Active beta-globin gene transcription occurs in methylated, DNase I-resistant chromatin of nonerythroid chicken cells.

Abstract

The nature and function of the transcriptionally competent state of chromatin remains an enigma despite much work. Two properties are generally characteristic of competent chromatin: an increased sensitivity to digestion by DNase I and a reduced frequency of cytosine methylation at CpG positions (63). However, the notion of "competent" chromatin is poorly defined. Generally it is taken to mean a region of chromatin that is committed, by virtue of a special conformation, to the present or future transcription of one or more of its resident genes.Little is known about the nature of the DNase I-sensitive conformation of competent chromatin. The DNase I-sensitive state encompasses both the active genes themselves and the nontranscribed regions of chromatin flanking them. This contrasts with the situation for transcriptionally induced perturbations of chromatin, for which several structural features have been described (3,14,18,22,40,58,67,79). These perturbations are limited both temporally and spatially to the regions actually undergoing the transcription and are therefore distinct from the all-inclusive DNase I sensitivity of competent chromatin. The large DNase I-sensitive regions characteristic of competent chromatin may include several genes and, in the most completely mapped instances, cover discrete domains that extend uniformly through 10 to 100 kilobase pairs (kb) of chromatin (2, 39, 49). These domains can even include more nontranscribed flanking chromatin than actively transcribed gene chromatin. Moreover, the nontranscribed regions are just as sensitive to DNase I digestion as are the transcribed regions, provided the last-cut approach to the measurement of DNase I sensitivity is used (2,39,49,67,72).

CpG methylation remodels chromatin structure in vitro

“…Cytosine methylation in the region 5 H of the chicken adult b-globin gene conforms, in general, to a pattern of hypermethylation where the gene is inactive and hypomethylation where the gene is active (Ginder & McGhee, 1981;Ginder et al, 1983;Groudine & Conkin, 1985;Lois et al, 1990). These studies, which have concentrated on the methylation status at HpaII sites, have also revealed that this pattern is not all-encompassing (see below).…”

Integration of Cot Analysis, DNA Cloning, and High-Throughput Sequencing Facilitates Genome Characterization and Gene Discovery

“…However, methyl filtration and similar approaches such as PstI cloning are based on the assumption that hypermethylated sequences represent DNA that is nongenic whereas hypomethylated sequences represent low-copy DNA. In most (if not all) instances, using cloning/sequencing techniques centered on differential sequence methylation will result in the loss of many important and interesting genes: (1) it is common knowledge that methylation is one of the primary means by which genes are regulated, and that the methylation status of genes (or portions of genes) differs markedly between tissues and/or developmental stages (Siegfried and Cedar 1997;Heslop-Harrison 2000), (2) some genes are normally active when hypermethylated (Lois et al 1990;Wölfl et al 1991;HeslopHarrison 2000) and may not function if they are demethylated (Li et al 1993), (3) in some genes methylation at one site enhances transcription whereas methylation at another site reduces transcription (Li et al 1993;Riesewijk et al 1996), and (4) some species normally possess hypermethylated genes and hypomethylated repeat sequences (Simmen et al 1999).…”

“…Repetitive DNA is often more highly methylated than lowcopy DNA, and consequently some researchers have used methylation-sensitive restriction enzymes (e.g., McCouch et al 1988) or bacterial host strains that preferentially restrict methylated DNA (Rabinowicz et al 1999) to produce genomic libraries enriched in low-copy (ostensibly genic) DNA. However, cloning strategies involving the preferential exclusion of hypermethylated DNA may result in the loss of important/ interesting genes because the pattern and significance of DNA methylation can differ markedly between species (e.g., Simmen et al 1999), genes within an organism (e.g., Lois et al 1990;Wölfl et al 1991), developmental stages (for review, see Heslop-Harrison 2000), and different regions of the same gene (e.g., Li et al 1993;Riesewijk et al 1996). Clearly, alternative strategies for isolating and sequencing the unique elements of genomes are needed.…”

Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes.

“…Examples of RNA polymerase II genes which are controlled at the level of transcription elongation include proto-oncogenes c-myc, c-fos, c-myb, L-myc, and N-myc (2,12,26,36,37,57), the human adenosine deaminase gene (10), the epidermal growth factor receptor gene (14), the 13-globin gene (30), the Drosophila hsp7O gene (28,46), and a number of genes of viruses such as the human immunodeficiency virus (HIV) (22,27), adenovirus (25), simian virus 40 (43), and polyomavirus (50). For reviews, see references 23 and 53.…”