DNA methylation, transcription and chromatin assembly in vitro

Johnson, Colin Anfimov (1995) DNA methylation, transcription and chromatin assembly in vitro. PhD thesis, University of Glasgow.

Abstract

Histone HI is implicated in the establishment of stable and tissue-specific gene repression. It is presumed to repress transcription by binding to the internucleosomal, linker DNA leading to chromatin compaction and the formation of the 30 nm chromatin fibre. Histone H1 is abundant in heterochromatin and is associated with nucleosomes containing 5-methylcytosine. Conversely, it is absent from CpG island chromatin which is characteristic of active autosomal housekeeping genes and it is depleted in chromatin that contains active genes. DNA methylation correlates with the inactivity of many genes in vertebrates. It has been proposed that methylation may direct the formation of an inactive chromatin structure that is inaccessible to transcription factors, in a process requiring the participation of proteins that bind preferentially to methylated DNA. This study is an attempt to understand the molecular mechanisms that underlie the repression of gene activity by a combination of DNA methylation and chromatin. In vitro systems for transcription and the formation of chromatin are used as simplified models. The extent of in vitro transcription from unmethylated or methylated template is assayed in the presence of varied levels of total histone HI. Two templates are used: plasmid pArg/Leu contains two tRNA genes, which are transcribed by RNA polymerase III (pol III), and plasmid pVHCk contains the SV40 promoter linked to a reporter gene, which is transcribed by RNA polymerase III (pol III). A nuclear extract of HeLa cells is used for in vitro transcription assays of both types of template. Histone H1 forms characterised complexes on DNA, under the conditions used for these studies. Histone H1-DNA complexes are presumed to be a valid model of inactive chromatin. Transcriptional inactivation by histone HI is effective at lower levels with methylated templates, in comparison with unmethylated templates. Complete inactivation of all types of template is obtained with a further increase in histone HI levels. Different somatic variants of histone HI show differing degrees of preferential inhibition. Furthermore, histone H1 is a contaminant of the nuclear extract. The extract can be depleted of endogenous histone H1 either by the addition of competitor DNA or by fractionation of the extract with ammonium sulphate. Both treatments increase the level transcription from the methylated pol III template to that of the unmethylated template. The effect is reversed by the addition of exogenous histone H1 to the pol III template. The preferential inhibition of transcription from methylated templates by histone HI does not appear to be due to a greater binding affinity of the protein to methylated DNA, in comparison to unmethylated DNA. Instead, the conformation of the complex between histone HI and methylated DNA is changed, which prevents the formation of initiation and elongation transcription complexes on the methylated pol III template. Endogenous histone HI in the nuclear extract therefore prevents fully methylated pol III and pol II templates from being transcribed as efficiently as the unmethylated templates. This effect is most obvious when only the promoter region of the pol II template is methylated. Fully methylated DNA is intrinsically resistant to limited digestion with the restriction enzyme Msp I, in comparison to unmethylated DNA. This differential effect of DNA methylation is also observed when these templates are reconstituted as chromatin using Xenopus SI50 egg extract. Chromatin reconstituted on fully methylated or regionally-methylated DNA shows a greater resistance to digestion with Mspl than chromatin reconstituted on unmethylated DNA. The preferential resistance to Mspl, which is enhanced by the addition of histone HI during the chromatin reconstitution, occurs even on regions of unmethylated DNA if another region of that DNA is patch- methylated prior to chromatin reconstitution. This is consistent with DNA methylation acting as a focus for the formation of inactive chromatin. The transcriptional activity of unmethylated, patch-methylated and fully methylated pol II templates supports these observations.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the Medical Research Council.
• Keywords: Biochemistry.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Adams, Dr. Roger and Goddard, Dr. John
• Date of Award: 1995
• Depositing User: Enlighten Team
• Unique ID: glathesis:1995-71531
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 24 Aug 2021 14:59
• Thesis DOI: 10.5525/gla.thesis.71531
• URI: https://theses.gla.ac.uk/id/eprint/71531