The role of chromatin in estrogen action in the uterus, I. The control of template capacity and chemical composition and the binding of H3-estradiol-17 beta.

It is now clearly established that genetic transcription in the mammalian uterus is regulated by the ovarian hormone estradiol-17f3 (for reviews see ref. 1). Stimulation of nuclear RNA synthesis in vivo has been found to be one of the earliest biochemical responses of the uterus of the ovariectomized or immature rat to a single injection of the hormone. This stimulation has been demonstrated for nuclear RNA rapidly labeled in vivo,2 for RNA synthesis assayed in vitro in isolated nuclei,2b 3 and by the assay in vitro of RNA synthesis directed by isolated chromatin and catalyzed by added RNA polymerase.4a The question thus arises: Through what molecular mechanisms of activity or synthesis does estradiol-17f3 result in an increase in the genetic material available for transcription in the uterus? In an attempt to obtain information of possible value in answering this question, we have undertaken an investigation of the role of uterine chromatin in the early action of the hormone in the ovariectomized rat. We find an early stimulatory effect on the nuclear synthesis and accumulation of chromatin-associated RNA. This effect is manifest 15 minutes after hormone treatment and occurs either prior to or simultaneously with a stimulation of the template activity of the isolated and purified chromatin. We further find that at two minutes after treatment in vivo H3-estradiol-17,3 is bound to the isolated chromatin. The time course for the amount of binding of the hormone in vivo parallels nicely the time course for the hormonal stimulation in vivo of the template activity of the chromatin, both parameters being maximal at eight hours after treatment. We conclude that estrogen regulates the chemical composition as well as the template capacity of uterine chromatin. The causal relations between these changes in template capacity, chemical composition, and binding of hormone remain to be unraveled. Materials and Methods.-The intact and ovariectomized adult rats used and their management for hormone treatment were as previously described.2 The remaining details of experimentation were as follows or appear in the text and relevant figures and tables. Preparation of purified chromatin: Chromatin was isolated and purified according to two methods. Method 1 was a modification of the procedure of Marushige and Bonner.5 About 250 mg of minced uterine tissue or 5 gm of minced liver was homogenized at -100C in 10 ml of saline-ethylenediaminetetraacetate (EDTA) (0.05 31 NaCi, 0.016 31 Na2 EDTA (pH 8.0)). The Ultra Turrax Tp 20/2 tissue disintegrator (Brinkman Instruments) was operated for 30 sec at 110 v and then for 2 min at 60 v. The following preparative steps were at 20C. The homogenate was filtered through four layers of nylon bolting cloth (110-mesh) and centrifuged at 1500 X g for 15 min. The filtrate was washed twice by sedimentation in 12 ml of the saline-EDTA and then twice in 12 ml of tris buffer (0.05 A!, pH 8.0). The sediment was then suspended in 12 ml of the tris buffer by homogenization in a Dounce homogenizer (Kontes Glass Co.) and centrifuged at 10,000 X g for 15 min. The pellet was suspended in 5 ml of the tris buffer, stirred for 30 min by a