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. 1998 Aug 18;95(17):9855-60.
doi: 10.1073/pnas.95.17.9855.

Acetylation and modulation of erythroid Krüppel-like factor (EKLF) activity by interaction with histone acetyltransferases

W Zhang  1 , J J Bieker
Affiliations

Acetylation and modulation of erythroid Krüppel-like factor (EKLF) activity by interaction with histone acetyltransferases

W Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Erythroid Krüppel-like factor (EKLF) is a red cell-specific transcriptional activator that is crucial for consolidating the switch to high levels of adult beta-globin expression during erythroid ontogeny. EKLF is required for integrity of the chromatin structure at the beta-like globin locus, and it interacts with a positive-acting factor in vivo. We find that EKLF is an acetylated transcription factor, and that it interacts in vivo with CBP, p300, and P/CAF. However, its interactions with these histone acetyltransferases are not equivalent, as CBP and p300, but not P/CAF, utilize EKLF as a substrate for in vitro acetylation within its trans-activation region. The functional effects of these interactions are that CBP and p300, but not P/CAF, enhance EKLF's transcriptional activation of the beta-globin promoter in erythroid cells. These results establish EKLF as a tissue-specific transcription factor that undergoes post-translational acetylation and suggest a mechanism by which EKLF is able to alter chromatin structure and induce beta-globin expression within the beta-like globin cluster.

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Figures

Figure 1
Figure 1
Tests of EKLF interaction with CBP, p300, and P/CAF in vivo. Combinations of EKLF and HAT coactivator expression plasmids (10 μg each) as indicated were transfected into COS7 cells and whole cell extracts were subjected to immunoprecipitation with anti-CBP, anti-HA, anti-P/CAF, or anti-EKLF antibodies. (A) Immunoprecipitated complexes were resolved, blotted, and probed with anti-EKLF monoclonal antibody 4B9. Location of co-electrophoresed EKLF in each gel is shown. Asterisks indicate nonspecific (Ig heavy chain) signals from the immunoprecipitating antibodies. (B) Immunoprecipitated complexes were resolved, blotted, and probed with anti-HA monoclonal antibody for detection of p300. Location of co-electrophoresed HA-p300 is shown.
Figure 2
Figure 2
Tests of EKLF acetylation by CBP, p300, and P/CAF in vitro. (A) COS7 cells were transfected and extracts were immunoprecipitated with the indicated antibodies. IP-HAT assays utilized 5 μg of GST-EKLF(76–376). Samples were electrophoresed and the dried gel was exposed in autoradiography (Upper). GST-EKLF(76–376) alone was tested for autoacetylation in lane 9. Locations of p300/CBP, P/CAF, and GST-EKLF are indicated. Protein samples were also stained to show that equivalent amounts were used (Lower). (B) Endogenous CBP from COS7 cells was immunoprecipitated, and IP-HAT assays were performed with 5 μg of various GST-EKLF fusion proteins as diagrammed on the Right, which also shows the locations and sequences of lysines conserved between mouse and human EKLF [amino acid residues 47, 74, 177, 279, 288; mouse numbering is based on initiator methionine being residue 19 (9)]. Proteins were resolved and subjected to autoradiography (Upper Left) or stained for protein (Lower Left). Asterisks show the location of nondegraded GST-EKLF fusion proteins. Molecular mass markers (on the left) are 70, 55, 33, 25, and 15 kDa (top to bottom).
Figure 3
Figure 3
Status of EKLF acetylation in vivo. COS7 cells transfected with 10 μg of pSG5 or pSG5/EKLF as indicated were labeled with sodium [3H]acetate in the presence of TSA and extracts were immunoprecipitated with anti-EKLF monoclonal antibody 6B3. Immunoprecipitated samples were resolved on SDS/PAGE and blotted and probed with anti-EKLF (Left) or processed and exposed in autoradiography (Right). Asterisk indicates a nonspecific signal from the immunoprecipitating antibody. Locations of molecular mass markers (70, 50, and 33 kDa, top to bottom) and EKLF are shown.
Figure 4
Figure 4
Analyses of EKLF transactivation of the β-globin promoter. K562 erythroleukemic cells were transfected with 1 μg of HS2/β/luc reporter and the indicated amounts (μg) of expression or vector plasmids, and extracts were processed for luciferase activity. TSA was added where indicated to a final concentration of 2 μM at the time of transfection. Multiple experiments were averaged after normalization of luciferase activity to cotransfected growth hormone levels. The effects of CBP (A), p300 (C), and P/CAF (D) are shown along with a dose–response test of CBP (B).
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