Proximal and Long-Range Alterations in Chromatin Structure Surrounding the Chinese Hamster Dihydrofolate Reductase Promoter

Pemov, Alexander; Bavykin, Sergei G.; Hamlin, Joyce L.

doi:10.1021/bi00007a034

Cited by 10 publications

(9 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In agreement with earlier studies (36,42), the promoter region of the DHFR gene, which serves as an internal control in these experiments, is characterized by an irregular nucleosomal array and a number of hypersensitive and protected sites (arrowheads in left-hand panel of Fig. 2), most of which have been shown to correspond to transcription factor binding elements (43).…”

Section: Micrococcal Nuclease Hypersensitive Sites Cannot Be Detectedsupporting

confidence: 90%

“…Micrococcal nuclease digestions were performed as described (36), with minor modifications. In brief, cells were lysed with 0.1% digitonin, and the nuclei were pelleted and washed once with cell wash buffer (5 mM Tris⅐HCl, pH 7.4͞50 mM KCl͞0.5 mM EDTA͞0.05 mM spermine͞0.125 mM spermidine͞0.5% thiodiglycol͞0.25 mM phenylmethylsulfonyl fluoride).…”

Section: Methodsmentioning

confidence: 99%

“…In Figs. 2 and 5, the digests shown were from reactions that gave the clearest hypersensitive pattern in the DHFR promoter (which served as an internal control) (36).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

Pemov

Bavykin

Hamlin

1998

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-␤ and ori-␥) being somewhat preferred. We show here that the chromatin in the ori-␤ and ori-␥ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G 1 ͞S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.

show abstract

Section: Micrococcal Nuclease Hypersensitive Sites Cannot Be Detectedsupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

Pemov

Bavykin

Hamlin

1998

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Inhibitor studies show that the effect does not require translation of RNA (33), and genetic studies suggest that the mechanism linking transcription and origin specification acts in cis at the DHFR locus rather than through a trans-acting, noncoding RNA. Since a sufficient fraction of the total amplified DHFR promoters are occupied with transcription factors to generate an in vivo footprint (54,67), it is possible that some aspect of the transcriptionally active state, rather than the act of transcription itself, renders the entire transcription unit inaccessible to replication initiation factors. Alternatively, it is possible that only those gene copies that are actively engaged in transcription are competent to initiate replication and that those are also the copies for which transcription has suppressed intragenic initiation.…”

Section: Discussionmentioning

confidence: 99%

The Chinese Hamster Dihydrofolate Reductase Replication Origin Decision Point Follows Activation of Transcription and Suppresses Initiation of Replication within Transcription Units

Sasaki

Ramanathan

Okuno

et al. 2006

Molecular and Cellular Biology

View full text Add to dashboard Cite

Chinese hamster ovary (CHO) cells select specific replication origin sites within the dihydrofolate reductase (DHFR) locus at a discrete point during G 1 phase, the origin decision point (ODP). Origin selection is sensitive to transcription but not protein synthesis inhibitors, implicating a pretranslational role for transcription in origin specification. We have constructed a DNA array covering 121 kb surrounding the DHFR locus, to comprehensively investigate replication initiation and transcription in this region. When nuclei isolated within the first 3 h of G 1 phase were stimulated to initiate replication in Xenopus egg extracts, replication initiated without any detectable preference for specific sites. At the ODP, initiation became suppressed from within the Msh3, DHFR, and 2BE2121 transcription units. Active transcription was mostly confined to these transcription units, and inhibition of transcription by alpha-amanitin resulted in the initiation of replication within transcription units, indicating that transcription is necessary to limit initiation events to the intergenic region. However, the resumption of DHFR transcription after mitosis took place prior to the ODP and so is not on its own sufficient to suppress initiation of replication. Together, these results demonstrate a remarkable flexibility in sequence selection for initiating replication and implicate transcription as one important component of origin specification at the ODP.In their replicon model (29), Jacob et al. proposed that replication initiation involves the recognition of a cis-acting DNA sequence or replicator by a trans-acting positive regulatory factor called the initiator. Since then, this model has been validated in bacteria and virus systems, but in eukaryotes a more complex model has been proposed to explain the diversity of replicator structures (22,66). With the exception of budding yeast, a specific sequence element that defines the replication origin in eukaryotes has not been identified (2,6,7,21,23). Instead, it appears that a complex combination of primary DNA sequence and epigenetic factors dictates where replication initiates, and a unique combination of these factors may define the position of each initiation site (22). Although replication in most eukaryotic organisms does not initiate at random with respect to DNA sequence, the size and distribution of these sites are highly variable (for a review, see reference 23). In gene-dense regions, replication can initiate at very defined sites (1, 5), while in large intergenic regions multiple initiation sites are found throughout large (5-to 50-kb) initiation zones (11,13,28,39,45,50).A large (50-kb) region downstream of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells is the most extensively studied initiation zone, yet the precise distribution of initiation sites within this region has still not been resolved. High-resolution mapping of the locations of small nascent DNA strands within a 12-kb region detected two very specific initiation sit...

show abstract

“…5 and 6), we could not predict a priori how the MCM proteins might be distributed in the genome. We therefore opted for a more unbiased approach in which we could determine the distribution of MCMs throughout the 120-kb region encompassing the DHFR locus, which has been previously characterized vis à vis origins (5), transcription units (54), 3 matrix attachment regions (55), and chromatin structure (56,57).…”

Section: Subunits In Hamster Cells Are Part Of a Multisubunitmentioning

confidence: 99%

A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin

Alexandrow¹,

Ritzi²,

Pemov³

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-␤, ori-␤, and ori-␥) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.Although much is known about the mechanism of initiation at the origins of mammalian viruses, little is known about these processes at mammalian chromosomal origins of replication. In viral, yeast, and bacterial replicons, initiation is confined to genetically defined replicator sequences, which direct the loading of initiation factors followed by localized melting of adjacent DNA sequences (reviewed in Ref. 1). This interaction allows access to the origin by primases, polymerases, and other factors involved in the elongation steps of replication. In these relatively simple systems, the term origin is often used to refer to both the replicator (initiation protein binding site) and the local site(s) where nascent strand synthesis begins.Considerable evidence suggests that initiation at mammalian origins is much more complex. A case in point i...

show abstract

Proximal and Long-Range Alterations in Chromatin Structure Surrounding the Chinese Hamster Dihydrofolate Reductase Promoter

Cited by 10 publications

References 55 publications

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The Chinese Hamster Dihydrofolate Reductase Replication Origin Decision Point Follows Activation of Transcription and Suppresses Initiation of Replication within Transcription Units

A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin

Contact Info

Product

Resources

About