Functional and Structural Characterization of a Human H4 Histone Gene Promoter: a Thesis

Abstract

Expression of the cell cycle dependent FO10S human H4 histone gene is regulated at both the transcriptional and post-transcriptional levels. We have investigated the 5' promoter elements mediating the transcriptional aspects of its regulation. A detailed in vivo and in vitro transcriptional analysis of promoter deletion mutants from this gene has identified three positive regulatory elements and two potentially negative regulatory elements within the first 1000 base pairs upstream of the transcription initiation site. In addition, the minimal promoter located within the first 70 base pairs is required for accurate transcription initiation and contains one of two in vivo identified protein-DNA interactions, site II. Binding of the nuclear factor HiNF-D to this region was correlated with the turn-on of histone gene transcription following stimulation of quiescent normal diploid fibroblasts to re-enter the proliferative phase. The most proximal positive regulatory element contains the other in vivo identified protein-DNA interaction, site I. Results from a series of in vitroprotein-DNA interaction studies revealed the binding of two nuclear factors to this element. One protein, HiNF-C, is indistinguishable from the transcription factor Sp1 while the other, HiNF-E, is a novel, potentially histone-specific member of the ATF transcription factor family. Binding of HiNF-C was required to stabilize the interaction of HiNF-E and together this region stimulated transcription 5 fold. The near-distal transcription activator region lies between -418 and -213 base pairs and forms a single protein- DNA complex, H4UA-1. The interaction domain for H4UA-1 contains recognition sequences for both the thyroid hormone receptor and the nuclear factor CTF/NF-1. The far-distal activator region (-730 and -589 base pairs) was the strongest positive regulatory element identified in the H4 promoter. This region increased transcription 10 fold and contains three protein-DNA interactions. One of the factors, H4UA-2, is an ATF transcription factor closely related to the HiNF-E interaction in the proximal positive element. These studies have defined the functional human H4 histone promoter to be a complex, modular structure extending at least 1000 base pairs.