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千葉大学大学院人文社会科学研究科研究プロジェクト報告書第257集『都市コミュニティにおける相互扶助と次世代育成』水島治郎 編Sustainable Urban Communities: Communality and Generativity Report on the Research Projects No.25

Regulation of Gγ-Globin Gene by ATF2 and Its Associated Proteins through the cAMP-Response Element

<div><p>The upstream Gγ-globin cAMP-response element (G-CRE) plays an important role in regulating Gγ-globin expression through binding of ATF2 and its DNA-binding partners defined in this study. ATF2 knockdown resulted in a significant reduction of γ-globin expression accompanied by decreased ATF2 binding to the G-CRE. By contrast, stable ATF2 expression in K562 cells increased γ-globin transcription which was reduced by ATF2 knockdown. Moreover, a similar effect of ATF2 on γ-globin expression was observed in primary erythroid progenitors. To understand the role of ATF2 in γ-globin expression, chromatographically purified G-CRE/ATF2-interacting proteins were subjected to mass spectrometry analysis; major binding partners included CREB1, cJun, Brg1, and histone deacetylases among others. Immunoprecipitation assays demonstrated interaction of these proteins with ATF2 and <i>in vivo</i> GCRE binding in CD34⁺ cells undergoing erythroid differentiation which was correlated with γ-globin expression during development. These results suggest synergism between developmental stage-specific recruitments of the ATF2 protein complex and expression of γ-globin during erythropoiesis. Microarray studies in K562 cells support ATF2 plays diverse roles in hematopoiesis and chromatin remodeling.</p></div

IPA analysis of ATF2-related molecular and cellular functions.

<p>IPA analysis of ATF2-related molecular and cellular functions.</p

Characterization of the G-CRE multi-protein complex.

<p>(<b>A</b>) A schematic diagram showing the purification protocol used to identify the G-CRE protein complex using nuclear extracts prepared from pCI-neo-ATF2 stable cells is shown (Material and Methods). Fractions from the 0.6 M NaCl elution were pooled and applied to a HisTALON and subsequently wild-type G-CRE oligonucleotide columns. The 0.4 M NaCl elution from the wild-type G-CRE column was subjected to MS/MS analysis. (<b>B</b>) Shown are the results for the EMSA with various protein fractions prior to and after affinity column purification. The following samples were tested: pooled fraction prior to the G-CRE affinity column (Input), pooled fractions at 0.4 M NaCl (0.4 M NaCl), fraction from the 0.2 M NaCl (Wash) and the unbound protein flow through (FT). The arrows indicate the ATF2 complex and non-specific band (NS). (<b>C</b>) Proteins associated to the G-CRE were purified, run on a 10% SDS-PAGE gel (shown in image) and stained with colloidal blue; the bands were excised, subjected to trypsin digestion and proteins identified by mass spectrometry. (<b>D</b>) Sucrose gradient was performed to demonstrate the association of the G-CRE proteins as a homogenous complex. Ovalbumin (3.5 S, 45 kDa), BSA (4.3 S, 69 kDa), Aldolase (7.3 S, 150 kDa), Catalase (11.3 S, 250 kDa), Thyroglobulin (19.2 S, 700 kDa) and Blue Dextran (2 mDa) were used as standard size markers and loaded in identical parallel gradient. Thirty micro-liters of fractions 10 through 17 were subjected to a 4–15% pre-casted acrylamide gel and analyzed by western blot. (<b>E</b>) Nuclear extracts prepared from K562 cells were IP with IgG (control), ATF2 and Brg1 antibodies followed by western blot.</p

Enrichment of bZIP proteins in the G-CRE during erythroid differentiation.

<p>Primary erythroid progenitors were generated in a two-phase <i>in vitro</i> expansion culture (Material and Methods). (<b>A</b>) ChIP assay was performed using control IgG, ATF2, CREB1 and cJun antibody. Purified chromatin was quantified by qPCR with G-CRE specific primer. ΔΔCT method was used to calculate the fold change of ATF2-, CREB1- and cJun-specific G-CRE enrichment. Results are represented as mean of two biological replicates with each having 2 technical replicates; shown is the mean ± SD. (<b>B</b>) Cellular lysates were prepared from primary cells and 50 µg of protein was IP with anti-CREB1 antibody. Control IgG was used as a background control. Precipitants were detected by western blot with the antibodies shown.</p

Mass spectrometry analysis of protein interacting with G-CRE affinity column after NaB treated in K562 cells.

<p>Functional classification of the GCRE multi-protein complex identified by MS/MS analysis.</p

ATF2 is a positive regulator of γ-globin expression in primary cells.

<p>Primary erythroid progenitors were cultured in a two-phase liquid system and transfected with Scr siRNA or siATF2. pMaxGFP was used to monitor transfection efficiency on day 11 (Materials and Methods). (<b>A</b>) The graph shows RT-qPCR analysis of ATF2 mRNA for three independent experiments. Western blot was conducted with ATF2 and β-actin antibody; a representative gel is shown. Abbreviations: UN, untransfected cells. (<b>B</b>) RT-qPCR analysis was completed for γ-globin and β-globin genes expression in primary cells. The fold change of γ-globin/GAPD and β-globin/GAPD was calculated for three independent experiments. (<b>C</b>) Cellular lysates from the siRNA-transfected cells were used for ELISA to measure HbF levels. The relative level of HbF/total Hb/total protein was calculated.</p

Genes affected by siATF2 treatment in K562 cells.

<p>Genes affected by siATF2 treatment in K562 cells.</p

ChIP assay demonstrate <i>in vivo</i> binding of protein in the G-CRE complex.

<p>K562 cells were treated with 2-chromatin complexes were IP with the antibodies indicated and qPCR analysis completed. (<b>A</b>) Shown in the graph is steady-state binding of the factors to the G-CRE in K562 cells. (<b>B</b>) The G-CRE chromatin enrichment for NaB-treated samples was studied. The white bars represent untreated and black bars showing data for NaB-treated K562 cells. (<b>C</b>) Similar studies were conducted for HS2. Baseline HS2 chromatin fold enrichment is shown in the graph. (<b>D</b>) HS2 chromatin enrichment for NaB-treated (black bars) compared to untreated (white bars) cells is shown. (<b>E</b>) Oligonucleotides pull-down assay. K562 cells were treated with 2 mM NaB and nuclear extracts prepared. Biotinylated probes were used to pull-down the G-CRE interacting proteins using streptavidin agarose beads. Two oligonucleotides were investigated - wild type G-CRE (G-CREwt) and mutant G-CRE (G-CREmt). Nuclear extracts (50 µg) from the different conditions were loaded as Input NE.</p

siATF2 treatment decreases γ-globin gene expression in K562 cells.

<p>(<b>A</b>) Transient transfections of K562 cells with a scrambled siRNA molecule (Scr, 50 nM) or siATF2 were performed. Total RNA and whole cell lysates were prepared and subjected to RT-qPCR and western blot respectively. The top graph shows the ATF2 levels for the mean ± SEM from three independent experiments. **p<0.01 and ***p<0.001. Shown on the bottom is a representative gel for western blot with the antibodies indicated. (<b>B</b>) EMSA assay was performed using nuclear extracts from untreated (Control NE), NaB-treated (NaB-NE), Scr (Scr-NE) or siATF2 (siATF2-NE) treated K562 cells. Antibody-mediated supershift studies were conducted using NaB-NE with anti-ATF2 (NaB+α-ATF2 Ab) or without ATF2 antibody (NaB+control Ab). The ATF2-containing protein-DNA complex is indicated along with non-specific (NS) bands. (<b>C</b>) γ-Globin expression in siATF2 treated cells was determined by RT-qPCR analysis. (<b>D</b>) RT-qPCR was performed for RNA samples prepared from K562, pCI-neo and pCI-neo-ATF2 stable cells. Copy number of stably integrated ATF2 was determined using an approach previously published from our lab <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078253#pone.0078253-Yao1" target="_blank">[20]</a>. (<b>E</b>) Western blot (WB) results for K562 and ATF2 stable lines analyzed with anti-FLAG-ATF2 and actin antibodies are shown. The relative γ-globin/GAPD ratio in the ATF2 stable cells was calculated after subtracting the γ-globin/GAPD value produced in the empty vector pCI-neo stable line. (<b>F</b>) The pCI-neo-ATF2 stable cells were transfected with siATF2 and western blot was performed (see Materials and Methods). Shown are representative gels with anti-FLAG and actin antibody for two independent experiments. (<b>G</b>) RT-qPCR analysis was performed to determine the levels of γ-globin expression after siRNA transfection of the pCI-neo-ATF2 stable line.</p