Abstract
We have observed changes in the chromatin structure of a human histone gene promoter that may be functionally related to variations in transcription during the cell cycle. A detailed analysis of the chromatin structure of a cell cycle-dependent human H4 histone gene and its flanking sequences was performed using DNase I, S1 nuclease, and restriction endonucleases. This gene was previously shown to have a DNase I- and S1-sensitive site for which the boundaries varied with the cell cycle, and we have now precisely mapped these modifications. During S phase, the entire coding region of this gene and the 5'-flanking region up to approximately -600 base pairs are sensitive to both DNase I and S1, while during mitosis/G1, accessibility to these enzymes is greatly decreased in regions from -250 to -600 base pairs and downstream of +100 base pairs. DNase I- and S1-hypersensitive sites in the proximal promoter region (which contains two sites of protein-DNA interaction as well as sequence elements necessary for the correct initiation of transcription) are present throughout the cell cycle, as is an additional site sensitive to both DNase I and S1, located at -700 to -800 base pairs. Restriction enzyme analysis confirmed the general openness of the promoter region and relative insensitivity of the 3'-flanking region, while salt wash experiments indicated several discrete sites in the promoter that are candidates for regulatory interactions. The chromatin structure of the proximal promoter region of this H4 gene is different during early S phase when it is maximally transcribed, as indicated by the ability of a high salt wash to render this region inaccessible to the restriction enzyme MspI only at this time of the cell cycle.
Highlights
We have observed changes in the chromatin struc- chromatin, making thegene accessible for transcription, and ture of a human histone gene promoter that may be thepresence of localized structuraldisruptionsthatmay functionally related to variations in transcription dupr-ermit the interaction of regulatory molecules with the exing thecell cycle
S1-sensitive site for which the boundaries varied with thecell cycle,and we havneow precisely mapped mining regions that may be involved in transcriptional control
The chromatin structuroef the proximal centered in the proximal promoterregion of this gene which promoter region of this H 4 gene is different during issensitivetobothDNase
Summary
The first 100 base pairs of the coding region pairs is accessible to both DNase I and S1 throughout the cell cycle, while the areaof about 100 basepairs just3' to this site is relatively well protected (Fig. 1A). Region from -50 to +80 base pairs showing a consistent level The restrictionenzyme MnlI confirms the relative sensitivof accessibility, while the area from -200 to -150 base pairs ity of the various portions of the F0108 H4 gene, with the is much less sensitive to S1 than DNase I (Fig. 1A). Show no obvious differences whenexamined a t early S phase, sites become increasinglymore resistant to digestion, with mid S phase, or mitosis/Gl. An additional site of nuclease the exception of two sites at the extrem3e'-end of the coding sensitivity extendingfrom approximately -800 to -700 base region (+300 base pairs) that relative to the cutting pattern.
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