Hemei Han

Developmental specificity of recruitment of TBP to the TATA box of the human γ-globin gene

It is unclear whether the core promoter is involved in developmental regulation. To address this question, we mutated the TATA box of the human γ-globin gene, produced transgenic mice, and examined the effect of the mutation during the course of mouse development. In our test system, the γ-globin gene is expressed at similar levels in the embryonic and adult erythroid cells. The TATA box mutation dramatically reduced expression of the γ-globin gene in the adult but not in embryonic erythroid cells. In addition, the disruption of the γ TATA box significantly reduced the recruitment of TATA box-binding protein (TBP) in the adult cells, but not in embryonic cells, suggesting that the recruitment of TBP to the γ gene promoter is developmentally specific. Similarly, the recruitment of transcription factor II B and RNA polymerase II to the γ promoter was affected in the adult but not in embryonic cells. The distinct effects of the TATA mutation in the embryonic and adult developmental stages suggest that the basal transcription apparatus can be recruited to a core promoter in a developmental stage-dependent manner. The TATA mutation resulted in a shift of transcription initiation site 6 bp or longer upstream to the cap site both in the embryonic and adult erythrocytes. We conclude that the TATA box determines the initiation site but not the efficiency of transcription of the γ-globin gene.

Developmentally Specific Role of the CCAAT Box in Regulation of Human γ-Globin Gene Expression

The CCAAT box is a widespread motif in eukaryotic promoters. In this study we demonstrate that the effects of the CCAAT box on γ-globin gene activation are developmentally distinct. Although this promoter element is essential for high level γ gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. The CCAAT mutation in the human γ-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 γ 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.

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The beta-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant beta-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.

Autonomous Silencing as Well as Competition Controls γ-Globin Gene Expression during Development

ABSTRACT To investigate the control of the γ-globin gene during development, we produced transgenic mice in which sequences of the β-gene promoter were replaced by equivalent sequences of the γ-gene promoter in the context of a human β-globin locus yeast artificial chromosome (βYAC) and analyzed the effects on globin gene expression during development. Replacement of 1,077 nucleotides (nt) of the β-gene promoter by 1,359 nt of the γ promoter resulted in striking inhibition of the γ-promoter/β-gene expression in the adult stage of development, providing direct evidence that the expression of the γ gene in the adult is mainly controlled by autonomous silencing. Measurements of the expression of the γ promoter/β-globin gene as well as the wild γ genes showed that gene competition is also involved in the control of γ-gene expression in the fetal stage of development. We conclude that autonomous silencing is the main mechanism controlling γ-gene expression in the adult, while autonomous silencing as well as competition between γ and β genes contributes to the control of γ to β switching during fetal development.

Transcriptional potential of the γ-globin gene is dependent on the CACCC box in a developmental stage-specific manner

To test the role of CACCC box on γ-globin gene activation, the CACCC box was deleted or mutated and γ-gene expression was monitored in transgenic mice. Disruption of the CACCC box had no effect on γ-gene expression in the cells of embryonic erythropoiesis but it strikingly reduced γ-gene expression in fetal erythropoiesis, and abolished γ-gene expression in adult erythroid cells. The CACCC mutation diminished HS formation, as well as TBP and polII recruitment at the γ-gene promoter; however, it only resulted in slight or no effects on histone H3 and H4 acetylation in adult erythropoiesis. Our findings indicate that each basic cis element of the proximal γ-gene promoter, i.e. CACCC, CCAAT or TATA box, can be disrupted without affecting the activation of γ gene in embryonic erythroid cells. We propose that the trans factors recruited by the three boxes interact with each other to form a ‘promoter complex’. In embryonic erythropoiesis the locus control region enhancer is able to interact with the complex even when components normally binding to one of the motifs are missing, but it can only activate an intact ‘promoter complex’ in adult erythroid cells.

Transcriptional environment and chromatin architecture interplay dictates globin expression patterns of heterospecific hybrids derived from undifferentiated human embryonic stem cells or from their erythroid progeny.

To explore the response of β globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged β globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas β globin was silent both in hESCs and in hESC-derived erythroblasts, β globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus β globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of β-globin, accompanied by a drastic change in the epigenetic landscape of γ and β globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching.