TRANSCRIPTION OF THE HISTONE H5-GENE IS REGULATED BY 3 DIFFERENTIATION-SPECIFIC ENHANCERS

Histone H5, an early marker of the avian erythroid lineage, is express ed 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 enh ancers, E1 (between - 2233 and - 1878 from the site of transcription i nitiation, +1) and E3 (between -1321 and -1163), and confirmed the pre sence 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 H5 gene activity i n the differentiated cells. The enhancers contain binding sites for se veral ubiquitous and erythroid cell-specific nuclear proteins, includi ng GATA-1, as demonstrated with GATA-1-specific antibodies. Although t he GATA sites were required for enhancer function, the concentration o f GATA-1, GATA-2, and GATA-3 decreased during cell differentiation, an d overexpression of these factors had little effect on H5 transcriptio n. 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. How ever, the UPE, a positive element in proliferating CFU(E) recognized b y the transcription factor H4TF2, was dispensable in the differentiate d cells. These results suggest that as the cells enter the final stage s of differentiation, there is a reprogramming of the regulatory facto rs that control H5 transcription and that the enhancers rescue and inc rease the activity of the promoter.