Transcription of the histone H5 gene is regulated by three differentiation-specific enhancers.

Abstract

Histone H5, an early marker of the avian erythroid lineage, is expressed at low levels in early erythroid precursors and at higher levels in more mature cells. We show that the increase in H5 expression is due to transcriptional activation of the H5 gene following differentiation of precursor CFU(E). We have found and characterized two upstream enhancers, El (between -2233 and -1878 from the site of transcription initiation, +1) and E3 (between -1321 and -1163), and confirmed the presence of a downstream enhancer (C. D.Trainor, S. J. Stamler, and J. D. Engel, Nature [London] 328:827-830, 1987) E7 (between +846 and + 1181) which are responsible for the increase in H5 gene transcription. The enhancers had a weak effect in nondifferentiated CFU(E) but a strong effect when the cells were induced to differentiate. Cooperation among the three enhancers, however, was not required for HS gene activity in the differentiated cells. The enhancers contain binding sites for several ubiquitous and erythroid cell-specific nuclear proteins, including GATA-1, as demonstrated with GATA-1-specific antibodies. Although the GATA sites were required for enhancer function, the concentration of GATA-1, GATA-2, and GATA-3 decreased during cell differentiation, and overexpression of these factors had little effect on H5 transcription. Hence, the differentiation-specific effect of the enhancers is not mediated by changes in relative levels of the GATA factors. Functional analysis of the H5 promoter indicated that the requirement of several elements, including a GC box necessary for transcription enhancement, did not change during the early stages of CFU(E) differentiation. However, the UPE, a positive element in proliferating CFU(E) recognized by the transcription factor H4TF2, was dispensable in the differentiated cells. These results suggest that as the cells enter the final stages of differentiation, there is a reprogramming of the regulatory factors that control H5 transcription and that the enhancers rescue and increase the activity of the promoter.Vertebrate erythropoiesis is characterized by the expression of sets of tissue-specific genes which are temporally regulated during maturation of the committed precursors. The chicken histone H5 gene is an interesting marker by which to study how these processes are regulated, because it responds both to early and late differentiation cues during erythropoiesis. Contrary to late erythroid markers, including the adult globin chains and cell surface proteins (2), H5 is already present in the early precursor CFU(E) (3, 45), and its expression increases further with the onset of the expression of these late genes (1). Transcription run-on analysis of erythroid cells at different stages of maturation have also indicated that the activity of the H5 gene is lower in proliferating erythroid precursors than in more mature cells (1). Thus, H5 expression during maturation is predominantly regulated at the transcription level.In an attempt to understand the molecular mechanisms accounting for the t...

Initiation Binding Receptor, a Factor That Binds to the Transcription Initiation Site of the Histone h5 Gene, Is a Glycosylated Member of a Family of Cell Growth Regulators

“…DNase I footprinting and gel retardation assays were carried out as described before (25,72). Where indicated, the nuclear extracts were incubated with immunoglobulin G (IgG) from preimmune or immune anti-IBR serum for 2 h at room temperature prior to incubation with the labeled oligonucleotide probes.…”

“…Chicken erythroid HD3 cells [ts34 AEV-transformed CFU(E)] and H32 cells (transformed quail fibroblasts) were grown as previously described (72,73). HD3 cells were induced to differentiate at 41ЊC with 15 M H7 (Sigma).…”

“…The mechanisms that bring about transcriptional activation of the h5 gene are not fully elucidated but appear to reflect alterations in the balance between positive and negative effectors. Transcription of the h5 gene is regulated by a complex assortment of ubiquitous and blood cell-specific factors acting at the promoter and three differentiation-specific enhancers located upstream and downstream of the gene (72,73). Although the majority of these factors contribute to transcriptional activity, one of them, IBR, was found to repress h5 transcription in vitro (25).…”

“…Although the h5 gene is already set in an active chromatin conformation in early erythroid precursors (69), its transcription is not activated until the preerythroblast-to-erythroblast transition (2,72), coincident with the decline in the proliferation potential of the cells. The mechanisms that bring about transcriptional activation of the h5 gene are not fully elucidated but appear to reflect alterations in the balance between positive and negative effectors.…”

Distribution of histone H3.3 in hematopoietic cell lineages

“…For each of the other genes subject to analysis for H3 variant incorporation and for histone modifications, we chose three sites. The first was somewhere in the transcribed region, the second (proximal promoter) was within one nucleosome on either side of the transcription start site, and the third (distal promoter) was usually within two nucleosomes upstream of the start site, except in the case of histone H5, for which we chose the distal enhancer Ϸ2.1 kb upstream of the gene (12).…”