Introduction Increasing evidence supports the view that eukaryotic chromatin is organized as independent loops (Heng et al., 2001). Following histone extraction, these loops can be visualized as a DNA halo anchored to the densely stained nuclear matrix or chromosomal scaffold (Vogelstein et al., 1980; Gerdes et al., 1994; Bickmore and Oghene, 1996). As a basic unit, the loop is essential for DNA replication, transcription regulation and chromosomal packaging (Gasser and Laemmli, 1987; Berezney et al., 1995; Bode et al., 1995; Nickerson et al., 1995; Razin et al., 1995; Jackson, 1997; Stein et al., 1999; Sumer et al., 2003). The formation of each loop is dependent on a specific chromatin segment that must function as an anchor to the nuclear matrix. Sequences that attach to the nuclear scaffold or matrix have been termed either ‘SARs’ or ‘MARs’ (collectively termed S/MARs) as characterized by their preparation from metaphase or interphase cells respectively (Mirkovitch et al., 1984; Izaurralde et al., 1988; Craig et al., 1997; Hart and Laemmli, 1998). The recent description of mobile nuclear proteins has raised the issue of whether all S/MAR sequences serve as anchors and whether these anchors are rigid or flexible (Hancock, 2000; Pederson, 2000), but this now appears to be reconciled (Nickerson, 2001; Jackson, 2003). Despite increasing acceptance of the concept of S/MARs, one issue of concern is related to the procedure used to release loops and whether or not the protein extraction method will alter the nature of native chromatin loop domains. Recent evidence supporting the use of released loops for the study of S/MARs showed that sperm nuclear halos did transform into normal chromosomes after injection into oocytes (Mohar et al., 2002). These interesting results suggest that sperm nuclear halos retain all the information necessary for normal chromosomal organization, and the procedure to prepare the nuclear matrix does not change the basic biological features of nuclear chromatin. By monitoring the loop size of different chromatin domains along the nuclear matrix, our recent data also demonstrated that the chromosome territory information is well maintained in this experimental system (H.H.Q.H. et al., unpublished). Another significant development linking structural elements (S/MARs) with chromatin remodeling and high-order folding has recently been achieved by the characterization of SATB1 (for ‘special AT-rich binding protein 1’). SATB1 is one of the best-characterized S/MAR-binding proteins and it is preferentially expressed in thymocytes (Dickinson et al., 1992). SATB1-deficient mice die soon after birth as they do not develop T cells (Alvarez et al., 2000). SATB1-binding S/MAR sequences are located at the base of chromatin loops functioning as loop anchors on the nuclear matrix in cells expressing SATB1 and are 999 The biological significance of nuclear scaffold/matrix- attachment regions (S/MARs) remains a topic of long- standing interest. The key to understanding S/MAR behavior relies on determining the physical attributes of in vivo S/MARs and whether they serve as rigid or flexible chromatin loop anchors. To analyze S/MAR behavior, single and multiple copies of the S/MAR-containing constructs were introduced into various host genomes of transgenic mice and transfected cell lines. These in vivo integration events provided a system to study the association and integration patterns of each introduced S/MAR. By utilizing FISH to visualize directly the localization of S/MARs on the nuclear matrix or chromatin loop, we were able to assign specific attributes to the S/MAR. Surprisingly, when multiple-copy S/MARs were introduced they were selected and used as nuclear matrix anchors in a discriminatory manner, even though they all contained identical primary sequences. This selection process was probably mediated by S/MAR availability including binding strength and copy number, as reflected by the expression profiles and association of multi-copy tandem inserted constructs. Whereas S/MARs functioned as the mediators of loop attachment, they were used in a selective and dynamic fashion. Consequently, S/MAR anchors were necessary but not sufficient for chromatin loops to form. These observations reconcile many seemingly contradictory attributes previously associated with S/MARs. Key words: Chromatin loop, Loop anchor, S/MAR, FISH, Gene expression, Chromosome structure Summary Chromatin loops are selectively anchored using scaffold/matrix-attachment regions Henry H. Q. Heng 1,2,3, *, Sandra Goetze 5 , Christine J. Ye 6 , Guo Liu 1 , Joshua B. Stevens 1 , Steven W. Bremer 1 , Susan M. Wykes 1 , Juergen Bode 5 and Stephen A. Krawetz 1,4 1 Center for Molecular Medicine and Genetics, 2 Department of Pathology, 3 Karmanos Cancer Institute, 4 Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI 48202, USA 5 German Research Center for Biotechnology, RDIF/Epigenetic Regulation, Mascheroder Weg 1, 38124 Braunschweig, Germany 6 SeeDNA Biotech Inc, Windsor, ON N9A 4J2, Canada *Author for correspondence (e-mail: hheng@genetics.wayne.edu) Accepted 00976 Journal of Cell Science 117, 999-1008 Published by The Company of Biologists 2004 doi:10.1242/jcs.00976 Research Article