We have examined the chromatin structure of a 22 kilobase-pair chromosomal region containing the lysozyme gene in laying hen. Nuclease-hypersensitive sites were probed with DNAase I by using an indirect end-labeling technique. Eight DNAase I-hypersensitive sites could be mapped in the flanking regions of the gene in oviduct cells, in which the gene is expressed. The same sensitive sites were detected by utilization of an endogenous nuclease activity present in oviduct nuclei. Only one hypersensitive site was detected in the chromatin from erythrocytes, in which the gene is not expressed. The 3'-terminus of the lysozyme gene is highly exposed in nuclei from both tissues. Of special interest is the hypersensitive site at the 5'-terminus of the actively transcribed gene since it maps at the region of multiple initiation sites of transcription and the putative control regions of steroid hormones. DNAase I-hypersensitive chromatin structures also at a greater distance from the gene may take part in the control of gene expression.

The structural organization of chromatin is thought to determine the state of differentiation and activity of eukaryotic genes. Local interruptions of the regular nucleosomal array, the so-called DNase-hypersensitive sites, may indicate regions of the genome which play a critical part in regulation of differential gene activity. We present here two new observations on the chromatin structure of the chicken lysozyme gene, which strongly support a regulatory function for these sites. First, different sets of DNase I-hypersensitive sites have been found upstream from the promoter, depending on whether the gene is constitutively expressed (cultured macrophages) or in the steroid hormone-controlled state (oviduct). It seems, therefore, that diverse modes of regulation of the same gene are associated with discrete patterns of DNase I hypersensitivity. Second, one of the DNase I-hypersensitive sites in the oviduct chromatin disappears and reappears on steroid hormone withdrawal and secondary induction. These reversible changes in a narrow chromatin region reflect the transition from the potentially active to the active state of the lysozyme gene.

Four DNaseI hypersensitive (HS) chromatin regions were found in the uteroglobin locus located at -3.7, -2.4, -0.1 and +4.1 kb with respect to the transcription start site of the gene. The three sites upstream of the gene are only detected in the hormonally stimulated endometrium and disappear after hormone withdrawal, whereas the site at +4.1 is also found in tissues that do not express uteroglobin. In the -2.4 HS region, which is strictly dependent on progesterone treatment, three DNaseI sites are clustered within a 240 bp DNA segment that contains 20 imperfect repeats of an octanucleotide motif. Upstream of the uteroglobin gene there are three regions containing binding sites for the glucocorticoid and the progesterone receptors, located at -3.7, -2.6/-2.7 and -2.4. The -2.4 region contains two binding sites for the hormone receptors flanking the central HS site. In footprinting experiments with naked DNA binding of the receptor also renders this site more susceptible towards digestion with DNaseI. The -2.6/-2.7 region contains three binding sites for the hormone receptors located 140 bp upstream of the HS -2.4. While the -3.7 HS is also located within a receptor binding fragment, there is no binding of the hormone receptors to the promoter region. Thus, interaction of the receptor with DNA sequences far upstream from the promoter alters the chromatin conformation of neighbouring sequences and results in transcriptional activation.

We have determined the DNase I sensitive chromatin domain of the lysozyme gene in the hen oviduct. When nuclei were digested with DNase I, about 14 kb of upstream and 6 kb of downstream sequences in addition to the 4 kb long transcribed region were preferentially degraded. The transcription start site is located near the center of the approximately 24 kb long sensitive domain. At the 3' boundary there is a rather abrupt transition from the DNase I sensitive to the resistant chromatin configuration whereas at the 5' border this transition occurs in a gradual fashion over 6-7 kb of DNA. No obvious correlation between the boundaries of the domain and repetitive sequences could be established. DNase I-hypersensitive sites are clustered within the boundaries of the sensitive domain which seems to represent a functional unit of the gene.

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