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.

Allele-specific DNA methylation, histone acetylation and histone methylation are recognized as epigenetic characteristics of imprinted genes and imprinting centers (ICs). These epigenetic modifications are also used to regulate tissue-specific gene expression. Epigenetic differences between alleles can be significant either in the function of the IC or in the cis-acting effect of the IC on 'target' genes responding to it. We have now examined the epigenetic characteristics of NDN, a target gene of the chromosome 15q11-q13 Prader-Willi Syndrome IC, using sodium bisulfite sequencing to analyze DNA methylation and chromatin immunoprecipitation to analyze histone modifications. We observed a bias towards maternal allele-specific DNA hypermethylation of the promoter CpG island of NDN, independent of tissue-specific transcriptional activity. We also found that NDN lies in a domain of paternal allele-specific histone hyperacetylation that correlates with transcriptional state, and a domain of differential histone H3 lysine 4 di- and tri-methylation that persists independent of transcription. These results suggest that DNA methylation and histone H3 lysine 4 methylation are persistent markers of imprinted gene regulation while histone acetylation participates in tissue-specific activity and silencing in somatic cells.

Allele-specific transcription is a characteristic feature of imprinted genes. Many imprinted genes are also transcribed in a tissue- or cell type-specific manner. Overlapping epigenetic signals must, therefore, modulate allele-specific and tissue-specific expression at imprinted loci. In addition, long-range interactions with an Imprinting Center (IC) may influence transcription, in an allele-specific or cell-type specific manner. The IC on human chromosome 15q11 controls parent-of-origin specific allelic identity of a set of genes located in cis configuration within 2 Mb. We have now examined the chromatin accessibility of the promoter region of one of the Imprinting Centre-controlled genes, NDN encoding necdin, using in vivo DNA footprinting to identify sites of DNA-protein interaction and altered chromatin configuration. We identified sites of modified chromatin that mark the parental alleles in NDN-expressing cells, and in cells in which NDN is not expressed. Our results suggest that long-lasting allele-specific marks and more labile tissue-specific marks layer epigenetic information that can be discriminated using DNA footprinting methodologies. Sites of modified chromatin mark the parental alleles in NDN-expressing cells, and in cells in which NDN is not expressed. Our results suggest that a layering of epigenetic information controls allele- and tissue-specific gene expression of this imprinted gene.