Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus.

Promyelocytic leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. By using specific approaches, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. Hence, we describe a role of PML NBs as nuclear depot centers to regulate HIRA distribution in the nucleus, dependent both on SP100 and DAXX/H3.3 levels. Upon IFN-I stimulation, PML is required for interferon-stimulated genes (ISGs) transcription and PML NBs become juxtaposed to ISGs loci at late time points of IFN-I treatment. HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of ISGs, well beyond the peak of transcription. Though, HIRA accumulation in PML NBs is dispensable for H3.3 deposition on ISGs. We thus uncover a dual function for PML/PML NBs, as buffering centers modulating the nuclear distribution of HIRA, and as chromosomal hubs regulating ISGs transcription and thus HIRA-mediated H3.3 deposition at ISGs upon inflammatory response.

The TUDOR domain of SMN is an H3K79me1 histone mark reader.

Spinal muscular atrophy is the leading genetic cause of infant mortality and results from depleted levels of functional survival of motor neuron (SMN) protein by either deletion or mutation of the SMN1 gene. SMN is characterized by a central TUDOR domain, which mediates the association of SMN with arginine methylated (Rme) partners, such as coilin, fibrillarin, and RNA pol II (RNA polymerase II). Herein, we biochemically demonstrate that SMN also associates with histone H3 monomethylated on lysine 79 (H3K79me1), defining SMN as not only the first protein known to associate with the H3K79me1 histone modification but also the first histone mark reader to recognize both methylated arginine and lysine residues. Mutational analyzes provide evidence that SMNTUDOR associates with H3 via an aromatic cage. Importantly, most SMNTUDOR mutants found in spinal muscular atrophy patients fail to associate with H3K79me1.

SMA-linked SMN mutants prevent phase separation properties and SMN interactions with FMRP family members.

Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of the SMN1 gene in most cases or mutations in rare cases. Interestingly, several SMN1 mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that in SMN1 mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMRFM). Importantly, SMA-linked SMNTUDOR mutant forms (SMNST) failed to interact with FMRFM In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.

Involvement of Type I Interferon Signaling in Muscle Stem Cell Proliferation During Dermatomyositis.

BACKGROUND AND OBJECTIVES: The idiopathic inflammatory myopathy dermatomyositis is an acquired disease that involves muscle, lung, and skin impairments. Patients with dermatomyositis show a wide range of severity of proximal skeletal muscle weakness, associated with inflammatory infiltrates, vasculitis, capillary dropout, and perifascicular myofiber atrophy. Muscles of patients with dermatomyositis show signs of muscle regeneration. Because muscle stem cells (MuSCs) are responsible for myofiber repair, we wondered whether the proliferative properties of MuSCs are altered in dermatomyositis muscle. We investigated the role of type I interferon (IFN-I) in this process because dermatomyositis is associated with sustained inflammation with high IFN-I levels. METHODS: MuSCs isolated from normal muscles and those from adult and juvenile patients with dermatomyositis were grown in culture and analyzed in vitro for their proliferating properties, myogenic capacities, and senescence. Gain- and loss-of-function experiments were performed to assess the role of IFN-I signaling in the proliferative capacities of MuSCs. RESULTS: MuSCs derived from 8 adult patients with dermatomyositis (DM-MuSCs) (5 severe form and 3 mild form, established from histologic evaluation), from 3 patients with juvenile dermatomyositis, and from normal muscle were used to analyze their myogenesis in vitro. DM-MuSCs exhibited strongly reduced proliferating capacities as compared with healthy MuSCs (-31% to -43% for mild and severe dermatomyositis, respectively), leading to poor myotube formation (-36% to -71%). DM-MuSCs were enriched in senescent, ß-galactosidase-positive cells, partly explaining the proliferation defect. Gain- and loss-of-function experiments were performed to assess the role of IFN-I on the proliferative capacity of MuSCs. High concentrations of IFN-I decreased the proliferation of healthy MuSCs. Similarly, conditioned medium from DM-MuSCs decreased the proliferation of healthy MuSCs (-15% to -22%), suggesting the delivery of an autocrine effector. Pharmacologic blockade of IFN signaling (using ruxolitinib or anti-IFN receptor antibodies) in DM-MuSCs rescued their proliferation up to the control values. DISCUSSION: These results show that autocrine IFN-I signaling prevents MuSC expansion, leading to muscle repair deficit. This process may explain the persistent muscle weakness observed in patients with severe dermatomyositis.

PML nuclear bodies and chromatin dynamics: catch me if you can!

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance.

Latent/Quiescent Herpes Simplex Virus 1 Genome Detection by Fluorescence In Situ Hybridization (FISH).

Fluorescence in situ hybridization (FISH) has been widely used to analyze genome loci at a single cell level in order to determine within a cell population potential discrepancies in their regulation according to the nuclear positioning. Latent herpes simplex virus 1 (HSV-1) genome remains as an episome in the nucleus of the infected neurons. Accordingly, depending on the location of the viral genomes in the nucleus, they could be targeted by different types of epigenetic regulations important for the establishment and stability of latency, and ultimately for the capacity of HSV-1 to reactivate. Therefore, it is important to take into consideration the interaction of the viral genomes with the nuclear environment to integrate this aspect in the overall set of physiological, immunological, and molecular data that have been produced, and which constitute the main knowledge regarding the biology of HSV-1. In this method chapter we describe in detail the procedure to perform FISH for the detection of HSV-1 genomes particularly during latency and also the combination of this approach with the detection of cellular and/or viral proteins.

Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis.

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.

Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment.

Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely "multiple-acute"). Viral genomes in the "multiple-acute" pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the "multiple-latency" pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual.

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism.

BACKGROUND: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage. RESULTS: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells. CONCLUSIONS: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork.

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.

The histone chaperone HJURP is a new independent prognostic marker for luminal A breast carcinoma.

BACKGROUND: Breast cancer is a heterogeneous disease with different molecular subtypes that have varying responses to therapy. An ongoing challenge in breast cancer research is to distinguish high-risk patients from good prognosis patients. This is particularly difficult in the low-grade, ER-positive luminal A tumors, where robust diagnostic tools to aid clinical treatment decisions are lacking. Recent data implicating chromatin regulators in cancer initiation and progression offers a promising avenue to develop new tools to help guide clinical decisions. METHODS: Here we exploit a published transcriptome dataset and an independent validation cohort to correlate the mRNA expression of selected chromatin regulators with respect to the four intrinsic breast cancer molecular subtypes. We then perform univariate and multivariate analyses to compare the prognostic value of a panel of chromatin regulators to Ki67, a currently utilized proliferation marker. RESULTS: Unsupervised hierarchical clustering revealed a gene cluster containing several histone chaperones and histone variants highly-expressed in the proliferative subtypes (basal-like, HER2-positive, luminal B) but not in the luminal A subtype. Several chromatin regulators, including the histone chaperones CAF-1 (subunits p150 and p60), ASF1b, and HJURP, and the centromeric histone variant CENP-A, associated with local and metastatic relapse and poor patient outcome. Importantly, we find that HJURP can discriminate favorable and unfavorable outcome within the luminal A subtype, outperforming the currently utilized proliferation marker Ki67, as an independent prognostic marker for luminal A patients. CONCLUSIONS: The integration of chromatin regulators as clinical biomarkers, in particular the histone chaperone HJURP, will help guide patient substratification and treatment options for low-risk luminal A breast carcinoma patients.

Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells.

Senescence is a stable proliferation arrest characterized by profound changes in cellular morphology and metabolism as well as by extensive chromatin reorganization in the nucleus. One particular hallmark of chromatin changes during senescence is the formation of punctate DNA foci in DAPI-stained senescent cells that have been called senescence-associated heterochromatin foci (SAHF). While many advances have been made concerning our understanding of the effectors of senescence, how chromatin is reorganized and maintained in senescent cells has remained largely elusive. Because chromatin structure is inherently dynamic, senescent cells face the challenge of developing chromatin maintenance mechanisms in the absence of DNA replication in order to maintain the senescent phenotype. Here, we summarize and review recent findings shedding light on SAHF composition and formation via spatial repositioning of chromatin, with a specific focus on the role of lamin B1 for this process. In addition, we discuss the physiological implication of SAHF formation, the role of histone variants, and histone chaperones during senescence and also elaborate on the more general changes observed in the epigenome of the senescent cells.

Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.

The mechanism of activation of the alternative lengthening of telomeres (ALT) pathway of mammalian chromosome-end maintenance has been unclear. We have now discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced all hallmarks of ALT in both primary and cancer cells. These included the formation of ALT-associated PML (promyelocytic leukemia) bodies (APBs), the presence of extrachromosomal telomeric DNA species, an elevated frequency of telomeric sister chromatid exchanges (t-SCE) events and intertelomeric exchange of an integrated tag. The induction of ALT characteristics in this setting led to the simultaneous suppression of telomerase. We determined that ALT induction is positively regulated by the proteins RAD17 and BLM and negatively regulated by EXO1 and DNA2. The induction of ALT phenotypes as a consequence of ASF1 depletion strongly supports the hypothesis that ALT is a consequence of histone management dysfunction.

Dynamics of histone H3.3 deposition in proliferating and senescent cells reveals a DAXX-dependent targeting to PML-NBs important for pericentromeric heterochromatin organization.

Oncogene-induced senescence is a permanent cell cycle arrest characterized by extensive chromatin reorganization. Here, we investigated the specific targeting and dynamics of histone H3 variants in human primary senescent cells. We show that newly synthesized epitope-tagged H3.3 is incorporated in senescent cells but does not accumulate in senescence-associated heterochromatin foci (SAHF). Instead, we observe that new H3.3 colocalizes with its specific histone chaperones within the promyelocytic leukemia nuclear bodies (PML-NBs) and is targeted to PML-NBs in a DAXX-dependent manner both in proliferating and senescent cells. We further show that overexpression of DAXX enhances targeting of H3.3 in large PML-NBs devoid of transcriptional activity and promotes the accumulation of HP1, independently of H3K9me3. Loss of H3.3 from pericentromeric heterochromatin upon DAXX or PML depletion suggests that the targeting of H3.3 to PML-NBs is implicated in pericentromeric heterochromatin organization. Together, our results underline the importance of the replication-independent chromatin assembly pathway for histone replacement in non-dividing senescent cells and establish PML-NBs as important regulatory sites for the incorporation of new H3.3 into chromatin.

Subfunctionalization via adaptive evolution influenced by genomic context: the case of histone chaperones ASF1a and ASF1b.

Gene duplication is regarded as the main source of adaptive functional novelty in eukaryotes. Processes such as neo- and subfunctionalization impact the evolution of paralogous proteins where functional divergence is frequently key to retain the gene copies. Here, we examined antisilencing function 1 (ASF1), a conserved eukaryotic H3-H4 histone chaperone, involved in histone dynamics during replication, transcription, and DNA repair. Although yeast feature a single ASF1 protein, two paralogs exist in most vertebrates, termed ASF1a and ASF1b, with distinct cellular roles in mammals. To explain this division of tasks, we integrated evolutionary and comparative genomic analyses with biochemical and structural approaches. First, we show that a duplication event at the ancestor of jawed vertebrates, followed by ASF1a relocation into an intron of the minichromosome maintenance complex component 9 (MCM9) gene at the ancestor of tetrapods, provided a different genomic environment for each paralog with marked differences of GC content and DNA replication timing. Second, we found signatures of positive selection in the N- and C-terminal regions of ASF1a and ASF1b. Third, we demonstrate that regions outside the primary interaction surface are key for the preferential interactions of the human paralogs with distinct H3-H4 chaperones. On the basis of these data, we propose that ASF1 experienced subfunctionalization shaped by the adaptation of the genes to their respective genomic context, reflecting a case of genomic context-driven escape from adaptive conflict.

Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer.

Mammalian cells possess two isoforms of the histone H3-H4 chaperone anti-silencing function 1 (Asf1), Asf1a and Asf1b. However to date, whether they have individual physiological roles has remained elusive. Here, we aim to elucidate the functional importance of Asf1 isoforms concerning both basic and applied aspects. First, we reveal a specific proliferation-dependent expression of human Asf1b unparalleled by Asf1a. Strikingly, in cultured cells, both mRNA and protein corresponding to Asf1b decrease upon cell cycle exit. Depletion of Asf1b severely compromises proliferation, leads to aberrant nuclear structures and a distinct transcriptional signature. Second, a major physiological implication is found in the applied context of tissue samples derived from early stage breast tumours in which we examined Asf1a/b levels. We reveal that overexpression of Asf1b mRNA correlate with clinical data and disease outcome. Together, our results highlight a distribution of tasks between the distinct Asf1 isoforms, which emphasizes a specialized function of Asf1b required for proliferation capacity. We discuss the implications of these results for breast cancer diagnosis and prognosis.

Replication stress interferes with histone recycling and predeposition marking of new histones.

To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows that replication stress interferes with predeposition marking and histone recycling with potential impact on epigenetic stability.

Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information.

Understanding the basic mechanisms underlying chromatin dynamics during DNA replication in eukaryotic cells is of fundamental importance. Beyond DNA compaction, chromatin organization represents a means to regulate genome function. Thus, the inheritance and maintenance of the DNA sequence, along with its organization into chromatin, is central for eukaryotic life. To orchestrate DNA replication in the context of chromatin is a challenge, both in terms of accessibility to the compact structures and maintenance of chromatin organization. To meet the challenge of maintenance, cells have evolved efficient nucleosome dynamics involving assembly pathways and chromatin maturation mechanisms that restore chromatin organization in the wake of DNA replication. In this review, we describe our current knowledge concerning how these pathways operate at the nucleosomal level and highlight the key players, such as histone chaperones, chromatin remodelers or modifiers, involved in the process of chromatin duplication. Major advances have been made recently concerning de novo nucleosome assembly and our understanding of its coordination with recycling of parental histones is progressing. Insights into the transmission of chromatin-based information during replication have important implications in the field of epigenetics to fully comprehend how the epigenetic landscape might, or at times might not, be stably maintained in the face of dramatic changes in chromatin structure.