The expression of one of the human main type H1 histone genes (termed H1.2) appears to be regulated by several trans-acting factors. Upstream of consensus regulatory regions, such as the TATA-, CCAAT-and H1-box (AAACACA) sequences, a crucial control site is located between nucleotide positions -536 and -412 (relative to the ATG initiation site). Removal of this promoter portion causes in chloramphenicol acetyl transferase reporter gene constructs a loss of the S-phase control function of the H1.2 promoter in HeLa cells. Electrophoretic mobility-shift assay and DNase I footprinting analysis suggest that the H1-box variant AAACAGA is a potential control element within the distal promoter region.The H1 group of mammalian histones comprises several subtypes, which differ in primary structure. Developmental control and cell-type-specific composition of the H1 complement has been described [l]. Compared with core histones, H1 proteins represent the most variable class of the otherwise highly conserved histone protein family. Five main type H1 histone genes have been described [2-51 in addition to genes which are solely expressed in specific cell types, such as the H1° gene [6], which is only expressed in highly differentiated cells [7, 81, and the Hlt gene [9, 101, which is transcribed during the spermatocyte stages of spermatogenesis [111.Despite the different transcriptional regulation, all vertebrate H1 genes (except the avian red blood cell H1 subtype H5) share a promoter element with the sequence AAACACA [12, 131. Its involvement in the S-phase-dependent transcription of chicken [12] and human [13] H1 genes has been demonstrated by deletion analysis [12] and factor titration throughout the cell cycle [13]. We have extended the functional analysis of the human H1.2 (for nomenclature, see [2]) gene promoter including more distal promoter sites. A combination of a series of promoter deletions, reporter gene constructs and footprint analysis revealed that promoter sites upstream of the consensus H1 box contribute to the S-phasespecific expression of the H1.2 gene.
MATERIALS AND METHODS
Cell cultureHeLa cells were grown in minimal essential medium at 37°C with 10% (by vol.) fetal calf serum in the presence of 5% CO,. The cells were grown to a density of 3-4X105 cells/ml before harvesting. Subsequently they were rinsed with NaCW, (0.14 M NaC1, 2.5 mM KC1, 8.1 mM Na2HP04, 1.5 mM KH2P04, pH 7.4) and treated with trypsinBDTA [0.05% trypsin (mass/vol.), 0.02% EDTA (mass/vol.), in Puck salt (solution A)] for 2-3 min at 37°C. 16-20 h prior to transfection, cells were seeded at a density of 4-6X104 cells/cm2 in order to reach logarithmic growth at the time of transfection.
Cloning and stepwise deletion of 5' flanking sequences of the human H1.2 histone geneThree fragments (termed KO, K1 and K2, see Fig. 2) were excised by restriction enzyme cleavage from the H1.2 promoter [4] and inserted into a Bluescript KS vector, which had been cut with EcoRI and HincII. After insertion into Bluescript vector, XbaVXhoI-flanked fragments ...