Excision close to matrix attachment regions of the entire chicken alpha-globin gene domain by nuclease S1 and characterization of the framing structures.

Abstract

Nuclease Sl-hypersensitive sites in a 40-kb region of the chicken genome including the domain of the a-globin genes were mapped. Brief treatment of isolated chicken erythroid cell nudei with nuclease SI allowed separation ofan %20-kb genomic DNA fragment containing the whole a-globin gene duster. No Sl-hypersensitive sites were observed in the internal part of the domain. The upstream SI site was found in a DNA fragment of 1.7 kb where the origin of replication and several protein binding sites were identified previously. Precise mapping of the positions of SI cleavage in this fragment and "in vivo" footprinting of DNA-protein interactions in isolated nuclei showed a correspondence with some of these protein binding sites. The possible signfcance of all these observations is di in connection with the replication origin and the nuclear matrix attachment regions in the aming structures.In the nuclei of eukaryotic cells, the DNA is organized into large loops fixed to the nuclear skeleton (1-5). The specificity of loop organization-i.e., the specificity of distribution of the loop borders in the genome-has been intensively studied but the experimental results remain controversial (1,4 To investigate loop organization, we have now mapped, in interphase nuclei of erythroid cells, sites of preferential S1 cleavage within the chicken DNA fragment containing the domain of the a-globin genes. This domain represents one of the best studied genomic areas in higher eukaryotes in terms of the arrangement of up to 35 kb of DNA elements involved in chromatin structure, mechanisms of transcription, and control of gene expression. From a DNase I-hypersensitive site 14.5 kb upstream of the ir-,aD-, and aA-globin genes (8, 9) to a hypersensitive site 3 kb downstream of the gene cluster, multiple DNA elements such as matrix attachment regions (MARs) (8, 10, 11), topoisomerase II sites (6,8,12), a replication origin (13), enhancers (14-18), and a silencer (17) all thought to represent putative control elements, have been identified, in addition to the promoters of the individual genes (19,20) and repetitive DNA sequences (21). This region seems to be transcribed into a full-domain transcript which spans the entire domain between the most distal DNase I-hypersensitive sites (8,22,23), running right through all the DNA in the area between the external MARs, and also through an internal MAR placed upstream of the first gene.For the present investigation it was important that the positions of MARs (8,10) and topoisomerase II cleavage sites (24) had been mapped in this area and that multiple sites of protein-DNA interaction were identified within the 1.7-kb fragment including the putative origin of replication (13) and a replication-related MAR (10,11,25). We found that nuclease Sl-sensitive regions are located both upstream and downstream of the domain and that, in the upstream area, they coincided with the permanent site of DNA attachment to the nuclear matrix. In the upstream area, the exact positions of S1 cleavage and DNA-pro...

Higher Order Chromatin Structures in Maize and Arabidopsis

“…Many of the MARs that define topologically discrete sections in the genome contain topoisomerase II recognition motifs and subsequently are vulnerable to the actions of cytotoxic drugs (such as VM26) that generate a cleavable complex with topoisomerase II (Liu et al, 1983;Chen et al, 1984) in addition to being hypersensitive to DNase I (e.g., Cockerill and Garrard, 1986b;Rowe et al, 1986;Kas and Laemmli, 1992;Razin et al, 1993;Gromova et al, 1995b;Iarovaia et al, 1996). Razin and co-workers have used several approaches to define chromatin domain loops (Targa et al, 1994;Gromova et al, 1995aGromova et al, , 1995bLagarkova et al, 1995b;Iarovaia et al, 1996). In a review of the data addressing genome organization and attachments to the matrix, they tendered the idea that there are at least two types, or classes, of MARs that organize the eukaryotic genome: those that are the result of a transient functional activity of the genome (such as those that contribute to creating transcriptionally competent chromatin-the classic MAR) and those that might represent more permanent associations of DNA to the nuclear matrix that partition the genome into larger structural loops (Razin and Gromova, 1995;Razin, 1996).…”

“…Our approach is modeled after Gromova et al (1995a) and incorporates in situ treatments of nuclei and in vivo treatments of cells to release domains, with direct transfer and hybridization of specific single-copy genes. It has been demonstrated in several animal systems that a chromatin loop basement can be cleaved by nucleases (Filipski et al, 1990;Targa et al, 1994;Gromova et al, 1995a;Lagarkova et al, 1995b) and through the action of cytotoxic drugs that interact with the topoisomerase II component of the nuclear matrix (Cockerill and Garrard, 1986b;Rowe et al, 1986;Kas and Laemmli, 1992;Razin et al, 1993;Gromova et al, 1995b;Iarovaia et al, 1996). However, information about the organization of plant genomes at this scale is extremely limited (Espinas and Carballo, 1993).…”

“…The sites where the chromatin fiber is attached are referred to as matrix attachment regions (MARs) or scaffold attachment regions (SARs), although this latter term now tends to be reserved for attachments within the protein scaffold of individual mitotic chromosomes (Gasser et al, 1989;Laemmli et al, 1992). MARs often coincide with chromatin features associated with gene function, such as nuclease hypersensitive sites, non-B-DNA structures, origins of replication, and gene regulatory elements (Villeponteau et al, 1984;Cockerill and Garrard, 1986;Rowe et al, 1986;Bustos et al, 1989;Jackson et al, 1990;Bode et al, 1992;Avramova and Bennetzen, 1993;Paul and Ferl, 1993;Targa et al, 1994). In addition, transgenic analyses with both animals and plants have demonstrated that MARs can contribute to the normalization of transgene expression, further suggesting that MARs play a functional role in creating or maintaining transcriptional domains in vivo (Stief et al, 1989;Slatter et al, 1991;Allen et al, 1993Allen et al, , 1996Breyne et al, 1994;Phi-Van and Stratling, 1996).…”

Clusters of S1 Nuclease-Hypersensitive Sites Induced In Vivo by DNA Damage

“…On the other hand, there is indeed growing evidence for the existence of unpaired DNA in specific regions of chromatin (6,35,52,61) and for the presence within the cell of S1-hypersensitive sites that were mapped close to SARs (MARs) (24,74), previously defined as scaffold or matrix attachment regions (10,39,56). Single-stranded DNA was detected in SARs of the chicken ␣-globin gene domain (74) and in cis elements of the active human c-myc gene (52) and was implicated in the binding of lamina to SARs (50). On the other hand, there is accumulating evidence from the field of radiobiology that both the extent and distribution of DNA damage appear to depend on chromatin structure (for a review, see reference 65).…”

Memory mechanisms of active transcription during cell division

“…These sites correspond to the segments of the transcriptionally active genes. (13,40) Maybe there exists the binding of some specific factors to the promoters of previously active genes during mitosis to serve as 'molecular bookmarks' and to aid in the restoration of the parental gene expression patterns after mitosis. This was verified by the finding that a subpopulation of TFIID (about 10-20%) remains tightly associated with the condensed mitotic chromosomes in spite of the displacement of majority of TFIID (including TBPs and TAFs) from the disassembling prophase nucleus to the mitotic cytoplasm around the time of nuclear envelope breakdown.…”