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Every nucleosome across the genome must be disrupted and reformed when the replication fork passes, but how chromatin organization is re-established following replication is unknown. To address this problem, we have developed Mapping In vivo Nascent Chromatin with EdU and sequencing (MINCE-seq) to characterize the genome-wide location of nucleosomes and other chromatin proteins behind replication forks at high temporal and spatial resolution. We find that the characteristic chromatin landscape at Drosophila promoters and enhancers is lost upon replication. The most conspicuous changes are at promoters that have high levels of RNA polymerase II (RNAPII) stalling and DNA accessibility and show specific enrichment for the BRM remodeler. Enhancer chromatin is also disrupted during replication, suggesting a role for transcription factor (TF) competition in nucleosome re-establishment. Thus, the characteristic nucleosome landscape emerges from a uniformly packaged genome by the action of TFs, RNAPII, and remodelers minutes after replication fork passage.

Current models for epigenetic gene silencing envision a static relationship between histone modifications and transcription. However, evidence for nucleosome mobility and replacement favors a dynamic model that may explain phenomena ranging from variegation to the neural restriction of Rett syndrome.

Closely linked repeats of a Drosophila P transposon carrying a white transgene were found to cause white variegation. Arrays of three or more transgenes produced phenotypes similar to classical heterochromatin-induced position-effect variegation (PEV), and these phenotypes were modified by known modifiers of PEV. This effect on the repeated transgenes was much stronger for a site near centric heterochromatin than it was for a medial site, and it strengthened with increasing copy number. Differences between variegated phenotypes could be accounted for if different topological structures were generated by pairing between closely linked repeat sequences. We propose that pairing of repeats underlies heterochromatin formation and is responsible for diverse gene silencing phenomena in animals and plants.

Centromeres are chromosomal elements that are both necessary and sufficient for chromosome segregation. However, the puzzlingly broad range in centromere complexity, from simple “point” centromeres to multi-megabase arrays of DNA satellites, has defied explanation. We posit that ancestral centromeres were epigenetically defined and that point centromeres, such as those of budding yeast, have derived from the partitioning elements of selfish plasmids. We further propose that the larger centromere sizes in plants and animals and the rapid evolution of their centromeric proteins is the result of an intense battle for evolutionary dominance due to the asymmetric retention of only one product of female meiosis.

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1α is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.

Variegation is a common feature of gene silencing phenomena, yet the basis for stochastic on/off expression is unknown. We used a conditional system that allows probing of heterochromatin at a reporter GFP gene by altering GAL4 transcription factor levels during Drosophila eye development. Surprisingly, the frequency of gene silencing is exquisitely sensitive to GAL4 levels, as though binding site occupancy affects the silenced state. The silent state is plastic, as spontaneous derepression occasionally occurs in both mitotically active and differentiating cells. By simultaneously assaying expression of a nearby gene, we further show that the size of an activated region within heterochromatin is small. We propose that variegation occurs because heterochromatin inhibits the transient exposure of factor binding sites.

Our study in Cell demonstrated that centromeric nucleosomes induce positive supercoils. We showed this using both the Drosophila CenH3 histone variant assembled in vitro by its native chaperone (RbAp48) and yeast minichromosomes in vivo (Furuyama and Henikoff, 2009xFuruyama, T. and Henikoff, S. Cell. 2009; 138: 104–113Abstract | Full Text | Full Text PDF | PubMed | Scopus (127)See all ReferencesFuruyama and Henikoff, 2009). To verify the in vivo results, we used yeast centromere mutants and conditional mutant kinetochore proteins. We showed that positive supercoiling is an inherent property of functional centromeres at mitosis and depends on deposition of CenH3 (Cse4). In their Correspondence, Lavelle et al. accept our conclusions with respect to the topology of centromeric DNA and agree that this is a provocative result. However, they raise a key question that was only indirectly addressed by our study, namely, which protein core structure can impose such an extraordinary reversal of DNA wrapping? Our views and theirs on this subject are largely in agreement, in that conventional octameric nucleosomes are inconsistent with the right-handed wrapping that can lead to positive supercoiling. However, we favor hemisomes as the candidate core structure, based on previous direct in vivo biochemical evidence. The Drosophila CenH3 histone variant is a stoichiometric component of stable tetrameric nucleosomes purified in their native form, which are proposed to be hemisomes based on protein content, DNA wrapping, and direct measurements of dimensions and conformation at the single-molecule level (Dalal et al., 2007xDalal, Y., Wang, H., Lindsay, S., and Henikoff, S. PLoS Biol. 2007; 5: e218Crossref | PubMed | Scopus (65)See all References, Wang et al., 2008xWang, H., Dalal, Y., Henikoff, S., and Lindsay, S. Epigenetics Chromatin. 2008; 1: 10Crossref | PubMedSee all References). In contrast, Lavelle et al. argue that a particle derived from a “reversome” is an alternative possibility (Bancaud et al., 2007xBancaud, A., Wagner, G., Conde, E.S.N., Lavelle, C., Wong, H., Mozziconacci, J., Barbi, M., Sivolob, A., Le Cam, E., Mouawad, L. et al. Mol. Cell. 2007; 27: 135–147Abstract | Full Text | Full Text PDF | PubMed | Scopus (71)See all ReferencesBancaud et al., 2007). Reversomes are transient structures that require sustained high torsional stress in order to flip a left-handed octamer into a right-handed configuration. Lavelle et al. propose that such an unstable intermediate might become stabilized upon loss of H2A/H2B dimers via unknown protein-protein interactions. However, our ability to induce positive supercoils using purified histones, RbAp48, and relaxed plasmid circles, without the addition of any machinery for generating torsional stress, demonstrates that no such elaborate mechanism is required. In addition, key features of CenH3 nucleosomes that have been documented in vitro and in vivo strongly argue against structures for CenH3 particles other than hemisomes.Lavelle et al. refer to an important in vitro finding by the Prunell group in which the human CenH3 histone variant (CENP-A) and histone H4 failed to assemble into distinct tetrasomes (H4/CenH3/CenH3/H4-containing particles), despite the fact that these particles are readily formed using H3 instead of CenH3 (Conde e Silva et al., 2007xConde e Silva, N., Black, B.E., Sivolob, A., Filipski, J., Cleveland, D.W., and Prunell, A. J. Mol. Biol. 2007; 370: 555–573Crossref | PubMed | Scopus (79)See all ReferencesConde e Silva et al., 2007). They concluded that the addition of H2A and H2B provides the hydrophobic environment necessary for CenH3 octamers to form under conditions of 2M salt. This implies that the assembly intermediate for CenH3 octameric nucleosomes, namely the tetrasome, is inherently unstable. Therefore, there is no plausible assembly pathway for CENP-A tetrasomes in vivo, which according to their flipping model would be an obligatory intermediate. So, although we accept the possibility that flipped particles may be transient intermediate structures produced by torsional stress, we find their existence at centromeres to be implausible.In addition, Lavelle et al. refer to evidence in yeast that argues for a noncanonical Cse4 structure based in part on depletion of H2A/H2B (Mizuguchi et al., 2007xMizuguchi, G., Xiao, H., Wisniewski, J., Smith, M.M., and Wu, C. Cell. 2007; 129: 1153–1164Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all ReferencesMizuguchi et al., 2007). The original interpretation of these experiments was that the non-histone protein Scm3 took the place of H2A/H2B dimers. However, recent work shows that viable yeast with CenH3 at their centromeres can be obtained in the absence of Scm3 (Camahort et al., 2009xCamahort, R., Shivaraju, M., Mattingly, M., Li, B., Nakanishi, S., Zhu, D., Shilatifard, A., Workman, J.L., and Gerton, J.L. Mol. Cell. 2009; 35: 794–805Abstract | Full Text | Full Text PDF | PubMed | Scopus (104)See all ReferencesCamahort et al., 2009). Although it is conceivable that a tetrasome instead occupies the yeast centromere, we are struck by the fact that yeast Cse4 can support centromere function in human cells depleted of CENP-A (Wieland et al., 2004xWieland, G., Orthaus, S., Ohndorf, S., Diekmann, S., and Hemmerich, P. Mol. Cell. Biol. 2004; 24: 6620–6630Crossref | PubMed | Scopus (94)See all ReferencesWieland et al., 2004). H2A/H2B has been observed in human centromeric chromatin (Foltz et al., 2006xFoltz, D.R., Jansen, L.E., Black, B.E., Bailey, A.O., Yates, J.R. 3rd, and Cleveland, D.W. Nat. Cell Biol. 2006; 8: 458–469Crossref | PubMedSee all ReferencesFoltz et al., 2006), such that Lavelle et al. would need to suppose that Cse4 is part of a right-handed tetrasome in yeast but can nevertheless replace its orthologous copy in humans to form a completely different yet functional particle. Rather than consider such an unlikely possibility, we note that centromeres with CenH3 histones likely shared a common evolutionary origin (Malik and Henikoff, 2009xMalik, H.S. and Henikoff, S. Cell. 2009; 138: 1067–1082Abstract | Full Text | Full Text PDF | PubMed | Scopus (123)See all ReferencesMalik and Henikoff, 2009), so that the most parsimonious interpretation is that all eukaryotic CenH3 nucleosomes have similar structures and so wrap DNA in a right-handed manner.In summary, Lavelle et al. accept our surprising discovery of a right-handed wrap around centromeric nucleosomes but disagree as to the nature of the histone core that is responsible. However, we find that their evidence for flipping of a left-handed octamer does not apply to CenH3 nucleosomes, and we await further biochemical evidence to definitively resolve this issue. Although we recognize that there is a lively debate in the centromere field concerning this issue (Dechassa et al., 2009xDechassa, M.L., D'Arcy, S., and Luger, K. Cell. 2009; 138: 22–24Abstract | Full Text | Full Text PDF | PubMed | Scopus (9)See all References, Hill and Williams, 2009xHill, E. and Williams, R. J. Cell Biol. 2009; 186: 453–456Crossref | PubMed | Scopus (2)See all References), we note that there is general agreement that the extraordinary epigenetic inheritance of centromeres depends ultimately on the properties of CenH3 nucleosomes (Bernad et al., 2009xBernad, R., Sanchez, P., and Losada, A. Exp. Cell Res. 2009; 315: 3233–3241Crossref | PubMed | Scopus (17)See all ReferencesBernad et al., 2009). We are excited about the prospects for resolution of perhaps the oldest unsolved problem in genetics (Flemming, 1882xZellsubstanz, Kern und Zelltheilung. Flemming, W. See all ReferencesFlemming, 1882).

Eukaryotic transcription is a dynamic process in which RNA polymerases read DNA templates that are tightly wrapped around histones. In this issue of Cell, Janicki et al. (2004) provide a glimpse of the events that follow transcriptional induction, using an integrated cytological system observed in real time. They find that local decondensation of the template is accompanied by profound changes in chromatin composition.

In drosophila and other dipterian insects ,homologous pairing of chromosomes in mitotic cells is a well established phenomenon.Of particular interest here are the significance of homologous pairing for gene expression in drosophila and its wider implications for mammals .

Transcription at two heat shock loci in Drosophila melanogaster, in subdivisions 87A and 87C, was investigated by hybridization in situ with 3 H-labeled messenger, nuclear RNA and whole cell RNA from cells cultured at elevated temperature. What appears to be the same 9 × 10 5 dalton heat shock message hybridizes at both sites. At 87A, little additional hybridization is obtained with nuclear or whole cell RNA. In contrast, at 87C the saturation level of hybridization by nuclear and whole cell RNA is much higher than that obtained with the message alone. This evidence for extensive hybridization at 87C but not at 87A by RNA distinct from the message is confirmed by the finding that excess nonradioactive message competes away most of the hybridization by 3 H-labeled nuclear and whole cell RNA at the latter locus but not at the former. The noncompetable RNA migrates on an electrophoretic gel as a heterogeneous population of molecules, extending to sizes both larger and smaller than the message. These and other observations lead to the conclusion that at 87A transcription includes little more than sequences complementary to the 9 × 10 5 dalton message, while at 87C, there are sequences complementary to the same message and extensive additional sequences complementary to some other species of RNA.

We have investigated transcription termination on a segment of Drosophila DNA that complements a yeast adenine-8 mutation. Poly(A) + RNA transcribed from this segment in yeast terminates at multiple sites clustered just beyond an AAUAAA sequence implicated in polyadenylation of higher eucaryotic messages. Deletion analysis indicates that, in yeast, this sequence is not required for polyadenylation. Rather, transcription termination is signalled by a region that is upstream of the AAUAAA sequence. At least part of the control region appears to be an 8-base pair (bp) sequence also found in the termination control region of the yeast CYC1 gene. Termination sites for the various deletions show a clear sequence preference. These sites occur in clusters at least 50 bp downstream of the control region, suggesting similarities between termination in yeast and ρ-dependent termination in bacteria.

We have determined the organization and transcription of a Drosophila DNA segment coding for the purine pathway enzyme, GAR transformylase. Because the transcript encoding this enzyme is very rare, we used a novel method for exon mapping without isolating a cDNA. Our results suggest a long polypeptide having multiple domains, with GAR transformylase at the COOH terminus preceded by an extensive repeat. Part of the same DNA segment specifies a shorter transcript, which consists of the same 5′ exons but lacks the last three 3′ exons. A polyadenylation signal within an intron allows this single gene to encode two polypeptides. We hypothesize that altemative processing of a transcript is a basis for channeling metabolic intermediates into two different pathways.

Close examination of the P31-fed bwD survivors provided intriguing evidence for multiple homeotic transformations (Janssen et al. 2000axJanssen, S, Cuvier, O, Muller, M, and Laemmli, U.K. Mol. Cell. 2000; 6: 1013–1024Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesJanssen et al. 2000a). Extra bristles revealed a transformation of the sixth abdominal segment into the fifth, fewer sex combs revealed a Sex-combs-reduced-like transformation, and larger halteres in combination with an Ultrabithorax gain-of-function mutation revealed enhanced transformation of the third thoracic segment into the second. This unusual combination of phenotypes had been seen before: partial loss-of-function mutations in the gene encoding GAF cause the same syndrome (Farkas et al. 1994xFarkas, G, Gausz, J, Galloni, M, Reuter, G, Gyurkovics, H, and Karch, F. Nature. 1994; 371: 806–808Crossref | PubMed | Scopus (284)See all ReferencesFarkas et al. 1994). So the question became, does P31 interact with bwD in such a way that GAF levels are reduced?An unexpected connection between bwD and GAF had previously been reported (Platero et al. 1998xPlatero, J.S, Csink, A.K, Quintanilla, A, and Henikoff, S. J. Cell Biol. 1998; 140: 1297–1306Crossref | PubMed | Scopus (106)See all ReferencesPlatero et al. 1998). GAF appears to bind exclusively to GA-rich satellites, including bwD, during mitosis (Figure 2AFigure 2A), but does not bind at all during interphase. The absence of GAF from interphase heterochromatin is seen most clearly in polytene chromosomes, where thousands of euchromatic sites have GAF, yet the heterochromatic chromocenter and bwD are completely devoid of GAF (Figure 2BFigure 2B). Therefore, the GA-rich satellites provide a potential binding reservoir for GAF that fills up only at the onset of mitosis, during which time the euchromatin condenses and loses all detectable GAF. These observations provide the connection between bwD and GAF that can potentially explain the P31-bwD syndrome. Perhaps the binding of P31 to bwD opens it up, resulting in the unscheduled transfer of GAF from euchromatic sites to bwD at interphase, a process that normally occurs only during mitosis.Figure 2Relocalization of GAF from Euchromatin to bwD(A) Cycling of GAF from dispersed sites in euchromatin to GA-rich satellites in pericentric heterochromatin and to bwD, which is located near the distal tip of chromosome arm 2R. (B) GAF is found at numerous sites throughout euchromatin, but is undetectable at bwD during interphase. After treatment with P31, but not P9, GAF relocalizes to bwD in polytene nuclei.View Large Image | View Hi-Res Image | Download PowerPoint SlideA critical test of this scenario is to treat with P31 and look for transfer of GAF from euchromatic sites to bwD by staining of polytene chromosomes with anti-GAF antibody. Laemmli's group performed this test, and the result was stunning (Figure 2BFigure 2B). bwD normally shows no anti-GAF staining, whereas nearly all detectable GAF in P31-treated nuclei was found at bwD. Concomitantly, anti-GAF staining of euchromatic bands was nearly abolished (Janssen et al. 2000axJanssen, S, Cuvier, O, Muller, M, and Laemmli, U.K. Mol. Cell. 2000; 6: 1013–1024Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesJanssen et al. 2000a). Thus, it appears that P31-induced opening of bwD provides a sink for GAF, which leaves its normal sites. Consistent with this interpretation, the authors note that P31 binding and GAF binding to GAGAA repeats are not mutually exclusive. Furthermore, normal GAF binding sites, which are typically GAGAG, are not targeted by P31, so that P31 is expected to open up bwD preferentially to euchromatic GAF binding sites. Unscheduled GAF binding to bwD after P31 treatment provides compelling evidence for Laemmli's model that minor-groove binding exerts its effect on chromatin by opening it up.GAF is not the only mitosis-specific satellite binding protein. Drosophila Prod protein, which binds to the AT-rich AATAAGATAC decameric satellite, shows similar cycling behavior (Platero et al. 1998xPlatero, J.S, Csink, A.K, Quintanilla, A, and Henikoff, S. J. Cell Biol. 1998; 140: 1297–1306Crossref | PubMed | Scopus (106)See all ReferencesPlatero et al. 1998), and reservoirs for other proteins might exist in heterochromatin. We wonder if titration by a drug-opened satellite sink had occurred for the AT binders, resulting in PEV suppression. AT-rich satellites are especially abundant in the Drosophila genome, and so it seems reasonable to suspect that if these satellites open up, they would become massive sinks for proteins needed for heterochromatic silencing. Thus, titration of silencing factors by satellites could have led to derepression of the white reporter gene in wm4. This explanation may have precedent in the well-known PEV suppressing effect of extra Y chromosomes, which are thought to derepress wm4 and other PEV mutations by titrating out heterochromatin-specific factors, reducing their availability at sites subject to PEV (Dimitri and Pisano 1989xDimitri, P and Pisano, C. Genetics. 1989; 122: 793–800PubMedSee all ReferencesDimitri and Pisano 1989).The human genome also has satellites in abundance. Indeed, the Ikaros regulatory protein binds to human gamma satellites, which have been proposed to function as Ikaros storage sites in lymphocytes (Cobb et al. 2000xCobb, B.S, Morales-Alcelay, S, Kleiger, G, Brown, K.E, Fisher, A.G, and Smale, S.T. Genes Dev. 2000; 14: 2146–2160Crossref | PubMed | Scopus (173)See all ReferencesCobb et al. 2000). It might not be too far-fetched to imagine that human satellite sinks will someday provide therapeutic drug targets. When converted to dye-conjugated compounds, minor-groove binders have an added advantage, as demonstrated by Laemmli's group, in that they illuminate the DNA target that they open up. Potentially, a therapeutic drug can reveal its DNA target, analogous to illumination of proteins linked to GFP in living organisms. Perhaps future decongestants will not only clear your nose, but also light it up!*To whom correspondence should be addressed (e-mail: steveh@fhcrc.org).

A pupal cuticle protein gene has been found within an intron of a Drosophila gene that encodes three purine pathway enzymatic activities. The intronic gene is encoded on the DNA strand opposite the purine pathway gene and is itself interrupted by an intron. Whereas the purine pathway gene is active throughout development, the intronic cuticle protein gene is expressed primarily over a 3 hr period in the abdominal epidermal cells of prepupae that secrete the pupal cuticle. Therefore, a housekeeping gene and a developmentally regulated gene function in a nested arrangement.