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Blood, 15 April 2005, Vol. 105, No. 8, pp. 3330-3339. Prepublished online as a Blood First Edition Paper on December 23, 2004; DOI 10.1182/blood-2004-08-2988.
RED CELLS SATB1 family protein expressed during early erythroid differentiation modifies globin gene expressionFrom the Laboratory of Chemical Biology and Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD; and Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, CA.
Special AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with  -globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases  -globin and down-regulates SATB1 family protein and -globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the  -globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the -globin promoter. SATB1 overexpression increases -globin and decreases -globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the -globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the -globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in -promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the -globin gene by interaction with specific sites in the -globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation. (Blood. 2005;105:3330-3339)
The human -globin gene cluster on chromosome 11 consists of 5 developmentally specific genes for embryonic (), fetal (G, A), and adult ( , ) globins. A strong enhancer, located in the far upstream region of the cluster called the locus control region (LCR), contains 5 DNase I hypersensitive (HS) sites and is able to enhance tissue-specific globin gene expression and provide a high level of transcription activity from human globin gene constructs in transgenic mice. Transcription factors such as erythroid Krüppel-like factor (EKLF), GATA-1, and NF-E2, that bind to the LCR and other regulatory elements, and promoters in the globin gene locus, have been reported to regulate chromatin histone acetylation by associating with histone acetyltransferases.1-3 The LCR is required to increase the rate of transcription but may be dispensable for formation of an open chromatin domain of a downstream active globin gene in erythroid cells.4,5 For globin gene expression, spatial organization of the -globin cluster requires special interactions between distal transcriptional elements in the LCR and downstream active globin genes. Some developmental specificity between individual hypersensitive sites in the LCR and downstream globin genes is evident such as the interaction between HS2 and -globin for transcription activation.6
Complex packaging of eukaryotic chromosomes in nuclei creates chromatin loops and matrix/scaffold attachment regions (MARs/SARs; the term MARs is used here), originally identified as gDNA fragments that remain tightly associated with salt-extracted and DNase I-digested nuclei, have been postulated to be localized at the base of chromatin loops.7 MARs identified by such criteria often contain a base-unpairing region (BUR), the DNA bases of which become continuously unpaired when subjected to negative superhelical strain.8,9 Many candidate MARs in the
Special AT-rich binding protein 1 (SATB1) binds to double-stranded BUR sequences, specifically recognizing a specialized DNA context (an ATC sequence context), characterized by a cluster of sequence stretches with well-mixed As and Ts but either Cs or Gs exclusively on one strand (designated as ATC sequences).17 SATB1 has roles in tissue-specific organization of DNA sequences, in regulation of gene expression by acting as a "landing platform" for chromatin-remodeling enzymes and in designation of the region-specific histone modification in vivo.18,19 In vitro studies have indicated that SATB1 family protein can bind to some of the MARs in the
Cell culture Human erythroleukemia K562 cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1650/10% fetal bovine serum.20 Human primary erythroid progenitor cells were purified using Ficoll-Hypaque (BioWhittaker, Walkersville, MD) from blood obtained from consenting healthy volunteers through the National Institutes of Health (NIH) Department of Transfusion Medicine and cultured as described.21 Approval was obtained from the NIH institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki. Cell transfection
An SATB1 expression vector was constructed by excising SATB1 cDNA (EcoR1) from pECHAT1146,17 ligating into pIRES2-EGFP (Clontech, Palo Alto, CA) to give pEGFP/SATB1; accuracy was confirmed by DNA sequencing. For stable cell lines, pEGFP/SATB1 or pIRES2-EGFP was transfected by electroporation into K562 cells.22 Clones were selected using geneticin (500 µg/mL; Gibco, Grand Island, NY). For reporter gene assays, 5.0 x 105 HeLa cells or 5.0 x 106 K562 cells were transfected using Superfect reagent (Qiagen, Valencia, CA). After 72 hours, cells were harvested and assayed. PSV- Reporter gene construction
The RNA isolation and quantitative RT-PCR analysis
Total RNA was isolated and first-strand cDNA was synthesized using MuLV reverse transcriptase (RT) and oligo-d (T)16 (Applied Biosystems, Foster City, CA). For quantitative real-time polymerase chain reaction (PCR) analysis, gene-specific primers and fluorescent labeled TaqMan probes (6-carboxy fluorescein [FAM] as the 5' fluorescent reporter, tetramethylrhodamine [TAMRA] as 3' end quencher) were used in a 7700 Sequence Detector (Applied Biosystems, Foster City, CA) as described.24 All results were normalized to human Western blot analysis Cell lysates were obtained by adding RIPA buffer (10 mM Tris [tris(hydroxymethyl)aminomethane] HCl, 1 mM EDTA [ethylenediaminetetraacetic acid], 0.1% sodium dodecyl sulfate [SDS], 0.1% Na3VO4, 1% Triton-X 100) and protease inhibitor (Roche Diagnostics, Mannheim, Germany) into the cell pellet, incubated on ice for 30 minutes and centrifuged at 17 000g for 10 minutes. The protein sample was run on NuPAGE 4% to 12% Bis-Tris Gel (Invitrogen, Carlsbad, CA) for 1 hour at 200 V. Protein was transferred to nitrocellulose by standard methods. The blot was blocked with 5% nonfat milk in Tween 20-Tris-buffered saline (TTBS) buffer for 1 hour at room temperature, probed with primary antibody for 1 hour at room temperature, washed in TTBS buffer, probed with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature, and rinsed in TTBS buffer for chemiluminescent detection. Nuclear extract isolation and DNA-binding assay
K562 and K562/SATB1 nuclear extracts were prepared for electromobility shift assay (EMSA). Nuclei were extracted from washed cells using hypotonic buffer (10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT], and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) and centrifuged. The cytoplasm containing supernatant was discarded, the pellet resuspended in 2 x volume of extraction buffer (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF), placed on ice for 30 minutes, and centrifuged (10 000rpm; 10 minutes), and the supernatant containing the nuclear extract collected. For EMSA, DNA probes were labeled with ChIP Chromatin extracts were prepared as described.25 In brief, 2 x 107 cells were fixed with formaldehyde and incubated at 37°C for 3 minutes, washed in phosphate-buffered saline (PBS), resuspended in 15 mL Triton buffer (0.1 M Tris HCl, 0.05 M EDTA, 0.01 M EGTA [ethylene glycol tetraacetic acid], 0.25% Triton X-100) and incubated for 15 minutes. Triton-washed cells were centrifuged (1000g for 5 minutes), resuspended in 15 mL NaCl buffer (0.1 M Tris HCl, 0.01 M EGTA, 0.05 M EDTA, 5 M NaCl), and incubated for an additional 15 minutes. The samples were centrifuged, resuspended in 1 mL sonication buffer (0.1 M Tris HCl, 0.05 M EDTA, 0.01 M EGTA, 1% SDS), and sonicated for 10 bursts of 10 seconds. Cell debris was removed by centrifugation (17 000g for 5 minutes) and supernatant stored at -80°C as chromatin extracts. For chromatin immunoprecipitation (ChIP) analysis, specific antibodies against SATB1, acetylated or methylated isoforms of histone 3 and 4 (Upstate Biotechnology, Lake Placid, NY), or preimmune serum and 40 µL protein A-Sepharose suspension were added to the chromatin extract, incubated at 4°C overnight, and washed. Bound and input chromatin samples were placed in 0.5% (wt/vol) SDS and incubated overnight at 65°C to reverse the formaldehyde cross-linking. DNA was further purified by phenol-chloroform extraction and ethanol precipitated using glycogen (10 µg) as a carrier. For DNA sequence-specific quantification by real-time PCR, primers and fluorescent-labeled TaqMan probes were used. DNA (2 ng) from the ChIP selected (IP) fraction and 2 ng gDNA as a reference control (Ref) were used as templates. Using sequence-specific primers and TaqMan probes for quantitative real-time PCR, at low amplification the threshold cycle number (Ct) is directly proportional to the amount of corresponding specific DNA in the sample and is in the linear range. Each cycle represents a 2 x amplification of the amount of product. Enrichment of the specific sequences in IP was compared with Ref and calculated from the difference of the threshold cycle number (Ct) for the respective DNA pools. Specifically, IP/Ref = 2(Ct (Ref) - Ct (IP)) is used to determine the fold difference.26 Coimmunoprecipitation K562 nuclear extract (100 µg) was incubated with SATB1 or CBP antibodies, or preimmune serum and protein A-Agarose (Santa Cruz, Biotechnology, Santa Cruz, CA) in 1 mL binding buffer (0.5% NP-40, 10 mM Tris HCl, 150 mM NaCl, 2 mM EDTA, 10% glycerol, protease inhibitor) for 3 hours at 5°C. The reaction mixture was briefly centrifuged, the pellet washed in 1 mL binding buffer 3 times at 5°C for 5 minutes, and the immunoprecipitated SATB1/CBP or CBP/SATB1 complexes were separated on a polyacrylamide gel. Anti-SATB1 or anti-CBP antibody was used for Western blot analysis of bound proteins. Treatment with antisense oligonucleotide SATB1 sense and antisense oligonucleotides flanking the translation start site (5'-GCCTCGTTCAAATGATCCATACTCAGTC-3') were synthesized with a phosphorothioate backbone and purified by high-performance liquid chromatography (HPLC; Synthegen, Houston, TX). Fresh antisense or sense (control) oligonucleotide was added to the primary erythroid progenitor cells at day 1 and day 3 of phase 2 culture and the cells were harvested at day 5. Statistical methods Statistical analysis was carried out by standard methods. Error bars used throughout indicate SD of the mean.
SATB1 increases embryonic globin production
To investigate the influence of SATB1 on globin gene regulation, a SATB1 expression vector was stably transfected into K562 cells that endogenously express a low level (compared with T lymphocytes) of SATB1 (or its isoform),14,27 to generate K562/SATB1. Western blotting confirmed the increase in SATB1 expression (Figure 1A). Surprisingly, a red cell pellet clearly indicated a marked elevation in hemoglobin production in the K562/SATB1 cells without hemin (Figure 1A). Benzidine staining shows hemoglobinization increasing from 4% (control K562 population) to 48% in the K562/SATB1 cells (Figure 1B). Additional stable K562/SATB1 clones were isolated and analyzed. SATB1 levels, determined by Western blotting, correlated with hemoglobin production measured spectrophotometrically (Figure 1C). Because globin protein expression is primarily transcriptionally regulated, quantification of globin mRNA reflects the amount of globin produced. Globin gene expression was determined using gene-specific primers and TaqMan probes (Table 1). K562 cells express predominantly
We previously showed that increasing GATA-1 in K562 cells decreases -globin expression, whereas increasing GATA-2 increases - and -globin expression.28 SATB1 did not affect GATA-1 or GATA-2 production. Western blot analysis of K562, K562/SATB1, and K562 mock cells showed comparable amounts of GATA-1 and GATA-2 (Figure 1F). EMSA determined GATA-1 binding in nuclear extract from K562 cells and several clones of K562/SATB1 cells and indicated that increasing SATB1 only modestly affected GATA-1 binding to DNA in K562/SATB1 clones (Figure 1G, lanes 8-11), compared to the reported 10-fold or more increase by hemin.29-31 As controls for GATA-1 binding, cold probe (G) and GATA-1 antibody ( G) displaced GATA-1 binding, whereas a mutant GATA-1 competitor ( G) had no effect (Figure 1G, lanes 1-7). Hemin-induced erythroid differentiation
Hemin induction of K562 cell erythroid differentiation increases hemoglobin production and
SATB1 interacts with MARs localized in the -globin cluster in vivo
To identify new SATB1-binding sites in the
ChIP assay was used to assess SATB1 binding in living cells. SATB1-bound chromatin complexes from K562 cells were isolated using the anti-SATB1 antibody. We examined binding in vivo to ATC sequence-rich regions previously reported to bind to SATB1 in vitro or to nuclear matrix proteins as well as selected ATC sequence-rich regions from -globin 5' (39081-39310), -globin 5' (60661-60870), HS4 (853-1525), HS3 (4239-4909), and HS2 (M1 (8127-8327), M2 (8346-8546), M3 (8525-8725), M4 (8579-8797), and M5 (8832-9032) as shown in Figure 3A. Specific primer pairs and corresponding probes (Table 1) were used for quantitative real-time PCR analyses of chromatin fragments immunoprecipitated with the anti-SATB1 antibody (SATB1-ChIP DNA). All primer pairs yielded PCR products with control gDNA (Figure 3C,E-F). With SATB1-ChIP DNA, only HS2-M1 and M produced PCR products indicating SATB1 binding in vivo at these sites (Figure 3C, lanes 7, 12). No PCR products were produced using preimmune serum (Figure 3C, lanes 13-18). Increasing amounts of SATB1-ChIP DNA produced corresponding increases in PCR products for HS2-M1 and M (19149-19346) in the -globin 5' but not HS2-M2 primer pairs (Figure 3C, lanes 19-28). Quantitative real-time PCR demonstrated an increased binding of M1 and M to SATB1 in the K562/SATB1 cells compared with K562 cells (Figure 3D). No binding of SATB1 was observed for MARs in the 3' A-globin enhancer region (1 [41334-41441] and 2 [41549-41654]) or in the -globin IVS2 (63006-63114; Figure 3E), showing that SATB1 binding in vitro to the 3' A-globin enhancer or -globin IVS2 does not correlate with binding in living cells. In addition, no binding of SATB1 was observed for other ATC sequence-rich regions localized at HS3, HS4, A-globin 5' (-386 bp), and -globin 5' (-1526 bp; Figure 3F). SATB1 enhances transcriptional activity via binding to specific MARs
SATB1 activation of
These constructs were further analyzed in K562/SATB1 cells with elevated SATB1 expression (Figure 1B). Additional constructs were examined including direct mutation of M in pREP4/ to give pREP4/mut-, and mutation of M1 in pREP4/HS2- to give pREP4/mut-HS2-. Transfection of pREP4/ into K562/SATB1 cells resulted in robust transcription activity. This activity decreased by mutation of M in pREP4/mut- and was comparable to the reduction obtained with pREP4/. Addition of HS2 (pREP4/HS2-) increased transcription activity by 3.4-fold compared with pREP4/. The effect of mutating HS2-M1 was comparable to deletion of HS2-M1, and the transcription activity of pREP4/mut-HS2- and pREP4/HS2- was reduced to about 0.5 that of pREP4/HS2-. These results in the K562/SATB1 cells are comparable to those obtained for cotransfection of the reporter gene with the SATB1 expression vector in the control K562 cells.
SATB1 overexpression contributes to the formation of active chromatin structure at specific loci in the
We determined the acetylation and methylation states of core histones in HS2 and
Hemin induction decreases SATB1 immunoreactive protein with concomitant decreasing -globin expression and increasing -globin expression. ChIP analysis showed that hemin induction decreased histones H3 and H4 acetylation in chromatin associated with HS2 by 2-fold, and with the -globin promoter by 3- to 4-fold following 48 hours of hemin induction (Figure 5C). These changes, in addition to increases in associated H3-MeK9, are indicative of a shift of HS2 and the -globin promoter to a less transcriptionally active state, consistent with the reduced expression. Conversely, hemin increases H3 and H4 acetylation and decreases H3-MeK9 in chromatin associated with the -globin promoter, as expected for transcription activation and the marked increase in -globin expression (Figure 5C). CBP increases SATB1 transcriptional activity
CBP/p300 is known to interact with a variety of DNA-binding transcription factors and to possess intrinsic histone acetyltransferase activity.36,37 CBP cooperates with GATA-1 and is required for erythroid differentiation.38 Using coimmunoprecipitation analysis and antibodies specific for SATB1 and CBP, we found that a complex containing CBP and SATB1 in K562 cells could be immunoprecipitated by both CBP- and SATB1-specific antibodies but not by preimmune serum (Figure 6A, lanes 1-6). In contrast, no SATB1 and p300 protein complex in K562 cells was detected (Figure 6A, lane 8). E1A is known to be able to repress CBP transcriptional activity and has been used to test the requirement of CBP.39 In K562 cells, cotransfection of pEGFP/SATB1 and an E1A expression vector with pREP4/
Human primary erythroid progenitor cells
To determine expression of SATB1 during erythroid differentiation of human adult erythroid progenitor cells, human primary hematopoietic progenitor cells were isolated from peripheral blood and stimulated for erythroid differentiation. Western blotting with anti-SATB1 antibody detected protein early during erythroid differentiation at day 5 of erythropoietin stimulation (Figure 7A). By day 8 with erythropoietin, this protein band was markedly decreased to low levels. We have previously shown
To investigate the effect of SATB1 on globin gene transcription, a SATB1 expression vector was transfected into human primary adult erythroid progenitor cells, and the cells were harvested at day 8. Maintaining SATB1 expression at a high level beyond day 5 resulted in an increase in -globin expression (3-fold) with a reduction in -globin expression (3-fold; Figure 7C). These changes in -globin and -globin expression are concomitant with increases in histone H3 and H4 acetylation in the -globin promoter and with decreases in histone H3 and H4 acetylation in the -globin promoter (Figure 7D). These data suggest that, as observed in K562 cells, manipulation of the SATB1 level during differentiation of adult early erythroid progenitor cells can alter chromatin associated with -globin and -globin and change the balance of globin gene expression, particularly between -globin and -globin. To down-regulate SATB1 family protein expression, an SATB1 antisense oligonucleotide was synthesized. Transfection into cells down-regulated anti-SATB1 immunoreactive protein when compared with the sense control (Figure 7E). As observed with hemin induction in K562 cells, the antisense oligonucleotide resulted in a decrease in SATB1, a decrease in -globin, and an increase in -globin expression compared with the sense control (Figure 7E).
SATB1 is required for coordinating gene expression during T-cell development40 and can target multiple chromatin-remodeling complexes to specific genomic sites to regulate chromatin structure.18 We found that SATB1 family protein, which is expressed in K562 cells,27 is down-regulated by hemin induction, which is concomitant with increased -globin expression and decreased -globin expression. Conversely, increased SATB1 expression in transfected K562 cells results in activation of -globin and a decrease in -globin expression, with a marked induction of total hemoglobin production in the absence of hemin.
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Abstract
Introduction
) and cells stably transfected with a vector control (
; right panels as indicated). (E) For K562/SATB1 clones, 
)
(
), and 48 hours (
