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Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ε- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ε- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ε- and γ-globin genes and locus control region in KLF1(-/-) embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos.

The competition model of globin gene regulation states that the gamma-globin gene precludes expression of the beta-globin gene in early development by competing for the enhancing activity of the locus control region. The gamma-globin gene with a -161 promoter is sufficient for suppressing beta-globin gene expression, and the gamma-globin TATA and CACCC elements are necessary for this effect. In this work, stable transfection and transgenic mouse assays have been performed with constructs containing HS3 and HS2 from the locus control region, the gamma-globin gene with promoter mutation(s), and the beta-globin gene. The data indicate that the gamma-globin TATA and CACCC elements together have at least an additive effect on the beta/gamma-globin mRNA ratio in early erythroid cells, suggesting that the elements work coordinately to suppress beta-globin gene expression. The TATA and CACCC are the major gamma-globin promoter elements responsible for this effect. Transgenic mouse experiments indicate that the gamma-globin TATA element plays a role in gamma-globin expression and beta-globin suppression in the embryo and fetus; in contrast, the CACCC element has a stage-specific effect in the fetus. The results suggest that, as is true for the erythroid Krüppel-like factor (EKLF) and the beta-globin promoter CACCC, a protein(s) binds to the gamma-globin CACCC element to coordinate stage-specific gene expression.

A large nuclear protein complex, termed gammaPE (for gamma-globin promoter and enhancer binding factor), binds to five sites located 5' and 3' of the human y-globin gene. Two proteins, SATB1 (special A-T-rich binding protein 1) and HOXB2, can bind to yPE binding sites. SATB1 binds to nuclear matrix-attachment sites, and HOXB2 is a homeodomain protein important in neural development that is also expressed during erythropoiesis. The present work showed that antisera directed against either SATB1 or HOXB2 reacted specifically with the entire gammaPE complex in electrophoretic mobility shift assays (EMSAs), suggesting that the two proteins can bind to the gammaPE binding site simultaneously. When SATB1 or HOXB2 was expressed in vitro, they could bind independently to gammaPE binding sites in EMSA. Interestingly, the proteins expressed in vitro competed effectively with each other for the gammaPE binding site, suggesting that this may occur under certain conditions in vivo. Transient cotransfections of a HOXB2 cDNA and a y-globin-luciferase reporter gene construct into cells expressing SATB1 suggested that SATB1 has a positive and HOXB2 a negative regulatory effect on transcription. Taking into account their potentially opposing effects and binding activities, SATB1 and HOXB2 may modulate the amount of gamma-globin mRNA expressed during development and differentiation.

The human gamma-globin gene competitively inhibits beta-globin gene expression in early erythroid development. To identify the gamma-globin gene sequences required for this effect, transgenic mice and stable transfection analyses with constructs containing 5'HS2 from the locus control region, modified gamma-globin genes, and the beta-globin gene were used. The -136 to +56 region of the gamma-globin promoter is necessary for competitive inhibition, as the beta-globin gene was inappropriately expressed in mouse embryos and in K562 and HEL cells containing constructs in which this region was deleted. Independently, the -140 to +56 region of gamma-globin gene was not sufficient to inhibit beta-globin transcription in mouse embryos or in cultured cells. Competitive inhibition of beta-globin gene expression was observed in K562 and HEL cells having a gamma-globin gene with a -161 promoter. The data suggest that the -161 gamma-globin promoter, which includes the CACCC box, two CCAAT boxes, the stage selector element (SSE), and TATA box, has a major role in suppressing beta-globin transcription early in development. Proteins binding to these or other gamma-globin promoter elements may interact with those binding to the locus control region, consequently precluding beta-globin transcription.

Transgenic mice have been used extensively to study elements governing the erythroid-specific developmental switch from human fetal gamma to human adult beta globin. Previous work demonstrated that a small construct composed of hypersensitive site 2 (HS2) of the locus control region (LCR) linked to the gamma and beta globin genes (HS2-gamma-beta) is sufficient for correct tissue and temporal expression of these genes, whereas HS2-beta alone is inappropriately expressed in the embryo. Two models, which are not mutually exclusive, have been proposed to explain these results and those of other constructs in transgenic mice. One model emphasizes the conserved polarity in the globin locus and suggests a distance effect whereby the beta globin gene must be removed from the LCR/HS2 to prevent an early and incorrect activation of this gene in the embryonic compartment. A second hypothesis proposes a competition between the gamma and beta globin gene promoters for interaction with the LCR/HS2. The active gamma globin gene promoter positioned between the LCR/HS2 and the beta globin gene thereby interacts with the HS2 elements early in erythroid development and is expressed until a change in putative stage-specific nuclear factors makes an interaction with the adult beta globin gene more favorable. In an effort to test the competition model, a construct has been prepared in which a small deletion was produced in the promoter region of the gamma globin gene while in the context of the HS2-gamma-beta plasmid. Analysis of this construct in transgenic mice reveals a constitutive unregulated expression of the human beta globin gene during erythroid development. To determine if this competition effect is specific for globin genes, a heterologous reporter gene has been substituted for the gamma globin gene in the construct HS2-gamma-beta. In this case, the beta globin gene exhibits correct developmental expression. This data is consistent with a model in which transcription from a promoter upstream of the beta globin gene in some manner protects this adult gene from activation by the LCR/HS2 during early development.

A DNA region (site II) in the promoter of the human A gamma-globin gene (-182 to -168) is involved in transcriptional regulation. At least two nuclear proteins bind to this region: the erythroid-specific factor NF-E1/GF-1 and another factor present in many cell lines. In the present study, we demonstrate that the ubiquitous factor binding to site II has immunological identity with the octamer transcription factor OTF-1, which has been implicated in the regulation of expression of genes such as histone H2b and small nuclear RNA. In addition, we show that OTF-1 binds to site I (-291 to -267), a purine-rich region upstream of site II. Interestingly, OTF-1 binds to sites I and II with equal affinity. This was unexpected since the 14 bp site I binding site AAGAATAAATTAGA (-291 to -278), determined by methylation interference, does not show obvious similarities to the canonical octamer binding site for OTF-1 in site II (ATGCAAAT). Interaction of OTF-1 with functionally active binding sites in the gamma-globin promoter suggests that this factor has a role in gamma-globin transcription.

Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator.