Bookmarking genes foractivation in condensed mitoticchromosomesSam John and Jerry L. Workman*

Summary

A hallmark feature of mitosis is the extinction of bulk cellular transcription. Themechanism by which transcription is abrogated is likely linked to mitotic specificevents such as chromosome condensation. Recent studies1,2 that probe thestructure of genes that can be reactivated rapidly after mitotic repression (earlyG1) suggest that there are structural distortions in the promoter regions of thesegenes. These distortions are absent in genes that are typically repressed orreactivated in later phases of the cell cycle (late G1, S, or G2). Such changes in thechromatin structure of these genes may create a transient window for transcriptionfactor binding and rapid reactivation of genes in subsequent phases of the cellcycle. BioEssays 20:275–279, 1998. r 1998 John Wiley & Sons, Inc.

INTRODUCTIONThe period of cell division during the cell cycle is calledmitosis, often referred to as the M phase. The observationthat bulk transcription in cells undergoing mitosis is inhibitedwas made nearly three decades ago.3–7 Seminal studieshave suggested that the transcriptional and translationalmachinery is intact in mitotic cells, since exogenously intro-duced DNA is competent to undergo transcription.4

When the nuclear structure dissociates during mitosis, thenuclear envelope collapses and the mitotic spindle forms.The chromosomes condense into their most compact formand orient themselves on the spindle at the equator of thecell. The resulting compact structure of DNA, histones, andnonhistone proteins creates a highly refractory state to thetranscription process.37

Mitotic phosphorylation events are thought to play animportant role in chromosome condensation and transcrip-

tional abrogation. It has been suggested that phosphoryla-tion of H1 may be a requirement for mitotic chromosomecondensation.8 Other studies suggest that mitosis-specificphosphorylation of transcription factors, such as Oct-1,TFIIIB, and TFIID, can inhibit the DNA binding and transcrip-tion potential of these factors.9–11 Thus, phosphorylationevents could be contributory mechanisms to the generalinhibition of transcription in mitosis through the promotion ofchromatin condensation and factor inactivation.

Many in vitro and in vivo studies have demonstrated thedifficulty in transcription factor binding in presence of nucleo-somes located over factor-recognition sequences. There aremany pathways by which this chromatin-mediated repres-sion can be overcome.12 One mechanism uses chromatinremodeling activities that can perturb chromatin structureand promote factor binding. The cell has evolved multipro-tein complexes molecules that allow the transcriptionalmachinery to overcome the repressive influence of chroma-tin. These include ATP-driven chromatin remodeling ma-chines (e.g., swi/snf) and histone-modifying complexes suchas histone acetyltransferases and the opposing deacety-lases.13–19

It is possible that the restoration of the programmedpattern of gene expression after mitosis might involve themarking of genes through chromatin remodeling or modify-ing activities. Recent observations from the laboratory ofLevens1 suggest a novel mechanism that these workersdefine as ‘‘molecular bookmarking.’’ These investigators

Department of Biochemistry and Molecular Biology and the Center forGene Regulation, Pennsylvania State University, University Park,Pennsylvania.Contract grant sponsor: National Institutes of Health; Contract grantsponsor: National Science Foundation*Correspondence to: Jerry L. Workman, Department of Biochemistryand Molecular Biology and the Center for Gene Regulation, Pennsylva-nia State University, University Park, PA 16802-4500; E-mail:jlw10@psu.edu

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postulate that genes that are primed for rapid reactivationafter mitosis are ‘‘tagged or bookmarked’’ (i.e., structurallyperturbed). The ‘‘tag,’’ in turn, presumably serves to directthe transcriptional machinery to these promoters for there-establishment of postmitotic gene expression.

MITOTIC HYPERSENSITIVITY ON THE hsp70AND c-myc PROMOTERSSeminal papers by Weintraub,20 Weintraub and Groudine,21

and Struhl22 have shown that some hypersensitive sitesfound in chromatin can be propagated through the cellcycle, suggesting that some factors necessary for theinitiation and maintenance of hypersensitivity are stable toboth replication and chromatin condensation.

Work from Wu’s laboratory2 provides a clear example ofmitotic repression using the Drosophila hsp70 gene as amodel system. During mitosis, the heat-inducible expressionof the hsp70 gene is abolished. In vivo footprinting analysisdemonstrates that the factors that bind the hsp70 promoter,although unchanged in abundance in mitotic extracts, aredisplaced from their sites on the hsp70 promoter. Immunocy-tochemical studies demonstrate that these factors, whichwere nuclear during interphase, become spread throughoutthe mitotic cytoplasm. Importantly, the hypersensitive siteover the hsp70 promoter is maintained during mitosis de-spite the apparent absence of promoter binding proteins orongoing transcription.

A recent paper from the laboratory of Levens1 suggeststhat the ‘‘tagging’’ of genes poised for transcriptional activa-tion after mitosis might involve structural perturbations aroundthe start site of transcription as measured by KMnO4 hyper-sensitivity, a chemical nuclease that reacts preferentially withsingle-stranded DNA. While the precise cause or the natureof the perturbation is unclear, Levens and colleagues pro-vide preliminary information suggesting that it may involvethe maintained presence of DNA binding activities at thosesites during the mitotic phase of the cell cycle.

It was previously observed that the single-stranded na-ture of mitotic chromatin increases dramatically relative tothat of interphase chromatin.23,24 Using supercoiled DNAtransfected into cells, Weintraub25 demonstrated that particu-lar DNA sequences that are known to adopt strong second-ary structures in vitro can maintain those structures ashypersensitive sites when stably integrated into chromatin,suggesting that structure imposed by sequence alone canbe manifested as single-stranded nuclease-sensitive re-gions in chromatin. The globin gene also demonstratessingle-stranded character, interestingly, only in cells in whichthe gene is active,26 and it has been postulated that the DNAsequence in this region allows for the generation of single-

stranded structures that may serve as recognition sites forcellular proteins.

Many DNA-binding proteins have been identified that canmodulate transcription from the c-myc promoter. Levens andcolleagues27–29 have implicated two proteins, hnRNP K andFBP, in c-myc transcription. Interestingly, both proteins aresequence-specific single-stranded DNA binding proteins. Itremains unclear as to how these factors interact preferen-tially with single-stranded DNA. These factors may activelypromote strand dissociation to generate KMnO4 sensitivestructures or alternatively may stabilize such structurescreated either by sequence or by the presence of secondarycofactors.

Levens and colleagues1 have suggested an elegant andtestable hypothesis regarding mitosis-specific structural per-turbations and the transcriptional potential of a gene. Theseinvestigators postulate that hypersensitivity, to single strandednucleases such as KMnO4, in promoter regions of genes inmitosis serves as a molecular landmark for genes primed forreactivation after mitosis. They look at several genes that arerapidly reactivated after mitosis and find that all these genesshare in common major regions of KMnO4 sensitivity in thepromoter regions only during mitosis. The same regions innon-mitotic phases of the cell cycle do not demonstrate asimilar sensitivity, suggesting that the bookmark dissociatesfrom these promoters postmitotically only to reassociate priorto the next round of cell division.

An extension of their hypothesis is that genes that aredormant will not manifest such hypersensitivities. Indeed, thepermanganate sensitive c-myc P2 promoter in HeLa cells,where the promoter is transcriptionally active lacks similarstructural perturbations in neuroblastoma cells, where c-mycexpression is repressed. In addition, Levens and colleaguesdemonstrate that the formation of the mitosis-specific hyper-sensitive site is sensitive to protein synthesis inhibitors.Nocodazole synchronized mitotic cells were treated withcycloheximide for various times and the c-myc P2 hypersen-sitive site monitored by sensitivity to KMnO4. They observedthat following a moderate incubation with cycloheximide, themitosis-specific hypersensitivity is lost. Using a differentapproach, salt-extracted condensed mitotic chromosomeswere isolated and subjected to buffers of increasing ionicstrength. At low ionic strength, the hypersensitivity aroundthe TATA box is readily observed; however, at elevated saltconcentrations, the KMnO4 hypersensitivity is lost and thepattern of sensitivity resembles that of nonmitotic cells. Thereare many possible explanations for the observed loss ofhypersensitivity, such as turnover of factors necessary formaintenance of the condensed chromosome or nucleosomemobility under elevated salt conditions. Taken together,however, these results point toward the possibility that thereis a factor(s) that could potentially play a role in the formation

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and maintenance of the observed structural perturbations atthe promoters of these genes. These structural landmarksmay, in turn, serve to highlight genes for rapid transcriptionalactivation in the postmitotic cell.

PERSPECTIVESIt is a generally accepted notion that the organization of agene into chromatin serves to impede transcription.30–34 Theformation of condensed chromatin during mitosis creates anespecially formidable task for transcription factor bindingand the formation of a pre-initiation complex. These repres-sive effects are thought to be reversed by the active modifica-tion of nucleosomal structures by chromatin remodeling ormodifying activities. The paper by Levens and colleaguessuggests a novel mechanism whereby genes programmedfor rapid reactivation are denoted as such by virtue of aperturbed higher-order structure. Such a ‘‘molecular book-mark’’ serves to mark the gene for expression perhaps by therecruitment of the transcription machinery.

A working model is presented in Figure 1. The modelsuggests that the bookmark is placed on promoters in apremitotic phase of the cell cycle (perhaps G2). The mecha-nism of targeting of bookmarks to particular sites in chroma-tin is unclear and may involve sequence-specific promoter–factor interactions as well as interactions between thebookmark and components of the transcription complex orother cofactors. This protein(s) is able to withstand chromo-some condensation during mitosis, whereas most otherfactors (e.g., the transcription machinery) are excluded ordisplaced from chromatin. As cells exit mitosis and thechromatin decondenses, these marked sites in chromatinserve as target sites for the reassociation of the transcriptionapparatus. Implicit in this model is the disassociation of thebookmark as cells enter the postmitotic phase of the cellcycle since bookmarked promoters fail to demonstrate simi-lar hypersensitivities in nonmitotic phases of the cell cycle.

This study poses several interesting questions. What arethe factors that contribute to KMnO4 sensitivity at a promoterin M phase? It is unlikely that the structural perturbation isdictated by a common primary sequence forming permanga-nate-sensitive secondary structures. The hypersensitive siteson the hsp70 and c-myc promoters appear to be sufficientlydivergent in that respect. However, it remains a formalpossibility that the local environment of different sequencesin chromatin may give rise to similar structures. A more likelypossibility is the role of DNA binding factors that activelycreate and maintain such hypersensitive sites. Interestingly,preliminary data from the Levens group indicates that thesingle-stranded DNA-binding proteins, hnRNP K and FBPremain bound to the myc promoter even during mitosis. Itremains to be proved whether these factors are the primarycontributors to the observed hypersensitivity. Similarly, the

AP-2 protein has also been shown to be tightly associatedwith mitotic chromosomes, whereas factors such as Oct-1,Oct-2, Ets-1, c-Fos, and other proteins become dispersedinto the mitotic cytoplasm.2

Why might some factors be more readily displaced frommitotic chromosomes than others? Condensed chromatin istypically composed of hypoacetylated histones. Hypoacety-lation arises from the action of histone deacetylases and orthe lack of activity of histone acetyltransferases. As has beenpreviously suggested (ref 2), deacetylated histone tails canpotentially interact more efficiently with negatively chargedDNA and act as an effective competitor for transcriptionfactor binding. The factors that withstand condensation-dependent displacement may therefore fall into a class ofproteins with an especially high affinity for nucleosomes. Thisis a model that remains to be formally tested. The mitotic-specific modification (phosphorylation) of transcription fac-tors may also account for their altered affinity for chroma-tin.11,35

What are the benefits of bookmark structures for geneexpression? The KMnO4 structures described for the c-mycand hsp70 genes suggest that the promoter regions of thesegenes are not organized into a typical chromatin structure. Ithas been suggested36 that single-stranded DNA is not apreferred substrate for association of histones consistentwith the atypical chromatin structure of these hypersensitivesites. These perturbed regions could potentially serve aspreferred sites for assembly of the transcription machinery.Indeed, immunocytochemical studies have shown that asubpopulation of TFIID can remain associated with con-densed mitotic chromosomes.11 It remains to be shownwhether this population of TFIID is associated with activegenes.

The observations discussed in this paper suggest amechanism by which transcriptional competence can bemaintained through mitosis. Such a mechanism may alsoplay a role in the inheritance of stable chromatin states(imprinting). The general applicability of such a mechanismis yet to be ascertained as the number of mitotic promotersanalyzed remains limited. Analysis of a broader range ofpromoters and the development of in vitro systems that canreconstitute the observed in vivo hypersensitivity will allowfor further dissection of the role of specific factors andchromatin remodeling, as well as modifying activities inmolecular bookmarking.

ACKNOWLEDGMENTSWe thank the members of the Workman laboratory for theirconstructive criticsms and helpful advice. This work wassupported by grants from the National Institutes of Healthand the National Science Foundation (to J.L.W.).

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Figure 1. Working model for molecular bookmark-directed gene activation. Active genes have bookmarks placed in the vicinity of theproximal promoter in a premitotic phase of the cell cycle. The bookmark withstands chromosome condensation during mitosis, whilemost other DNA-binding proteins become evicted. During chromosome decondensation in early G1, the bookmark serves as alandmark for the reassociation of the transcription machinery. The model assumes the subsequent disassociation of the bookmark,since the mitotic-specific hypersensitivity is lost in later stages of the cell cycle. (Kindly provided by Dave Levens.)

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