Distinct domains of erythroid Krüppel-like factor modulate chromatin remodeling and transactivation at the endogenous beta-globin gene promoter

Abstract

Characterization of the mechanism(s) of action of trans-acting factors in higher eukaryotes requires the establishment of cellular models that test their function at endogenous target gene regulatory elements. Erythroid Krüppel-like factor (EKLF) is essential for beta-globin gene transcription. To elucidate the in vivo determinants leading to transcription of the adult beta-globin gene, functional domains of EKLF were examined in the context of chromatin remodeling and transcriptional activation at the endogenous locus. Human EKLF (hEKLF) sequences, linked to an estrogen-responsive domain, were studied with an erythroblast cell line lacking endogenous EKLF expression (J2eDeltaeklf). J2eDeltaeklf cells transduced with hEKLF demonstrated a dose-dependent rescue of beta-globin transcription in the presence of inducing ligand. Further analysis using a series of amino-terminal truncation mutants of hEKLF identified a distinct internal domain, which is sufficient for transactivation. Interestingly, studies of the chromatin structure of the beta-promoter revealed that a smaller carboxy-terminal domain generated an open promoter configuration. In vitro and in vivo binding studies demonstrated that this region interacted with BRG1, a component of the SWI/SNF chromatin remodeling complex. However, further study revealed that BRG1 interacted with an even smaller domain of EKLF, suggesting that additional protein interactions are required for chromatin remodeling at the endogenous beta-promoter. Taken together, our findings support a stepwise process of chromatin remodeling and coactivator recruitment to the beta-globin promoter in vivo. The J2eDeltaeklf inducible hEKLF system will be a valuable tool for further characterizing the temporal series of events required for endogenous beta-globin gene transcription.

FIG. 1.

Exogenous expression of human EKLF-ER rescues β-globin expression in J2eΔeklf null cells. The coding region of hEKLF was fused in frame to the tamoxifen response element ER in a retroviral GFP⁺ cloning vector (see Materials and Methods). Viral supernatants were used to transduce the hEKLF-ER sequence into J2eΔeklf cells. A pool of GFP⁺ cells was selected by FACS and then characterized for hEKLF expression and function. (A) Schematic of the retroviral construct utilized to test hEKLF function in J2eΔeklf cells. Note that the hEKLF cDNA was fused in frame with an HA epitope tag at the NH₂ terminus to facilitate immunological detection and with the ligand-binding domain of the estrogen receptor (ER^TM) at the carboxy terminus to facilitate functional control. (B) EKLF expression increases with tamoxifen induction. Crude nuclear extracts were prepared from J2eΔeklf/irGFP (lane 1) and J2eΔeklf/ flEKLF cells after 48 h in growth media with varied tamoxifen concentrations (0, 10⁻³, 10⁻², 10⁻¹, 10¹, 10², and 10³, lanes 2 to 8, respectively) and analyzed by immunoblotting with HA-specific antisera. To test for loading equivalency, the blot was stripped and reprobed with anti-PCNA antibody. (C) An increase in βmaj transcripts correlates with increasing EKLF expression. Total RNA was isolated from the J2eΔeklf/flEKLF cells and subjected to RPA, utilizing βmaj and α-globin antisense riboprobes.

FIG. 2.

Expression of the EKLF cDNA results in restoration of DNase I hypersensitivity at the βmaj promoter. (A) Schematic representation of HS formation at the βmaj promoter. (B) Induction of DNase I hypersensitivity by tamoxifen treatment of J2eΔeklf/flEKLF cells. Cells were cultured for 48 h in the absence (−) or presence (+) of tamoxifen. Nuclei were isolated and exposed to increasing concentrations of DNase I as previously described. To determine DNase sensitivity of βmaj, DNA was harvested, digested with EcoRI, and probed with a βmaj promoter-specific probe. For DNase sensitivity of ala-s, DNA was digested with BamHI and probed with an ala-s promoter-specific probe. The DNase I concentration is 0 in the first lane of each panel and increases as shown by the shaded triangle. The autoradiographs are representative of three independent experiments.

FIG. 3.

Generation of a series of β-CACC-binding hEKLF mutants. (A) Schematic representation of EKLF mutants tested. NH₂-terminal mutants were chosen based on functional properties of EKLF defined in in vitro studies. Construction of these mutants is detailed in Materials and Methods. Above the diagram are the known protein-protein interactions of EKLF. The dotted area indicates the zinc finger DNA binding domain. (B) All amino-terminal mutants of EKLF bind the cognate βmaj CACC motif. Equal amounts of bacterially expressed recombinant EKLF polypeptides were incubated with a ³²P-radiolabeled β-CACC probe for 30 min at room temperature. The products of this reaction were electrophoresed through a 4% polyacrylamide gel and then gel dried and subjected to autoradiography. The representative autoradiograph shows an electrophoretic mobility shift assay for each mutant.

FIG. 4.

The DNA binding domain is required but is not sufficient for chromatin remodeling at the βmaj globin promoter. (A) Schematic diagram of flEKLF retrovirus and derivative mutants used to stably transduce J2eΔeklf cells. (B) Retrovirus-mediated transfer into J2eΔeklf cells results in comparable levels of EKLF transgene expression. Nuclear extracts were prepared from individual pools of J2eΔeklf cells transduced with EKLF mutant retroviruses after 48 h of culturing in the presence of tamoxifen. Equal amounts of protein were separated on SDS-PAGE gels, blotted, and probed with HA-specific antisera. Blots were subsequently stripped and reprobed with p38-specific antisera to confirm equivalency of gel loading. (C) Induction of DNase I hypersensitivity at the β-globin promoter requires a large carboxy-terminal domain. Cells expressing each EKLF mutant were cultured for 48 h in the presence of tamoxifen. Nuclei were isolated and exposed to increasing concentrations of DNase I as previously described. DNA was harvested, digested with EcoRI, and probed with a βmaj-specific probe. The DNase I concentration is 0 in the first lane of each panel and increases as shown by the shaded triangle. The autoradiograph is representative of several independent experiments.

FIG. 5.

BRG1, the core component of the SWI/SNF complex E-RC1, requires the zinc finger region of hEKLF for maximal interaction in vitro and in vivo. (A) The hEKLF DNA binding region directly interacts with BRG1. EKLF mutant polypeptides fused to GST or GST protein alone bound to glutathione beads was incubated with equal amounts of in vitro-translated ³⁵S-methionine-labeled BRG1. Beads were extensively washed, and bound protein was revealed by SDS-PAGE and fluorography. (B) A similar pattern of EKLF-BRG1 interaction is observed in vivo. SW13 cells were cotransfected with BRG1- and EKLF-expressing plasmids. Nuclear extracts were prepared 48 h posttransfection and incubated with HA-epitope tag antisera. Interacting proteins were precipitated, and the products were separated by SDS-PAGE. After blotting to nylon filters was done, BRG1-specific antiserum was utilized to detect the presence of EKLF-BRG1 specific interactions.

FIG. 6.

An internal domain of hEKLF is sufficient to activate β-globin gene expression to wild-type levels. (A) Addition of amino acids 164 to 221 is sufficient to confer wild-type activation potential on the DNA binding (Δ253EKLF) and chromatin remodeling (Δ221EKLF) domains of EKLF. RNA was harvested from J2eΔeklf clones expressing varying EKLF mutant moieties at 48 h post-tamoxifen induction. RPA was performed utilizing βmaj and α riboprobes. The numbers underneath the panel represent the mean fold induction of the β/α ratio for each construct assayed, using three independent experiments and the activity in cells transduced by vector alone as baseline. (B) The identical domain of hEKLF activates a β-promoter-regulated reporter gene in transient assays. EKLF mutant constructs lacking the ER element were subcloned into a mammalian expression vector. These constructs were cotransfected individually into K562 cells, along with a human β-globin promoter construct, HS2βLuc, and a Renilla luciferase control plasmid. Luciferase activity was determined by luminometry 48 h posttransfection. Fold activation of each construct is represented with respect to a control transfection of HS2βLuc and the empty expression vector. The effect on HS2β luciferase activity for each hEKLF construct was measured in six separate assays of three independent transfections. The results of the Renilla luciferase analysis were utilized to correct for variation in transfection efficiency.

FIG. 7.

BRG1 is required for EKLF function in vivo. (A) BRG1 is required for transactivation of an EKLF-dependent promoter. Plasmids expressing full-length EKLF or amino-terminal mutants of EKLF were cotransfected into SW13 cells with either wt BRG1-expressing plasmid (black bars) or the derivative pΔBRG1 alone (white bars). In addition, each transfection contained the HS2βLuc reporter construct. Luciferase activity was determined by luminometry 48 h posttransfection. Fold activation of each construct is represented with respect to a control transfection of HS2βLuc and the empty expression vector. The graph reflects HS2β luciferase activation for each hEKLF construct, measured in six separate assays of three independent transfections. (B) Dominant-negative form of BRG1 (DN-BRG1) represses specifically EKLF-dependent transcription in fetal erythroblasts. J2eΔeklf/flEKLF-irGFP/DN-BRG1-irYFP cells were generated in a fashion similar to that described for J2eΔeklf/flEKLF-irGFP cells (see Fig. 1 and Material and Methods). Total cellular RNA was harvested 48 h post-tamoxifen induction, and RPA was performed using βmaj, βhl, and α riboprobes. The autoradiograph represents the level of each transcript detected for the J2eΔeklf lines. Asterisks, nonspecific bands.