene expression is shown relative to <i>ACTB</i> and normalized to day 0 (mean ± SD, n = 3 cell culture replicates). P-values were calculated for day 4 using a two-tailed <i>t</i> test. *P<0.05 and ***P<0.0005 compared to vehicle treatment. Data is representative of experiments using cells from two independent donors. (G) Induction of <i>GATA2</i> mRNA by ACY-957 in cells from the four SCD donors described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g003\" target=\"_blank\">Fig 3A</a>. P-values were calculated using a two-tailed, paired <i>t</i> test. *P<0.05 compared to vehicle treatment.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3175972, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Effect_of_HDAC1_2_inhibition_on_gene_expression_in_erythroid_progenitors_/3175972", "title"=>"Effect of HDAC1/2 inhibition on gene expression in erythroid progenitors.", "pos_in_sequence"=>5, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945594"], "description"=>"<p>(A) Experimental design. BM cells were cultured for 3 days in CS1 expansion media, and then infected with lentivirus containing a control hairpin that does not target any human gene (shCtrl) or with two different <i>GATA2</i> targeting hairpins (shG2-1, shG2-2). Infected cells were selected by addition of puromycin to the culture for 2 days. Following removal of puromycin at day 0, cells expressing each hairpin were cultured for 4 days in CS1 expansion media with 1 μM ACY-957 or vehicle control. (B) <i>GATA2</i> mRNA levels were measured by QPCR and expressed relative to <i>ACTB</i> mRNA (mean ± SD, n = 2 QPCR replicates for each of n = 2 infection replicates). (C) GATA2 protein levels at day 4. Values represent GATA2 protein levels normalized to ACTB and relative to vehicle treated shCtrl. (D) <i>HBG</i> mRNA levels were measured by QPCR and expressed relative to <i>ACTB</i> mRNA (mean ± SD, n = 2 QPCR replicates for each of n = 2 infection replicates). For panel ‘D’ P-values were calculated on day 4 using a two-tailed <i>t</i> test, ***P<0.0005.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3176038, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g007", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_i_GATA2_i_knockdown_attenuates_i_HBG_i_induction_by_ACY_957_/3176038", "title"=>"<i>GATA2</i> knockdown attenuates <i>HBG</i> induction by ACY-957.", "pos_in_sequence"=>7, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945279"], "description"=>"<p>(A) Chemical structure of the HDAC1/2-selective inhibitor ACY-957. The HDAC active site consists of a tubular pocket and a zinc binding domain. The benzamide scaffold contains a linker/surface region that binds the tubular pocket and a region which coordinates zinc. The internal cavity binding group provides selectivity for HDAC1/2. (B) <i>In vitro</i> biochemical assay for HDAC1, HDAC2, or HDAC3 inhibition by ACY-957. Assay was repeated on separate days with duplicates for each concentration (mean ± SD, n = 4). (C) <i>In vitro</i> biochemical assay for HDAC4, 5, 6, 7, 8, and 9 inhibition by ACY-957. (D) Cellular HDAC2 inhibition. BM cells were cultured in CS1 expansion media for 6 days followed by treatment with ACY-957 for an additional 48 hours. Assay was performed in triplicate for each of two different donor cells cultured on different days (mean ± SD, n = 6). (E) Dose-dependent induction of histone acetylation by ACY-957. BM cells were cultured in CS2 expansion media for 6 days followed by treatment with drug for an additional 24 hours and then examined by western blot. Values represent relative abundance of each acetylation or methylation mark relative to total histone H4 and then normalized to vehicle control.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3175816, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/ACY_957_is_an_HDAC1_2_selective_inhibitor_/3175816", "title"=>"ACY-957 is an HDAC1/2-selective inhibitor.", "pos_in_sequence"=>1, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945630"], "description"=>"<p>BM cells were cultured in CS1 expansion media and then shifted to CS1 differentiation media with vehicle or 1 μM ACY-957 until reaching a differentiation stage similar to cells used in the GeneChip experiments of <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g005\" target=\"_blank\">Fig 5</a>. (A) Erythroid differentiation stage of cells used for ChIP in Fig 8C, 8E and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g009\" target=\"_blank\">Fig 9A</a>. (B) Erythroid differentiation stage of cells used for ChIP in Fig 8D and <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g009\" target=\"_blank\">Fig 9B</a>. (C) HDAC1 and HDAC2 ChIP-Seq profiles at the <i>GATA2</i> locus in erythroid progenitors. Chromatin was immunoprecipitated and sequenced using antibodies against HDAC1 or HDAC2 (black histogram tracks). Sequencing read count (y-axis) is plotted as a function of genomic region bin (x-axis). Publically available ENCODE Consortium ChIP-Seq data for HDAC1, HDAC2, and GATA2 in K562 cells is shown (gray histogram tracks) [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.ref049\" target=\"_blank\">49</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.ref050\" target=\"_blank\">50</a>]. The previously described <i>GATA2</i> enhancer regions (red text) map to GATA2 binding peaks in K562 cells. In both K562 and primary erythroid progenitors, HDAC1 and HDAC2 occupy a region bounded by the +9.5 kb and -3.9 kb enhancer regions (red dashed lines). (D) Histone acetylation at <i>GATA2</i> regulatory regions in vehicle and ACY-957 treated erythroid progenitors. Chromatin from each treatment was immunoprecipitated using anti-H3K9ac, anti-H2BK5ac, or anti-H3K27ac antibodies. A region near the INO80 gene (Ctrl), identified as a region of saturated acetylation across a wide variety of ENCODE cell lines, was used as a control (mean ± SD, n = 2 QPCR replicates for each of n = 3 IP replicates per antibody). P-values were calculated using a two-tailed <i>t</i> test. **P<0.005 and ***P<0.0005 for ACY-957 compared to vehicle control. (E) GATA2 binding at <i>GATA2</i> regulatory regions following ACY-957 treatment. IP with anti-GATA2 antibody and sequencing as described in ‘C’. ACY-957 treatment resulted in increased GATA2 occupancy (black histogram tracks) at the <i>GATA2</i> regulatory regions, indicated by solid red bars (wide view) or red arrows (magnified view). ACY-957-responsive regions localize to GATA2 binding peaks in K562 cells (gray histogram track).</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3176074, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g008", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/HDAC1_2_inhibition_by_ACY_957_leads_to_elevated_levels_of_histone_acetylation_and_GATA2_binding_at_i_GATA2_i_regulatory_regions_/3176074", "title"=>"HDAC1/2 inhibition by ACY-957 leads to elevated levels of histone acetylation and GATA2 binding at <i>GATA2</i> regulatory regions.", "pos_in_sequence"=>8, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945663"], "description"=>"<p>Changes in GATA2 binding due to ACY-957 treatment were surveyed across the 70 kb β-like globin gene cluster. Cell culture, IP with anti-GATA2, and sequencing were performed as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153767#pone.0153767.g008\" target=\"_blank\">Fig 8</a>. (A) The location of each β-like globin gene and LCR hypersensitivity sites (HS1-4) are shown in the ‘Gene’ track (blue). GATA2 occupancy is shown for vehicle or ACY-957 treated primary erythroblasts (‘vehicle’ and ‘ACY-957’ tracks, black). Only 6 regions had statistically significant GATA2 binding peaks in either vehicle or ACY-957 treated cells (indicated by triangles below the ‘Gene’ track). Within the 70 kb β-like globin locus, ACY-957 treatment lead to elevated levels of GATA2 binding only at a single region, found at the <i>HBD</i> promoter. ENCODE Consortium data for K562 cells shows this <i>HBD</i> region makes long-range looping interactions with the LCR (‘5C peaks’ track, gray), is a predicted enhancer (‘Chr state’ track, gray), is a region of open chromatin (‘DNase I’ track, gray), and is a region of GATA2 and GATA1 binding (‘GATA2’ and ‘GATA1’ tracks, gray). (B) Confirmation of GATA2 ChIP-Seq results by ChIP-QPCR. Two unique primer sets were used to detect GATA2 occupancy at the <i>HBD</i> promoter. A primer set at the <i>HBB</i> promoter, predicted by ChIP-Seq as a region where GATA2 had statistically significant binding that was unaltered by ACY-957 treatment, was used as a control (mean ± SD, n = 3 QPCR replicates for each of n = 3 IP replicates). P-values were calculated using a two-tailed <i>t</i> test. ***P<0.0005 for ACY-957 compared to vehicle control. (C) Summary of findings. ACY-957 inhibits HDAC1/2 leading to elevated histone acetylation at <i>GATA2</i> enhancer regions. Increased histone acetylation promotes occupancy of GATA2 at these regulatory regions, resulting in sustained activation of GATA2 during erythroid maturation through a positive autoregulatory loop. Elevated GATA2 contributes to <i>HBG</i> induction through an unknown mechanism, but may involve increased GATA2 binding at the <i>HBD</i> promoter (dashed line).</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3176107, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g009", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/HDAC1_2_inhibition_by_ACY_957_leads_to_elevated_levels_of_GATA2_binding_at_the_i_HBD_i_promoter_/3176107", "title"=>"HDAC1/2 inhibition by ACY-957 leads to elevated levels of GATA2 binding at the <i>HBD</i> promoter.", "pos_in_sequence"=>9, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945339"], "description"=>"<p>(A) Time-dependent increase in the percent <i>HBG</i> mRNA in CS1 cells. BM cells were cultured in CS1 expansion media, then shifted to CS1 differentiation media for 5 days with vehicle (dimethyl sulfoxide), 30 μM hydroxyurea (HU), or 1 μM ACY-957 (mean ± SD, n = 2 QPCR and n = 2 cell culture replicates). Data is representative of experiments using cells from three independent donors. (B) HbF protein induction in ACY-957 treated cells. Cells from day 5 of differentiation in ‘A’ were stained with an anti-HbF antibody and detected by flow cytometry. (C) Effect of ACY-957 on each β-like globin transcript. Samples from ‘A’ with each β-like globin transcript plotted relative to β-actin (<i>ACTB</i>). (D) Time-dependent increase in the percent <i>HBG</i> mRNA in CS2 cells. BM cells were cultured in CS2 expansion media, then shifted to CS2 differentiation media for 5 days with vehicle, 30 μM HU, or 1 μM ACY-957 (mean ± SD, n = 2 QPCR and n = 2 cell culture replicates). Data is representative of experiments using cells from two independent donors. (E) HbF protein induction in ACY-957 treated cells. Cells from day 5 of differentiation in ‘D’ were stained with an anti-HbF antibody and detected by flow cytometry. (F) Samples from ‘D’ with each β-like globin transcript plotted relative to <i>ACTB</i>. (G) Dose-dependent increase in percent <i>HBG</i> mRNA in BFU-E colonies derived from human bone marrow mononuclear cells cultured with ACY-957 (mean ± SD, n = 3 cell culture replicates). Data is representative of experiments using cells from two independent donors. (H) Samples from ‘G’ with each β-like globin transcript plotted relative to <i>ACTB</i>. In panels ‘A’, ‘C’, ‘D’, and ‘F’, P-values were calculated on day 5 only using a two-tailed <i>t</i> test. In panel ‘G’ and ‘H’ P-values were calculated using a two-tailed <i>t</i> test. For all panels *P<0.05, **P<0.005, and ***P<0.0005 compared to vehicle treatment. <i>HBG</i> mRNA (%) = [<i>HBG</i>/(<i>HBB</i>+<i>HBD</i>+<i>HBG</i>+<i>HBE</i>)]*100. MFI, mean fluorescent intensity. RFU, relative fluorescence units. RQ, relative quantity.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3175858, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/ACY_957_induces_i_HBG_i_mRNA_and_HbF_protein_in_primary_cells_from_healthy_donors_/3175858", "title"=>"ACY-957 induces <i>HBG</i> mRNA and HbF protein in primary cells from healthy donors.", "pos_in_sequence"=>2, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945399"], "description"=>"<p>(A) PBMC from four SCD donors were isolated, expanded, and then placed in differentiation media with vehicle or 1 μM ACY-957 for the indicated number of days. (B) Percent <i>HBG</i> in sickle cell patient cells in ‘A’ after 3 days of culture. (C) Hematopoietic progenitors were isolated from peripheral blood of a sickle cell patient, expanded, and then cultured for 3 days in differentiation media with 1, 2, or 3 μM ACY-957. HbF protein was detected by flow cytometry. P-values were calculated using a two-tailed, paired <i>t</i> test. **P<0.005 compared to vehicle treatment. <i>HBG</i> mRNA (%) = [<i>HBG</i>/(<i>HBB</i>+<i>HBD</i>+<i>HBG</i>+<i>HBE</i>)]*100.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3175891, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/ACY_957_induces_i_HBG_i_mRNA_and_HbF_protein_in_primary_cells_from_sickle_cell_disease_patients_/3175891", "title"=>"ACY-957 induces <i>HBG</i> mRNA and HbF protein in primary cells from sickle cell disease patients.", "pos_in_sequence"=>3, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/4945552"], "description"=>"<p>(A) Experimental design. BM cells were cultured for 3 days in CS1 expansion media, and then infected with lentivirus containing GFP (oeCtrl) or full length <i>GATA2</i> transcript (oeG2). Infected cells were selected for by addition of puromycin to the culture for 2 days. Cells were then shifted to CS1 differentiation media for the remainder of the experiment. (B) <i>GATA2</i> mRNA levels in oeG2 cells expressed relative to oeCtrl (mean ± SD, n = 2 QPCR replicates for each of n = 2 infection replicates). (C) GATA2 protein levels at day 5 of differentiation. Values represent GATA2 protein levels relative to ACTB and then normalized to oeCtrl. (D) <i>HBG</i> mRNA levels during differentiation of oeCtrl and oeG2 cells (mean ± SD, n = 2 QPCR replicates for each of n = 2 infection replicates). <i>HBG</i> mRNA (%) = [<i>HBG</i>/(<i>HBB</i>+<i>HBD</i>+<i>HBG</i>+<i>HBE</i>)]*100. (E) Samples from ‘D’ with each β-like globin transcript expressed relative to <i>ACTB</i> mRNA. (F) TFRC and GYPA cell surface expression at day 5 of differentiation. (G) Total β-like globin mRNA (sum of <i>HBB</i>, <i>HBD</i>, <i>HBG</i>, and <i>HBE</i>) in samples from ‘D’. For panels ‘D’, ‘E’, and ‘G’, P-values were calculated on day 5 using a two-tailed <i>t</i> test. *P<0.05, **P<0.005, ***P<0.0005 for oeG2 compared to oeCtrl. Data is representative of experiments performed using cells from three independent donors.</p>", "links"=>[], "tags"=>["GATA 2 protein", "HDAC 1", "HBB", "histone", "GATA 2 attenuated HBG induction", "Histone Deacetylases 1", "SCD", "gene expression", "GATA 2 autoregulatory regions", "2 Induces Fetal Hemoglobin", "HbF", "erythroid progenitors", "ACY", "knockdown", "GATA 2 gene", "GATA 2", "GATA 2 locus", "chemical", "GATA 2 Therapeutic intervention"], "article_id"=>3175993, "categories"=>["Biochemistry", "Microbiology", "Cell Biology", "Genetics", "Molecular Biology", "Pharmacology", "Chemical Sciences not elsewhere classified", "Biological Sciences not elsewhere classified", "Developmental Biology", "Cancer", "Hematology", "Virology"], "users"=>["Jeffrey R. Shearstone", "Olga Golonzhka", "Apurva Chonkar", "David Tamang", "John H. van Duzer", "Simon S. Jones", "Matthew B. Jarpe"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0153767.g006", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Overexpression_of_GATA2_induces_i_HBG_i_and_suppresses_i_HBB_i_in_erythroid_progenitors_/3175993", "title"=>"Overexpression of GATA2 induces <i>HBG</i> and suppresses <i>HBB</i> in erythroid progenitors.", "pos_in_sequence"=>6, "defined_type"=>1, "published_date"=>"2016-04-13 05:43:44"}

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  • {"unique-ip"=>"49", "full-text"=>"62", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"11"}
  • {"unique-ip"=>"24", "full-text"=>"31", "pdf"=>"8", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"9"}
  • {"unique-ip"=>"24", "full-text"=>"28", "pdf"=>"9", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"4"}
  • {"unique-ip"=>"26", "full-text"=>"21", "pdf"=>"10", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"5"}
  • {"unique-ip"=>"13", "full-text"=>"8", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"6"}
  • {"unique-ip"=>"22", "full-text"=>"26", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"7"}
  • {"unique-ip"=>"28", "full-text"=>"34", "pdf"=>"6", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"10"}
  • {"unique-ip"=>"23", "full-text"=>"23", "pdf"=>"7", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"8"}
  • {"unique-ip"=>"28", "full-text"=>"35", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"11", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"12"}
  • {"unique-ip"=>"32", "full-text"=>"41", "pdf"=>"10", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"11", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"2"}
  • {"unique-ip"=>"33", "full-text"=>"32", "pdf"=>"5", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"3"}
  • {"unique-ip"=>"27", "full-text"=>"32", "pdf"=>"6", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"4"}
  • {"unique-ip"=>"27", "full-text"=>"25", "pdf"=>"6", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"5"}

Relative Metric

{"start_date"=>"2016-01-01T00:00:00Z", "end_date"=>"2016-12-31T00:00:00Z", "subject_areas"=>[]}
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