Einstein Quart. J. Biol. and Med. (2001) 18(3):113-122 Chromatin Higher Order Structure and Chromosomal Positioning in the Nucleus Matthew Schmerer (Nelson, 1986). The nuclear matrix also seems to be a site Department of Developmental and Molecular Biology for chromatin attachment, which may provide a founda- Albert Einstein College of Medicine tion for the higher order structure of the chromatin Bronx, New York 10461 (Nelson, 1986; van Driel, 1995). This concept of nuclear compartmentalization alluded to the hypothesis that Abstract whole chromosomes or parts of chromosomes may occu- py certain regions of the nucleus (Manuelidis, 1985, While it has been known for some time that chromatin 1990). is highly packaged, it has only recently been discovered that chromatin is attached to the nuclear matrix. It has The existence of chromatin loops and chromosome also been recently shown that interphase chromosomes domains lend support to the contention that the nucle- remain separate from one another in so called, “chro- us is a highly structured organelle. The first level of chro- mosome domains.” This author suggests a functional matin organization is chromatin loops. The chromatin of relationship between chromatin attachment to the uncondensed chromosomes in interphase nuclei is nuclear matrix and chromosomal domains in the inter- attached to the nuclear matrix at regular intervals mak- phase nucleus. Herein, the evidence for the presence of ing loops of 5 to 100 kb in length (Cook and Brazell, matrix attachment sites and chromosomal domains is 1976; Paulson and Leammli, 1977). The second level of presented, and it is proposed that chromatin attachment chromatin organization is the location of the uncon- to the nuclear matrix provides the physical foundation densed chromosomes in the nucleus. Chromosomes for interphase chromosomes to be localized to chromo- occupy unique domains in the nucleus such that individ- some domains. ual chromosomes do not overlap (Manuelidis, 1985, 1990). This author would like to suggest that these two Introduction levels of organization are linked together, such that chromatin loops, which attach to the nuclear matrix pro- It has long been known, based on electron microscopy vide a foundation for the localization of interphase studies, that chromatin is packaged into nucleosomes chromosomes to unique domains. (Olins and Olins, 1974), then into 10 nm fibers (Cameron et al., 1979), and then into 30 nm solenoids (Finch and Chromatin Loops and SARs/MARs Klug, 1976; Felsenfeld and McGhee, 1986). This knowl- edge raised, among other issues, the question of how The chromatin of interphase nuclei is attached to the chromatin gets packaged into metaphase chromosomes. nuclear matrix at specific sites called matrix association Beyond the issue of chromatin packaging is the organi- regions (MARs) or scaffold attachment regions (SARs). zation of the interphase nucleus and where chromatin, These names refer to the same sequences that are asso- as linear uncondensed chromosomes, fits in. This ques- ciated with the nuclear matrix after high salt treatment tion was first addressed by David Comings in 1968 when or ionic detergent treatment (Razin and Gromova, 1995) he described, “The Rationale for an Ordered and will be hereto referred to as MARs only (see Fig. 1). Arrangement of Chromatin in the Interphase Nucleus” The purpose of the high salt treatment is to remove the (Comings, 1968). In this paper, Comings described the histones from chromatin, so that the DNA will be more evidence at that time for the non-random localization of accessible to nuclease treatment. Unfortunately, this chromatin in the nucleus. This includes some of the first preparation method leaves behind actively transcribing evidence that DNA replication occurs, to a greater and replicating DNA as well as nuclear matrix-associated extent, at specific points on the nuclear membrane and DNA sequences (Razin and Gromova, 1995). This makes it along the edge of the nucleolus, as well as some sugges- difficult to map specific sequences, but is sufficient for tive observations that chromatin is ordered in a specific the initial visualization of the chromatin loops them- way in the nucleus. Since then we have learned a great selves (Paulson and Laemmli, 1977; Hancock and Hughes, deal about the internal structure of the nucleus and how 1982). it is compartmentalized. We now have evidence for rRNA processing in the nucleolus (Sheer and Benavente, The presence of chromatin loops was also suggested 1990) and for RNA Polymerase II transcription at certain based on ultra-centrifugation of high salt-extracted locations in the nucleus, but not the nucleolus (van Driel, nuclei (Cook and Brazell, 1976). Based on those initial 1995). It is also known that the nucleus has an internal studies, a model was proposed where the chromatin skeleton, called the nuclear matrix, where the machinery loops are composed of the 30 nm solenoid, which is con- for DNA replication and transcription is anchored stricted between MARs (see Figure 2) (Marsden and 114 The Einstein Quarterly Journal of Biology and Medicine Figure 1: Mapping of relative positions of unique DNA sequences within the DNA loops by progressive detachment of DNA from the nuclear matrix. Three extended DNA loops (after histone removal) are shown, one of them bearing three sequence elements (a, b and c) whose relative positions in the loop are to be determined. After mild (A) or more intensive (B) treatment of the sample with a nuclease, the matrix-bound and cleaved-off DNA are separated by differential centrifugation. The distantly located sequence element a and b, while element c will still be recov- ered in a matrix-bound DNA fraction. Knowing the average loop length and the percentage of DNA in the nuclear matrix preparations possess- ing different subsets of probed sequence elements, one can estimate the approximate positions of these elements within the loop. (Adapted from Razin and Gromova, 1995). Laemmli, 1979). Firm evidence for the existence of these chicken β-globin domain (see Figure 4 for a schematic of loops came from the identification of the MAR the procedure). These data showing MAR sequences in sequences. These sequences were identified using different genes of one organism and in genes from dif- Exonuclease III (Exo III) sensitivity assays followed by ferent organisms provide strong evidence for the evolu- restriction mapping to identify the location of fragments tionary conservation of chromatin loops (see Cockerill that are resistant to Exo III digestion. Blasquez et al. and Garrard, 1986b for further discussion). (1989) used this technique to identify MAR sequences in the mouse light chain immunoglobulin gene (see Figure What is the significance of these loops? Do they have 3). The numbered bands (1-11) in the matrix lanes indi- any function other than as a means of packaging DNA in cate sequences that were protected from Exo III diges- the nucleus? The observation that the nuclear matrix tion, because they were bound to the nuclear matrix. It and replication origins are bound to each other was first is important to note the periodicity of the attachment made almost thirty years ago by David Comings (1968) sites. It is this periodicity that leads to the loop struc- and was demonstrated more recently by Razin et al. tures. Other groups used similar approaches to deter- (1986). These chromatin loops and their attachment to mine the MAR sites within the Drosophila histone gene the nuclear matrix through MARs may be important for cluster (Gasser and Laemmli, 1986) and the Drosophila initiating replication or for facilitating the process of hsp70 gene (Mirkovitch et al., 1984). A fortuitous obser- replication by bringing the DNA to the replication vation was that MARs often contain topoisomerase II machinery. They may also be important for localizing binding sites (Cockerill and Garrard, 1986a). Razin et al. certain sequences to specific compartments in the nucle- (1993) took advantage of the presence of topoisomerase us in order to silence them or bring them near the tran- II binding sites as a way to map MAR sequences in the scriptional machinery, but this is only speculation. Chromatin Higher Order Structure 115 Figure 2: DNA domains in nuclei. Elements that organize chromosomal loops in nuclei, termed “matrix association regions” (MARs), are depict- ed as solid rectangles (DNA sequences) and circles (proteins) (Adapted from Blasquez et al., 1989). Evidence for DNA localization to certain domains for the tion in the nucleus, it is clear that interphase chromo- purpose of silencing or activating stretches of the DNA is somes remain separate entities and the domains they present more at the level of chromosome domains. form remain exclusive of the nucleolus. Chromosome domains The most popular techniques for determining chromoso- mal domains are in situ hybridization with biotin labeled Uncondensed chromosomes in the interphase nucleus do probes followed by electron microscopy, fluorescent in not unravel and then randomly associate with each situ hybridization (FISH), pulse labeling of cells with other, but remain segregated from each other in their BrdU (or similar analog) followed by confocal microscopy own chromosomal domains (Manuelidis, 1990). The (see Figure 5), or injection of a fluorescent nucleotide domain organization of interphase chromosomes has analog (Cy3-AP3-dUTP) followed by video microscopy or been observed in Drosophila (Mathog and Sedat, 1989), confocal microscopy (Leitch et al., 1990; Sadoni et al., plants (Leitch et al., 1990), and mammals (Zink et al., 1999; Zink et al., 1998). The in situ hybridization tech- 1998; Manuelidis and Borden, 1988). This demonstrates niques are very powerful techniques for localizing DNA that these domains have been conserved throughout in the nucleus, but they require fixation of the cells at evolution in eukaryotic organisms. During metaphase, some point during the procedure, which could introduce the condensed chromosomes adopt what has become artifacts. These artifacts could distort the actual chromo- known as the Rabl orientation, where the centromeres somal positioning in vivo.