Centromeres are chromosomal loci that ensure proper chromosome segregation by providing a platform for kinetochore assembly and spindle force transduction during cell division. Human centromeres are defined primarily by a unique chromatin domain featuring the histone H3 variant, Centromere Protein A (CENP-A), that marks a single active centromere locus per chromosome. CENP-A chromatin typically occupies a small subregion of low DNA methylation within multi-megabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. However, the mechanisms defining and maintaining precise centromere position and domain size, and the role of the underlying alpha satellite DNA sequence, are poorly characterised. Using an experimentally-induced neocentromere in RPE1 cells, we discovered that the SUV39H1 and H2 methyltransferases tri-methylate H3K9 at neocentromere boundaries to maintain CENP-A domain size independent of DNA methylation or satellite sequences. Furthermore, we found that the CENP-A domain at canonical alpha-satellite-based centromeres is characterized by local depletion of H3K9me3-mediated heterochromatin, coinciding with the DNA methylation dip region. We identified the SETDB1 methyltransferase as key to maintaining H3K9me3 within flanking active higher order alpha satellite arrays while SUV39s and SUZ12 contribute to globally heterochromatinize both alpha satellites and neighbouring repeats. Loss of this heterochromatin boundary results in the progressive expansion of the primary CENP-A domain, erosion of DNA methylation, and the nucleation of new centromeres across alpha satellite sequences. Our study identifies the functional specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size and number. ### Competing Interest Statement The authors have declared no competing interest.

Centromeres provide the chromosomal scaffold for the assembly of the kinetochore complex, thereby linking replicated sister chromatids to the mitotic spindle, driving their segregation into nascent daughter cells. The location and maintenance of centromeres rely, in large part, on a unique conserved chromatin domain, defined by nucleosomes containing the histone H3 variant, Centromere Protein A (CENP-A), whose discovery 40 years ago we now celebrate. Current models place CENP-A, along with many of its orthologs, at the centre of a self-propagating epigenetic feedback loop that heritably maintains centromere position through mitotic and meiotic divisions. CENP-A is stably recycled through DNA replication but requires replenishment each cell cycle. In many organisms, assembly is restricted to G1 phase, indicating tight cell cycle control of the assembly machinery. Here, we provide a historical overview of the discoveries that led to current models of cell cycle control of centromere assembly, starting with early models of regulation to the intricate, multi-layered phosphoregulation revealed to date. Our review focuses primarily on the human and other animal systems, in which the current view is that negative and positive control through cyclin-dependent kinases and Polo-like kinase 1 combine to link CENP-A assembly to mitotic exit. Cell cycle-coupled CENP-A assembly has been attributed to so-called licensing or priming events. We discuss the validity of these models and terminology and highlight key outstanding questions that remain unanswered.

Interferon-γ (IFNγ) transiently activates genes related to inflammation and innate immunity. A subset of targets retain a mitotically heritable memory of prior IFNγ exposure, resulting in hyperactivation upon re-exposure through poorly understood mechanisms. Here, we discover that the transcriptionally permissive chromatin marks H3K4me1, H3K14ac and H4K16ac are established during IFNγ priming and are selectively maintained on a cluster of guanylate-binding protein (GBP) genes in dividing human cells in the absence of transcription. The histone acetyltransferase KAT7 is required for H3K14ac deposition at GBP genes and for accelerated GBP reactivation upon re-exposure to IFNγ. In naive cells, the GBP cluster is maintained in a low-level repressive chromatin state, marked by H3K27me3, limiting priming through a PRC2-dependent mechanism. Unexpectedly, IFNγ priming results in transient accumulation of this repressive mark despite active gene expression. However, during the memory phase, H3K27 methylation is selectively depleted from primed GBP genes, facilitating hyperactivation. Furthermore, we identified a cis-regulatory element that forms transient, long-range contacts across the GBP cluster and acts as a repressor, curbing hyperactivation of previously IFNγ-primed cells. Our results provide insight into the chromatin basis for the long-term transcriptional memory of IFNγ signaling, which might contribute to enhanced innate immunity.

Centromeres are defined by a unique single chromatin domain featuring the histone H3 variant, centromere protein A (CENP-A), and ensure proper chromosome segregation. Centromeric chromatin typically occupies a small subregion of low DNA methylation within multimegabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. Here, we define the molecular basis of how heterochromatin serves as a primary driver of centromere and neocentromere position, size and number. Using single-molecule epigenomics, we uncover roles for H3K9me3 methyltransferases SUV39H1/H2 and SETDB1, in addition to noncanonical roles for SUZ12, in maintaining H3K9me3 boundaries at centromeres. Loss of these heterochromatin boundaries leads to the progressive expansion and/or repositioning of the primary CENP-A domain, erosion of surrounding DNA methylation and nucleation of additional functional CENP-A domains across the same alpha-satellite sequences. Our study identifies the functional importance and specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size and suppressing ectopic centromere nucleation events.

Hintergrund Kopf-Hals-Plattenepithelkarzinome (HNSCC) werden meist in fortgeschrittenen Tumorstadien nach Symptombeginn diagnostiziert. Eine Früherkennung könnte den Behandlungserfolg verbessern. Die Analyse von fünf epigenetischen Tumormarkern in der aktuellen OncSaliva-Studie soll deren klinische Leistung in nicht invasiven Flüssigbiopsieproben zeigen.

Exposure of human cells to interferon-γ (IFNγ) results in a mitotically heritable yet reversible state called long-term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFNγ. Here, we discovered that in primed cells, both interferon-responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re-exposure to IFNγ, specifically at promotors of primed genes. Priming does not alter the degree of IFNγ-induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK-STAT signaling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that the memory of interferon exposure constitutes a STAT1-mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

Interferon-γ (IFNγ) transiently activates genes involved in inflammation and innate immunity. A subset of targets maintain a mitotically heritable memory of prior IFNγ exposure resulting in hyperactivation upon reexposure. Here we discovered that the active chromatin marks H3K4me1, H3K14Ac and H4K16Ac are established during IFNγ priming and selectively maintained on a cluster of GBP genes for at least 7 days in dividing cells in the absence of transcription. The histone acetyltransferase KAT7 is required for the accelerated GBP reactivation upon reexposure to IFNγ. In naïve cells, we find the GBP cluster is maintained in low-level repressive chromatin marked by H3K27me3 limiting priming in a PRC2-dependent manner. Unexpectedly, IFNγ results in transient accumulation of this repressive mark but is then selectively depleted from primed GBP genes during the memory phase facilitating hyperactivation of primed cells. Furthermore, we identified a cis-regulatory element that makes transient, long-range contacts across the GBP cluster and acts as a repressor, primarily to curb the hyperactivation of previously IFNγ-primed cells. Combined our results identify the putative chromatin basis for long-term transcriptional memory of interferon signalling that may contribute to enhanced innate immunity. ### Competing Interest Statement The authors have declared no competing interest.

The centromere is a unique chromatin domain that links sister chromatids and forms the attachment site for spindle microtubules in mitosis. Centromere inheritance is large-ly DNA sequence-independent but strongly reliant on a self-propagating chromatin domain featuring nucleosomes containing the H3 variant CENP-A. Unlike other histones, CENP-A is maintained with unusually high stability in chromatin. Previously, we have shown that mitotic maintenance of CENP-A and other constitutive centromere-associated network (CCAN) pro-teins is controlled by a dynamic SUMO cycle and that the deSUMOylase SENP6 is necessary for stable maintenance of CENP-A at the centromere. Here, we discover that the removal of SENP6 leads to a rapid loss of the CCAN, followed by a delayed loss of centromeric CENP-A, indicating that the CCAN is the primary SUMO target. We found that the ATP-dependent segregase p97/VCP removes centromeric CENP-A in a SUMO-dependent manner and inter-acts physically with the CCAN and CENP-A chromatin. Our data suggest a direct role of p97 in removing centromeric CENP-A via SUMOylated CCAN proteins, thereby ensuring centro-mere homeostasis and potentially preventing ectopic CENP-A accumulation.

Transcriptional memory is characterized by a primed cellular state, induced by an external stimulus that results in an altered expression of primed genes upon re-exposure to the inducing signal. Intriguingly, the primed state is heritably maintained across somatic cell divisions even after the initial stimulus and target gene transcription cease. This phenomenon is widely observed across various organisms and appears to enable cells to retain a memory of external signals, thereby adapting to environmental changes. Signals range from nutrient supplies (food) to a variety of stress signals, including exposure to pathogens (foes), leading to long-term memory such as in the case of trained immunity in plants and mammals. Here, we review these priming phenomena and our current understanding of transcriptional memory. We consider different mechanistic models for how memory can work and discuss existing evidence for potential carriers of memory. Key molecular signatures include: the poising of RNA polymerase II machinery, maintenance of histone marks, as well as alterations in nuclear positioning and long-range chromatin interactions. Finally, we discuss the potential adaptive roles of transcriptional memory in the organismal response to its environment from nutrient sensing to trained immunity.

Centromeres are unique chromosomal loci that form the anchorage point for the mitotic spindle during mitosis and meiosis. Their position and function are specified by a unique chromatin domain featuring the histone H3 variant CENP-A. While typically formed on centromeric satellite arrays, CENP-A nucleosomes are maintained and assembled by a strong self-templated feedback mechanism that can propagate centromeres even at non-canonical sites. Central to the epigenetic chromatin-based transmission of centromeres is the stable inheritance of CENP-A nucleosomes. While long-lived at centromeres, CENP-A can turn over rapidly at non-centromeric sites and even erode from centromeres in non-dividing cells. Recently, SUMO modification of the centromere complex has come to the forefront as a mediator of centromere complex stability, including CENP-A chromatin. We review evidence from different models and discuss the emerging view that limited SUMOylation appears to play a constructive role in centromere complex formation, while polySUMOylation drives complex turnover. The deSUMOylase SENP6/Ulp2 and the proteins segregase p97/Cdc48 constitute the dominant opposing forces that balance CENP-A chromatin stability. This balance may be key to ensuring proper kinetochore strength at the centromere while preventing ectopic centromere formation.

Background Head and neck squamous cell carcinoma (HNSCC) are mainly diagnosed at advanced tumor stage after the onset of symptoms. Timely detection could improve treatment outcome. In the current OncSaliva study we determine the clinical performance of five tumor-specific DNA methylation markers using non-invasive liquid biopsy specimen.

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

Background To date, no predictive or prognostic molecular biomarkers except BRCA mutations are clinically established for epithelial ovarian cancer (EOC) despite being the deadliest gynecological malignancy. Aim of this biomarker study was the analysis of DNA methylation biomarkers for their prognostic value independent from clinical variables in a heterogeneous cohort of 203 EOC patients from two university medical centers. Results The marker combination CAMK2N1/RUNX3 exhibited a significant prognostic value for progression-free (PFS) and overall survival (OS) of sporadic platinum-sensitive EOC ( n = 188) both in univariate Kaplan–Meier (LogRank p < 0.05) and multivariate Cox regression analysis ( p < 0.05; hazard ratio HR = 1.587). KRT86 methylation showed a prognostic value only in univariate analysis because of an association with FIGO staging (Fisher’s exact test p < 0.01). Thus, it may represent a marker for EOC staging. Dichotomous prognostic values were observed for KATNAL2 methylation depending on BRCA aberrations. KATNAL2 methylation exhibited a negative prognostic value for PFS in sporadic EOC patients without BRCA1 methylation (HR 1.591, p = 0.012) but positive prognostic value in sporadic EOC with BRCA1 methylation (HR 0.332, p = 0.04) or BRCA -mutated EOC (HR 0.620, n.s.). Conclusion The retrospective analysis of 188 sporadic platinum-sensitive EOC proved an independent prognostic value of the methylation marker combination CAMK2N1/RUNX3 for PFS and OS. If validated prospectively this combination may identify EOC patients with worse prognosis after standard therapy potentially benefiting from intensive follow-up, maintenance therapies or inclusion in therapeutic studies. The dichotomous prognostic value of KATNAL2 should be validated in larger sample sets of EOC.

AbstractBackgroundIn patients diagnosed with cervical cancer, the purpose of lymphadenectomy is the removal of lymph nodes for diagnosis and potential treatment of metastasized tumor cells. It is unclear if afferent lymphatic vessels harbor tumor cells and, thus, may pose additional risk for recurrence or progression if not removed.AimIn this feasibility study, we analyzed the lymphatic vessels afferent to sentinel lymph node (SLN) using a highly sensitive and specific molecular marker for cervical cancer cells.Methods and ResultsTwenty patients diagnosed with cervical cancer of FIGO stage IA1 to IIB2 underwent laparoscopic SLN removal. Labeling was done using patent blue and the afferent lymphatic vessels were harvested from the parametric tissue and frozen at −80°C. HPV DNA type was evaluated in the primary tumor. Lymphatic vessels afferent to the sentinel lymph nodes were analyzed for the presence of viral oncogene transcripts of the respective HPV type. In one of 18 patients, all with tumor stage ≤IBI and pN0 by conventional histopathology, HPV mRNA could be detected in two of four lymphatic vessels, whereas at least one of the lymphatic vessel biopsies of both patients with tumors ˃4 cm and pN1 status was HPV mRNA positive. No clinical correlation with recurrence after a median follow‐up of 9 years was noticed.ConclusionHPV mRNA indicative of disseminated tumor cells could be detected in lymphatic vessels. The relevance of harvesting lymphatic vessels afferent to SLN in order to increase oncologic safety will have to be investigated in a future prospective study.

Cervical cancer (CC) arises from premalignant cervical intraepithelial neoplasia (CIN) induced by a persistent infection with human papillomaviruses. The multi-stepwise disease progression is driven by genetic and epigenetic alterations. Our previous studies demonstrated a clear downregulation of inter-α-trypsin-inhibitor-heavy chain 5 (ITIH5) at mRNA and protein levels in CC compared to CIN2/3 and normal cervical tissue. Initial in vitro functional analyses revealed a suppressive effect of ITIH5 on relevant mechanisms for cancer progression in conventional two dimensional (2D) cell culture model systems. Based on these studies, we aimed to investigate the functional relevance of ITIH5 in multicellular tumor spheroid (MCTS) models, which resemble in vivo tumors more closely. We successfully established CC cell line-derived MCTS using the hanging-drop technique. ITIH5 was ectopically overexpressed in HeLa and SiHa cells and its functional relevance was investigated under three dimensional (3D) culture conditions. We found that ITIH5 re-expression significantly suppressed tumor spheroid growth and spheroid invasiveness of both HeLa and SiHa spheroids. Immunohistochemical (IHC) analyses revealed a significant reduction in Ki-67 cell proliferation index and CAIX-positive areas indicative for hypoxia and acidification. Furthermore, we observed an increase in cPARP-positive cells suggesting a higher rate of apoptosis upon ITIH5 overexpression. An effect of ITIH5 expression on the susceptibility of cervical MCTS towards cytostatic drug treatment was not observed. Collectively, these data uncover pronounced anti-proliferative effects of ITIH5 under 3D cell culture conditions and provide further functional evidence that the downregulation of ITIH5 expression during cervical carcinogenesis could support cancer development.

The cellular machinery that regulates gene expression can be self-propagated across cell division cycles and even generations. This renders gene expression states and their associated phenotypes heritable, independently of genetic changes. These phenotypic states, in turn, can be subject to selection and may influence evolutionary adaptation. In this review, we will discuss the molecular basis of epigenetic inheritance, the extent of its transmission and mechanisms of evolutionary adaptation. The current work shows that heritable gene expression can facilitate the process of adaptation through the increase of survival in a novel environment and by enlarging the size of beneficial mutational targets. Moreover, epigenetic control of gene expression enables stochastic switching between different phenotypes in populations that can potentially facilitate adaptation in rapidly fluctuating environments. Ecological studies of the variation of epigenetic markers (e.g. DNA methylation patterns) in wild populations show a potential contribution of this mode of inheritance to local adaptation in nature. However, the extent of the adaptive contribution of the naturally occurring variation in epi-alleles compared to genetic variation remains unclear. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαβDAI), which harbours two Zα domains, and for a ZαβDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαβDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαβDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.

Pluripotent stem cells (PSCs) are central to development as they are the precursors of all cell types in the embryo. Therefore, maintaining a stable karyotype is essential, both for their physiological role as well as for their use in regenerative medicine. Karyotype abnormalities in PSCs in culture are common but the underlying causes remain unknown. To gain insight, we explore the composition of the centromere and kinetochore in human embryonic and induced PSCs. Centromere function depends on CENP-A nucleosome-defined chromatin. We show that while PSCs maintain abundant pools of CENP-A, CENP-C and CENP-T, these essential centromere components are strongly reduced at stem cell centromeres. Outer kinetochore recruitment is also impaired to a lesser extent, indicating an overall weaker kinetochore while the inner centromere protein Aurora B remains unaffected. We further show that, similar to differentiated human cells, CENP-A chromatin assembly in PSCs requires transition into G1 phase. Finally, reprogramming experiments indicate that reduction of centromeric CENP-A levels is an early event during dedifferentiation, coinciding with global chromatin remodelling. Our characterization of centromeres in human stem cells suggests a possible link between impaired centromere function and stem cell aneuploidies.