Developmentally specific role of the CCAAT box in regulation of human gamma-globin gene expression

Abstract

The CCAAT box is a widespread motif in eukaryotic promoters. In this study we demonstrate that the effects of the CCAAT box on gamma-globin gene activation are developmentally distinct. Although this promoter element is essential for high level gamma gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. The CCAAT mutation in the human gamma-globin gene promoter impairs recruitment of TATA-binding protein (TBP), TFIIB, and RNA polymerase II in adult splenic erythroblasts but not in embryonic erythroid cells. We also show that the efficiency of gamma gene transcription is correlated with recruitment of TBP on the TATA box but that the level of TBP recruitment is not nuclear factor Y (NF-Y)-dependent. Our data also suggest that it is unlikely that transcriptional stimulation by the CCAAT box is exerted through direct protein-protein interaction between NF-Y and TBP.

Fig. 1. Human γ-globin RNA quantitation by RNase protection assay

A representative result is presented in this figure. Erythroid tissues were collected from four individual transgenic embryos (labeled as 1–4) and from four 3-week-old individual mice (labeled as 5–8) carrying the construct μLCR^Aγ (mut CCAAT). The day-12 blood (Bl) and yolk sac (YS) samples are composed of embryonic erythrocytes. The day-12 fetal liver (FL) and adult (Ad) blood is composed of definitive erythrocytes. Total RNA was isolated and hybridized with an RNA probe mix containing the antisense sequences of human γ-globin exon 2 and mouse α- and ζ-globin exon 1. Protected fragments and sizes are as follows: human ^Aγ-globin (Hu γ), 170 bp; mouse ζ-globin (Mu ζ), 151 bp; and mouse α-globin (Mu α), 128 bp. Intensities of the protected bands were measured by a PhosphorImager. Expression levels of the human γ gene were normalized by endogenous mouse α-like globin mRNAs and corrected by copy numbers (see “Materials and Methods”).

Fig. 2. Recruitment of NF-Y in transgenic mice carrying the CCAAT box-mutated γ-globin gene

NF-Y recruitment on the γ promoter was measured by real time PCR-based ChIP assay. The day-11 blood and yolk sac (marked as “Embryo” in A) and the adult spleen (marked as “Adult” in B) were harvested from transgenic mice carrying the wild-type γ promoter (WT) or the mutant γ construct (CCAAT mut). Formaldehyde was added to cell suspension, and cross-linked chromatin was solubilized by sonication. Soluble chromatin was subjected to immunoprecipitation with the antibody against NF-Y. The γCCAAT box in the recovered chromatin was quantitated by real time PCR. Data presented in this figure were generated from three independent immunoprecipitations and duplicate DNA quantitation. The endogenous ε^y promoter or β^maj promoter served as internal control in the embryonic or adult samples. Nonspecific IP control was performed in the chromatin of the wild-type mice and expressed as a percentage of immunoprecipitation from specific antibody. Non-expressing tissue control was performed in the brain of the wild-type mice immunoprecipitated by anti-NF-Y and expressed as a percentage of immunoprecipitation in erythroid tissues. The effect of the CCAAT box mutation on NF-Y recruitment was calculated by dividing NF-Y recruitment on the mutated promoter over that on the β^maj promoter in the CCAAT-mutant mice by the same ratio in the control mice. All data were corrected by the copy numbers of the transgenes.

Fig. 3. Recruitment of components of the basal transcription apparatus in transgenic mice carrying the CCAAT box-mutated γ-globin gene

TBP (A and B), TFIIB (C and D), and Pol II (E and F) recruitment on the γ promoter was measured by ChIP assay as described in the legend of Fig. 2. The embryonic and adult stages are indicated at the top of the left and right column panels, respectively. The antibodies used in each analysis are indicated inside the panels. WT, wild-type γ promoter; CCAAT mut, mutant γ construct.

Fig. 4. Analysis of NF-Y/TBP interaction by a modified ChIP assay

A, diagram showing the ChIP assay designed for detection of protein-protein interaction between NF-Y and TBP. If physical contact between NF-Y and TBP occurs in the cells, the two proteins would be cross-linked by formaldehyde. After NgoMI cleavage, both the TATA and CCAAT fragments should be detected in the chromatin immuno-precipitated either by the anti-TBP or by anti-NF-Y antibodies. If the contact is absent, the TATA fragment should be present only in chromatin pulled down by the anti-TBP antibody and absent in chromatin pulled down by the anti-NF-Y antibody and vice versa. B, measurements of the CCAAT and TATA fragments in the chromatin immuno-precipitated by anti-NF-Y antibody before (filled bars) and after (open bars) NgoMI digestion. C, measurements of the CCAAT and TATA fragments in the chromatin immunoprecipitated by anti-TBP antibody before (filled bars) and after (open bars) NgoMI digestion. D, measurements of the CCAAT and TATA fragments in the chromatin immuno-precipitated by anti-NF-Y antibody before (filled bars) and after (open bars) BamHI digestion. E, measurements of the CCAAT and TATA fragments in the chromatin immunoprecipitated by anti-TBP antibody before (filled bars) and after (open bars) BamHI digestion.