The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [3 H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.

A triplet of adjacent, highly similar GT motifs in the phyA promoter of rice functions to support maximal expression of this gene. We have obtained a recombinant clone that encodes a full‐length nuclear protein, designated GT‐2, which binds specifically to these target sequences. This novel protein contains acidic, basic and proline‐ + glutamine‐rich regions, as well as two autonomous DNA‐binding domains, one NH2‐terminal and the other COOH‐terminal, that discriminate with high resolution between the three GT motifs. A duplicated sequence of 75 amino acids, present once in each DNA‐binding domain, appears likely to mediate DNA target element recognition. Each copy of this duplicated protein sequence is predicted to form three amphipathic alpha‐helices separated from each other by two short loops. The absence of sequence similarity to other known proteins suggests that this predicted structural unit, which we term the trihelix motif, might be representative of a new class of DNA‐binding proteins.

Factor VII is a vitamin K-dependent coagulation protein essential for proper hemostasis. The human Factor VII gene spans 13 kilobase pairs and is located on chromosome 13 just 2.8 kilobase pairs 5 to the Factor X gene. In this report, we show that Factor VII transcripts are restricted to the liver and that steady state levels of mRNA are much lower than those of Factor X. The major transcription start site is mapped at ؊51 by RNase protection assay and primer extension experiments. The first 185 base pairs 5 of the translation start site are sufficient to confer maximal promoter activity in HepG2 cells. Protein binding sites are identified at nucleotides ؊51 to ؊32, ؊63 to ؊58, ؊108 to ؊84, and ؊233 to ؊215 by DNase I footprint analysis and gel mobility shift assays. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Sp1, are shown to bind within the first 108 base pairs of the promoter region at nucleotide sequences ACTTTG and CCCCTCCCCC, respectively. The importance of these binding sites in promoter activity is demonstrated through independent functional mutagenesis experiments, which show dramatically reduced promoter activity. Transactivation studies with an HNF-4 expression plasmid in HeLa cells also demonstrate the importance of HNF-4 in promoting transcription in nonhepatocyte derived cells. Additionally, the sequence of a naturally occurring allele containing a previously described decanucleotide insert polymorphism at ؊323 is shown to reduce promoter activity by 33% compared with the more common allelic sequence.

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.

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