TAL1 is a key hematopoietic transcription factor that binds to regulatory regions of a large cohort of erythroid genes as part of a complex with GATA-1, LMO2 and Ldb1. The complex mediates long-range interaction between the β-globin locus control region (LCR) and active globin genes, and although TAL1 is one of the two DNA-binding complex members, its role is unclear. To explore the role of TAL1 in transcription activation of the human γ-globin genes, we reduced the expression of TAL1 in erythroid K562 cells using lentiviral short hairpin RNA, compromising its association in the β-globin locus. In the TAL1 knockdown cells, the γ-globin transcription was reduced to 35% and chromatin looping of the Gγ-globin gene with the LCR was disrupted with decreased occupancy of the complex member Ldb1 and LMO2 in the locus. However, GATA-1 binding, DNase I hypersensitive site formation and several histone modifications were largely maintained across the β-globin locus. In addition, overexpression of TAL1 increased the γ-globin transcription and increased interaction frequency between the Gγ-globin gene and LCR. These results indicate that TAL1 plays a critical role in chromatin loop formation between the γ-globin genes and LCR, which is a critical step for the transcription of the γ-globin genes.

Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them.

GATA-1 and NF-E2 are erythroid specific activators that bind to the β-globin locus. To explore the roles of these activators in transcription of the human fetal stage specific γ-globin genes, we reduced GATA-1 and p45/NF-E2 using shRNA in erythroid K562 cells. GATA-1 or p45/NF-E2 knockdown inhibited the transcription of the γ-globin genes, hypersensitive site (HS) formation in the LCR and chromatin loop formation of the β-globin locus, but histone acetylation across the locus was decreased only in the case of GATA-1 knockdown. In p45/NF-E2 knockdown cells, GATA-1 binding was maintained at the LCR HSs and γ-globin promoter, but NF-E2 binding at the LCR HSs was reduced by GATA-1 knockdown regardless of the amount of p45/NF-E2 in K562 cells. These results indicate that histone acetylation is dependent on GATA-1 binding, but the binding of GATA-1 is not sufficient for the γ-globin transcription, HS formation and chromatin loop formation and NF-E2 is required. This idea is supported by the distinctive binding pattern of CBP and Brg1 in the β-globin locus. Furthermore GATA-1-dependent loop formation between HS5 and 3′HS1 suggests correlation between histone modifications and chromatin looping.

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