Maintenance of Long-Range DNA Interactions after Inhibition of Ongoing RNA Polymerase II Transcription Robert-Jan Palstra, Marieke Simonis, Petra Klous, Emilie Brasset ¤ , Bart Eijkelkamp, Wouter de Laat* Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands 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 b-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. Citation: Palstra R-J, Simonis M, Klous P, Brasset E, Eijkelkamp B, et al (2008) Maintenance of Long-Range DNA Interactions after Inhibition of Ongoing RNA Polymerase II Transcription. PLoS ONE 3(2): e1661. doi:10.1371/journal.pone.0001661 Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France Received December 13, 2007; Accepted January 21, 2008; Published February 20, 2008 Copyright: ß 2008 Palstra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Netherlands Organisation for Scientific Research (NWO) (912-04-082 and 815-02-013), and the Netherlands Genomics Initiative (050-71-324). E.B. was supported by a Rubicon grant from the Netherlands Organisation for Scientific Research (NWO) (825-07-012). The funding organizations didn’t have any role in preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *E-mail: w.delaat@erasmusmc.nl ¤ Current address: Centre National de la Recherche Scientifique (CNRS), UMR6247-GReD, Faculte ´ de Me ´ decine, Clermont Universite ´ , Institut National de la Sante ´ et de Recherche Me ´ dicale (INSERM), Clermont-Ferrand, France Introduction 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]. This spatial conformation is erythroid-specific and developmentally regulated [5] and depends on several (tissue- specific) transcription factors [6–8]. Comparable interactions between genes and cis-regulatory elements have been demonstrat- ed for several other gene loci (e.g.[9,10]). Large-scale nuclear architecture is also correlated with RNA polymerase II (RNAPII) transcription. For example, clusters of active genes on chromosomes preferentially locate at the edge or outside of their chromosome territory [11,12]. Moreover, actively transcribed genes tens of mega-bases apart on the chromosome or even on other chromosomes can come together in the nucleus [13,14]. Recently we have used novel 4C technology to analyze the genomic environments of several gene loci and found that active and inactive loci tend to separate in the nuclear space. The b-globin locus was found to switch its nuclear environment in relation to its expression status, with the active b-globin locus contacting active loci and the inactive b-globin locus contacting inactive loci elsewhere on the chromosome [15]. It is unclear how local interactions between regulatory DNA elements and long-range contacts between active genes are established. It has however been suggested that the process of RNAPII transcription itself plays an important role in shaping the genome [13,14,16–19]. In this study we tested the prediction made by these models that inhibition of transcription changes DNA folding and eliminates looping [18,20]. We demonstrate that ongoing RNAPII transcrip- tion or the presence of RNAPII at regulatory sites is not required to maintain the shape of the genome in the mammalian cell nucleus. In absence of transcription the overall chromatin state remains unaltered. This observation supports the notion that the organization and modification of nucleosomes along the DNA fiber and the binding of specific trans-acting factors, more than transcription or RNAPII-binding, determines the interaction between DNA loci and the formation of chromatin loops. Results Inhibition of transcription in the b-globin locus To study the role of 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. First, we extensively tested how drug treatment affected transcription at the b-globin locus. Single cell suspensions made from freshly dissected E14.5 fetal livers were cultured for five hours in the PLoS ONE | www.plosone.org 1 February 2008 | Volume 3 | Issue 2 | e1661