59HS5 of the Human b-globin Locus Control Region Is Dispensable for the Formation of the b-globin Active Chromatin Hub Ping Kei Chan1, Albert Wai2, Sjaak Philipsen2*, Kian-Cheng Tan-Un 1 * 1 Department of Zoology, Kadoorie Biological Science Building, The University of Hong Kong, Hong Kong Special Administrative Region (SAR), China, 2 Erasmus MC, Department of Cell Biology, Rotterdam, The Netherlands Abstract Hypersensitive site 5 (59HS5) of the b-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 59HS5 in the three dimensional organization of the b-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 59HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 59HS5 does not have an appreciable effect on the three dimensional organization of the b-globin locus. This rules out models in which 59HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 59HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 59HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 59HS5 in definitive erythroid cells. Thus, binding of CTCF to 59HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality. Citation: Chan PK, Wai A, Philipsen S, Tan-Un K-C (2008) 59HS5 of the Human b-globin Locus Control Region Is Dispensable for the Formation of the b-globin Active Chromatin Hub. PLoS ONE 3(5): e2134. doi:10.1371/journal.pone.0002134 Editor: Axel Imhof, University of Munich and Center of Integrated Protein Science, Germany Received September 10, 2007; Accepted March 6, 2008; Published May 7, 2008 Copyright: 2008 Chan 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 the HKU School of Professional and Continuing Education (HKU SPACE). Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.philipsen@erasmusmc.nl (SP) ; kctanun@hkucc.hku.hk (KT) Introduction The human b-globin locus contains five genes that are arranged in the same order as their developmental expression pattern: 59-e (embryonic) Gc Ac (foetal) d b (adult)-39. The major activating region, termed the Locus Control Region (LCR), is located 6 to 22 kb 59 to the e-globin gene. The most prominent charactistic of the human b-globin LCR is that it drives high level, tissue-specific, copy number-dependent and position-independent expression of linked transgenes in mice [1]. The core components of the LCR are the DNase I hypersensitive sites (59HS1-5) [2]. Each of the hypersensitive sites holds a unique array of transcription factor binding sites, including those for GATA1, NF-E2 and EKLF proteins. This suggests that the HS in the human LCR may have different stage-specific activities and cannot be replaced function- ally by each other [3,4]. Transcriptional activation of the b-like globin genes is thought to be a multi-step system. This model includes the active involvement of erythroid transcription factors in the spatial organization of the locus, and increased accessibility to trans-acting factors by recruitment of chromatin remodeling complexes [5]. The resultant transcriptionally active locus is arranged in a three dimensional structure termed the active chromatin hub (ACH), in which the LCR hypersensitive sites interact directly with the transcribed genes through a looping mechanism [6,7]. The deduced spatial arrangement of the regulatory elements and the genes in the ACH offers an explanation for the observation that a gene proximal to the LCR has a transcriptional advantage over a more distally located gene [8,9]. The mode of action of the LCR to the b-globin genes appears to be orientation dependent as an inverted LCR is incapable of activating downstream globin genes at high levels, and the e-gene can not be activated when placed upstream of the LCR [10]. This property of the LCR may be due to the spatial organization of its hypersensitive sites, but it is also possible that the locus contains elements that block LCR action in one of the directions [11]. Recently, we have shown that human 59HS5 acts as a developmental stage-specific border element in erythroid cells. In that study, we placed a marked b-globin gene upstream of the LCR and the genes, using PAC constructs of the human b-globin locus in which 59HS5 was flanked by loxP sites [11]. In the present study, we applied Chromosome Conformation Capture (3C) technology to further investigate the functional properties of 59HS5 in vivo. The rationale behind the use of the 3C technique was to delineate the spatial organization of the b-globin locus upon Cre/loxP-mediated deletion of 59HS5 in human b- globin transgenes in mice. To date, directionality of the LCR has not yet been studied using the 3C technique in the context of the three-dimensional structure of the human b-globin locus. 3C analysis of the human b-globin transgenes was performed at the primitive and definitive stages of erythropoiesis. Since 59HS5 functions as a border in primitive erythroid cells, any major alterations in the 3D organization of the locus related to this property should be revealed. We also performed 3C analysis in the PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2134