In this study, a determination was made of the chromatin structure of the rat growth hormone (GH) gene locus by DNase I sensitivity analysis using GC [GH+, prolactin (PRL)-], 235 (GH-, PRL+), GH3 (GH+, PRL+) and liver (GH-, PRL-) cells. From 7 kb upstream from the transcription start site to 19 kb downstream from the polyadenylation site, two major DNase I-hypersensitive sites (M-DHS; UIA, UIIA) and three M-DHS (DIA, DII, DIII) were found within 2 kb upstream and 7 kb downstream regions, respectively. Two minor DHS (m-DHS; UIB, UIIB) in the upstream region and one m-DHS (DIB) downstream were shown to be associated with M-DHS. Thus, a total of five M-DHS and three m-DHS were mapped on the rat GH gene locus. Among these, five (UIIB, UIA, UIB, DIB, DIA) including two (UIA, DIA) M-DHS were specific for GH-producing cells. UIIA and DIII were M-DHS only in PRL-producing 235 cells while the major hypersensitivity of DII was detected in GH-producing cells and liver cells. Assessment of the enhancing activity of the DHS regions indicated novel enhancers in one upstream and two downstream regions that function well with the GH promoter in GC cells. These enhancers, each appearing different, coincided with m-DHS but not M-DHS in GC cells, and were not activated by Pit-1. Based on these observations, the following functions of five M-DHS and three m-DHS regions were defined: enhancer; locus control region (LCR); switch region serving for conversion from GH/PRL-producing cells to PRL-producing cells; and a region having a structural function in chromatin.

Key wordssubclinical hypothyroidism, thyrotropin receptor mutation.Subclinical hypothyroidism is defined as an elevated serum TSH concentration, and normal serum free thyroxine (T 4 ) and 3,5,3 ′ -triiodothyronine (T 3 ) concentrations associated with few or no symptoms or signs of hypothyroidism. 1,2 Recently, not only infants with overt hypothyroidism but also those with subclinical or mild hypothyroidism have been found by neonatal screening for congenital hypothyroidism.We identified a missense mutation in the thyrotropin receptor (TSHR) gene in a patient with subclinical hypothyroidism found by neonatal screening. Case reportThe propositus (a girl) was the first child of non-consanguineous Japanese parents. She was born at 39 weeks of gestation following an uncomplicated pregnancy and delivery, with a weight of 2682 g. She was referred because of a TSH level of 15 mU/L on neonatal screening at day 5 of life. The family history revealed no thyroid disease. At recall examination at day 46 of life, a serum TSH concentration of 12.0 mU/L in conjunction with normal serum concentrations of T 4 (10.4 µ g/dL) and T 3 (181 ng/dL) were detected. She did not have a goiter nor abnormal physical findings. Though her serum thyroid hormone levels were within normal limits, she was followed up for hyperthyrotropinemia. At the age of 3 years, an excessive TSH response to intravenous administration of thyrotropin releasing hormone (TRH) was considered diagnostic of congenital primary hypothyroidism, and subsequently, she was treated with levothyroxine (L-T 4 ). Figure 1 shows the clinical course of the propositus until 13 years of age. Before treatment with L-T 4 , the serum TSH level fluctuated between 10 and 30 mU/L. It declined to within normal limits after treatment with L-T 4 . The serum T 3 and T 4 levels were almost within normal limits throughout the course. At the age of 8 years, she underwent 123 I scintigraphy and TRH testing after the discontinuation of L-T 4 treatment. 123 I scintigraphy showed a normal-sized eutopic thyroid gland. Her 24 h 123 I uptake value was 17%. The perchlorate discharge test was negative. After the intravenous administration of 300 µ g/m 2 of TRH, her serum TSH concentration increased from 18.8 mU/L to a peak of 93.7 mU/L, while serum T 3 did not increase from the basal level of 111 ng/dL to 100 ng/dL at 120 min.

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Cosmid clones from -32 kb to +74 kb region of the rat somatotropin gene locus were isolated for examination of the chromatin structure in the region from -39 kb to +47 kb by DNase I-sensitivity analysis using rat pituitary-derived GC (somatotropin+, prolactin-), and 235 (somatotropin-, prolactin+) cells, and liver-derived BRL (somatotropin-, prolactin-) cells. DNase I-hypersensitive sites (DHS) specific for somatotropin-producing cells were previously shown to be located exclusively in the -2 kb to +9 kb region [Aizawa, A., Yoneyama, T., Kazahari, K. & Ono, M. (1995) Nucleic Acids Res. 23, 2236. No other DHS having this specificity was found in the region examined in this study. Except for these and two other DHS located in a cluster in this region, no DHS could be found from -23 kb to +22 kb. DHS having no or less cell-type specificity were scattered about in the -39 kb to -23 kb and +22 kb to +47 kb regions. The polyadenylation site of the human skeletal-muscle Nachannel a-subunit gene has been shown present 22 kb upstream from the somatotropin gene [BennaniBaiti, I. M., Jones, B. K., Liebhaber, S. A. & Cooke, N. E. (1995) Cenornics 29, 647-6521. Polyadenylation site of the rat skeletal-muscle Na-channel gene was shown in this study to be at -15.7 kb. The skeletal-muscle Na-channel gene was specifically expressed in skeletal-muscle cells but not in somatotropin-producing cells, and thus the boundary region that ensures the cell-type-specific expression of each gene would appear to be situated between two genes. The region prerequisite for cell-type-specific expression of the rat soinatotropin gene was estimated based on the present findings.Keywords: chromatin; deoxyribonuclease I ; gene expression; sodium channel; somatotropin.Somatotropin and prolactin are typical vertebrate pituitary hormones. Similarity in amino acid sequence and gene organization suggests that the genes coding for these hormones are likely to have evolved from a common ancestor [I, 21. Somatotropin and prolactin are produced in somatotrophs and lactotrophs, respectively, both of which differentiate from Ratlike's pouch that arises from the oral ectoderm at the roof of the embryonic mouth [3, 41. The expression of somatotropin and prolactin in these tissues has been studied as a model system for elucidating the mechanism of cell-type-specific gene expression in vertebrates.Transient transfection studies indicated the region required for the cell-type-specific expression of the rat somatotropin gene to be as much as 235 bp upstream from the transcription-start site [ 5 ] . Two Pit-1/GHF-1 (referred as Pit-I in this article) binding sites, Spl and zinc-finger-protein factor(Zn-I5)-binding sites and the thyroid-hormone-responsive element were identified in this region 16, 71. Though transient transfection study can delimit the region required for cell-type-specific gene expression, in most transgenic mice studies, this region cannot confer celltype-specific gene expression either quantitatively or qualita-

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To elucidate the mechanisms underlying cell type-specific expression of the growth hormone (GH) and prolactin (PRL) genes, we used rat pituitary-derived cell lines producing exclusively GH (GC cells) or PRL (235 cells), and examined the following: expression of transcription factors essential for GH and/or PRL gene expression; promoter/enhancer activity of the GH and PRL genes transiently introduced by transfection; and chromatin structures of the GH and PRL genes. Even in PRL-nonproducing GC cells, the PRL promoter/enhancer was more active than the GH promoter, and the transcription factors, Pit-1 and estrogen receptor (ER), essential for PRL gene expression were functional. The PRL promoter/enhancer of GC cells was normal. On DNase I sensitivity analysis of the chromatin structure, two hypersensitive sites were detected in PRL gene chromatin of PRL-producing 235 cells but none in that of GC cells. It thus follows that the major reason for absence of the expression of the endogenous PRL gene in GC cells is neither the lack of transcription factors necessary for PRL gene expression nor an anomaly of the PRL gene itself, but that the chromatin structure of the PRL gene differs in PRL-nonproducing and -producing cells. It was shown in this study that neither Pit-1 nor ER is required for conversion of the structure of PRL gene chromatin to a DNase I-hypersensitive state.