Stage-specific repression by the EKLF transcriptional activator

Abstract

Dynamic changes in transcription factor function can be mediated by switching its interaction with coactivators and corepressors. Erythroid Kruppel-like factor (EKLF) is an erythroid cell-specific transcription factor that plays a critical role in beta-globin gene activation via its interactions with CBP/p300 and SWI/SNF proteins. Unexpectedly, it also interacts with Sin3A and histone deacetylase 1 (HDAC1) corepressors via its zinc finger domain. We now find that selected point mutants can uncouple activation and repression and that an intact finger structure is not required for interactions with Sin3A/HDAC1 or for transrepression. Most intriguingly, EKLF repression exhibits stage specificity, with reversible EKLF-Sin3A interactions playing a key role in this process. Finally, we have located a key lysine residue that is both a substrate for CBP acetylation and required for Sin3A interaction. These data suggest a model whereby the stage of the erythroid cell alters the acetylation status of EKLF and plays a critical role in directing its coactivator-corepressor interactions and downstream transcriptional effects.

FIG. 1.

Characterization of EKLF derivatives that are deficient in activation yet retain repression. (A) DNA binding (left) and transactivation (right) by wild-type EKLF (lane E), single-zinc-finger mutants (MZn1, MZn2, and MZn3), or a triple mutant (MZn1-3) were tested in vitro by a gel shift assay on a β-globin promoter CACCC element oligonucleotide (left) and in vivo by cotransfection with the natural β-globin promoter-luciferase reporter into K562 cells (right). Extracts for gel shift analysis were prepared from COS7 cells after transfection with the indicated constructs. The arrow indicates the novel shift seen upon EKLF transfection, and the protein levels of the wild type and mutant constructs are shown by anti-EKLF Western blot analysis below each lane. pSG5 is the empty EKLF expression vector (lane C). Luciferase activity in extracts was normalized to cotransfected growth hormone. IP, immunoprecipitation. (B) The wild type or mutants with EKLF mutations in each (MZn1, MZn2, and MZn3) or all (MZn1-3) of the zinc fingers were cotransfected into K562 cells with myc-Sin3A. Extracts were immunoprecipitated with anti-EKLF and blotted and probed with anti-myc after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A portion of the extract was saved as supernatant to monitor myc-Sin3A expression levels. pSG5 is the empty EKLF expression vector. (C) The wild type or EKLF mutants with mutations in each (MZn1, MZn2, and MZn3) or all (MZn1-3) of the zinc fingers or with deletion of all the zinc fingers (P) were cotransfected into K562 cells with HDAC1. Extracts were immunoprecipitated with anti-HDAC1, blotted, and probed with anti-EKLF after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A portion of the extract was saved as supernatant to monitor EKLF expression levels. pBJ5 is the empty HDAC1 expression vector. (D) Transrepression by wild-type GAL/EKLF, single-zinc-finger mutants (MZn1, MZn2, and MZn3), or triple mutant (MZn1-3) derivatives was tested by cotransfection with the pG5tkCAT reporter in K562 cells. Cotransfection with GAL 1-147 provides the baseline (nonrepressed) reporter activity. Chloramphenicol acetyltransferase (CAT) activity in extracts was normalized to cotransfected growth hormone.

FIG. 2.

Indirect interaction between EKLF and HDAC1. In vitro pull-down assays were performed with GST or GST/EKLF that was incubated with radiolabeled, in vitro-translated HDAC1 (top), in vitro-translated FL/HDAC1 (middle), or purified FL/HDAC1 (bottom). The top panel is the autoradiograph, and the other two panels are from Western blot analysis with anti-FLAG antibodies.

FIG. 3.

Selection and testing of an EKLF derivative that is deficient in repression yet retains activation. (A) A space-filling molecular model (17) of the three EKLF zinc fingers interacting with the double-strand CAC oligonucleotide (G-rich strand in red and C-rich strand in gold) is shown. The three XYZ amino acids (30) critical for binding within zinc finger 1 (K306, yellow; H309, green; A312, light blue) and the location of K302 (dark blue) are as indicated. (B) DNA binding (left) and transactivation (right) by wild-type (Wt) EKLF and K302R mutant EKLF were tested in vitro by a gel shift assay on a β-globin promoter CACCC element oligonucleotide (left) and in vivo by cotransfection with the natural β-globin promoter-luciferase (LUC) reporter into K562 cells (right). Extracts for gel shift analysis were prepared from COS7 cells after transfection with the indicated constructs. The novel shift seen upon EKLF transfection is indicated on the left. pSG5 is the empty EKLF expression vector. Luciferase activity in extracts was normalized to cotransfected growth hormone. (C) The wild type (WT) or K302R EKLF mutant was cotransfected into K562 cells with myc-Sin3A. Extracts were immunoprecipitated (IP) with anti-EKLF, blotted, and probed with anti-myc or anti-EKLF (as indicated) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A portion of the extract was saved as supernatant to monitor myc-Sin3A and EKLF expression levels. pSG5 is the empty EKLF expression vector. (D) Transrepression by wild-type GAL/EKLF, GAL/ZnF, or the GAL/EKLF K302R mutant was tested by cotransfection with the pG5tkCAT reporter in K562 cells. Cotransfection with GAL 1-147 provides the baseline (nonrepressed) reporter activity. Chloramphenicol acetyltransferase (CAT) activity in extracts was normalized to cotransfected growth hormone.

FIG. 4.

Cell and stage specificity of EKLF transrepression. (A) Transrepression by wild-type GAL/EKLF was tested in K562 (human fetal-type erythroid), NIH 3T3 (murine nonerythroid), and MEL (murine adult-type erythroid) cells and compared to basal levels of reporter activity after cotransfection with GAL 1-147. Chloramphenicol acetyltransferase (CAT) activity in extracts was normalized to cotransfected growth hormone. (B) HOX11-immortalized erythroid cells (EBHX11) growing in LIF (11L; left panel) or Epo (llE; right panel) were transfected with pG5tkCAT and GAL 1-147 or GAL/EKLF. The statuses of embryonic (βh1) and adult (βmaj) globins in these two cell lines are indicated below each panel. Chloramphenicol acetyltransferase activity in extracts was normalized to cotransfected growth hormone.

FIG. 5.

Endogenous EKLF-Sin3A protein interactions in EBHX11 cells grown under different conditions. Extracts were generated from EBHX11 cells grown in Epo (Hox11E) or LIF (Hox11L) or after cytokine replacement (2 days) as indicated. Extracts were immunoprecipitated (IP) and probed after Western blotting with anti-EKLF or -Sin3A antibodies as indicated. The statuses of adult βmaj (βm) globins in these two cell lines under the different conditions are indicated above each panel. The asterisk indicates a nonspecific signal on the Western blot.

FIG. 6.

Model for EKLF activation or repression. EKLF is modified by p300/CBP at two sites: K288 and K302. K288ac may play a role in optimal interaction with SWI/SNF via BRG1. At the same time, K302 is critical for EKLF-Sin3A interaction, raising questions about potential cross-regulation between these two pathways and the downstream targets that may be affected. The possibility that acetylated EKLF (K302ac) binds with higher affinity to HDACs and is deacetylated as a result of this interaction has not been tested but is also implied by this scheme.