Abstract
A relationship exists between nuclear architecture and gene activity and it has been proposed that the activity of ongoing RNA polymerase II transcription determines genome organization in the mammalian cell nucleus. Recently developed 3C and 4C technology allowed us to test the importance of transcription for nuclear architecture. We demonstrate that upon transcription inhibition binding of RNA polymerase II to gene regulatory elements is severely reduced. However, contacts between regulatory DNA elements and genes in the β-globin locus are unaffected and the locus still interacts with the same genomic regions elsewhere on the chromosome. This is a general phenomenon since the great majority of intra- and interchromosomal interactions with the ubiquitously expressed Rad23a gene are also not affected. Our data demonstrate that without transcription the organization and modification of nucleosomes at active loci and the local binding of specific trans-acting factors is unaltered. We propose that these parameters, more than transcription or RNA polymerase II binding, determine the maintenance of long-range DNA interactions.
Highlights
An intricate relationship appears to exist between chromosome folding and gene expression in the mammalian cell nucleus [1,2]
To study the role of RNA polymerase II (RNAPII) transcription in nuclear architecture, we investigated the intricate folding of the b-globin locus and its long-range contacts with other genes in primary erythroid cells treated with drugs that inhibit transcription
Single cell suspensions made from freshly dissected E14.5 fetal livers were cultured for five hours in the presence of very stringent concentrations of a-amanitin (100 mg/ ml), a drug that inhibits RNAPII transcription initiation and elongation
Summary
An intricate relationship appears to exist between chromosome folding and gene expression in the mammalian cell nucleus [1,2]. At the level of gene loci, regulatory DNA elements communicate with target genes located sometimes tens or even hundreds of kilobases away by contacting them, thereby looping out the intervening chromatin fiber. This was shown originally for the mouse b-globin locus, which extends over 180 kb and contains several cis-regulatory elements dispersed throughout the locus (Figure 1A). In expressing cells, these regulatory elements cluster with the active genes to form a so-called Active Chromatin Hub (ACH) [3,4]. Comparable interactions between genes and cis-regulatory elements have been demonstrated for several other gene loci (e.g.[9,10])
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