Nuclease sensitivity and DNA methylation in estrogen regulation of Xenopus laevis vitellogenin gene expression.

K Folger,J N Anderson,M A Hayward,D J Shapiro

doi:10.1016/s0021-9258(18)32143-4

K Folger, J N Anderson + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(18)32143-4

Copy DOI

Abstract

Estrogen activates transcription of the vitellogenin genes in livers of male Xenopus laevis. We have examined the conformation of the vitellogenin genes in chromatin and the methylation state of one vitellogenin gene during the process of estrogen stimulation and withdrawal. Sensitivity of the vitellogenin genes to DNase I digestion parallels transcription. The vitellogenin genes are insensitive to DNase I digestion in unstimulated liver cells, become more sensitive to DNase I digestion following estrogen activation of vitellogenin gene transcription, and are insensitive to DNase I digestion in liver cells withdrawn from estrogen. In contrast, the methylation state of nine potential methylation sites within the vitellogenin A1 gene is identical in red blood cells, unstimulated and withdrawn liver, estrogen-stimulated liver, and hepatocytes purified from estrogen-stimulated liver. Rapid transcription of the vitellogenin genes in estrogen-stimulated liver cells occurs with six of the nine methylation sites examined fully methylated.

Highlights

Estrogen activates transcription of the vitellogenin genes in livers of male Xenopus laevis
The methylation state of nine potential methylation sites within the vitellogenin A1 gene is identical inred blood cells, unstimulated and withdrawn liver, estrogen-stimulated liver, and hepatocytes purified from estrogen-stimulated liver
It has beenknown for several years that chromatin which is undergoing active transcription appears to possess a unique conformation which is reflected in enhancedsusceptibility to digestion by DNase I and by micrococcal nuclease [13,14,15,16] and by preferential solubility at low magnesium concentrations [17]

Summary

RESULTS

Ocytes pellet under these conditions [26]. The suspension of hepatocytes synthesizing vitellogenin was diluted with 4 volumes of TKSS containing 0.25 M sucrose and thecells were pelleted by centrifugation at 5,900 X g for 5 min at 2 "C. DNase Sensitivity of the Vitellogenin Genes in Xenopus Liver Chromatin-Administration of estrogen to male X . This results in cessation of vitellogenin gene transcription [4] and the degradation of all of the liver vitellogenin mRNA [5, 7] When these withdrawncells are restimulatedwith estrogen (secondary stimulation) the onseotf vitellogenin mRNA synthesis is ious size classes of DNase I-digested DNA were sonicated to reduce the length of the DNA fragments to a few hundred nucleotides in length prior to hybridization [16] and residual RNA was destroyed by incubation in0.3 M NaOH a t 37 "C for 12-18 h. Diately accessible for initiation of transcription, resulting in a shorter lag period In this model the lag period of several hours before appearance of vitellogenin mRNA following primary estrogen stimulation [5, 8] mightrepresentthetime.

Hpo II I I

UNINLDIVUECRED INDUCED LIVER INDUCED HEPATOCYTE

DNase Sensitivityand Methylation of the Vitellogenin Genes

Findings

DISCUSSION