Epigenetic centromere identity is precisely maintained through DNA replication but is uniquely specified among human cells.

Centromere identity is defined and maintained epigenetically by the presence of the histone variant CENP-A. How centromeric CENP-A position is specified and precisely maintained through DNA replication is not fully understood. The recently released Telomere-to-Telomere (T2T) genome assembly containing the first complete human centromere sequences provides a new resource for examining CENP-A position. Mapping CENP-A position in clones of the same cell line to the T2T assembly identified highly similar CENP-A position after multiple cell divisions. In contrast, centromeric CENP-A epialleles were evident at several centromeres of different human cell lines, demonstrating the location of CENP-A enrichment and the site of kinetochore recruitment vary among human cells. Across the cell cycle, CENP-A molecules deposited in G1 phase are maintained in their precise position through DNA replication. Thus, despite CENP-A dilution during DNA replication, CENP-A is precisely reloaded onto the same sequences within the daughter centromeres, maintaining unique centromere identity among human cells.

Chromothripsis drives the evolution of gene amplification in cancer.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.

Guarding the Genome: CENP-A-Chromatin in Health and Cancer.

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

DNA replication acts as an error correction mechanism to maintain centromere identity by restricting CENP-A to centromeres.

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.

Phosphorylation of CENP-A on serine 7 does not control centromere function.

CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function.

Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points.

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat-containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1-containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2-4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases.

Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis.

Liver cancer, which typically develops on a background of chronic liver inflammation, is now the second leading cause of cancer mortality worldwide. For patients with liver cancer, surgical resection is a principal treatment modality that offers a chance of prolonged survival. However, tumor recurrence after resection, the mechanisms of which remain obscure, markedly limits the long-term survival of these patients. We have shown that partial hepatectomy in multidrug resistance 2 knockout (Mdr2-/- ) mice, a model of chronic inflammation-associated liver cancer, significantly accelerates hepatocarcinogenesis. Here, we explore the postsurgical mechanisms that drive accelerated hepatocarcinogenesis in Mdr2-/- mice by perioperative pharmacological inhibition of interleukin-6 (IL6), which is a crucial liver regeneration priming cytokine. We demonstrate that inhibition of IL6 signaling dramatically impedes tumorigenesis following partial hepatectomy without compromising survival or liver mass recovery. IL6 blockade significantly inhibited hepatocyte cell cycle progression while promoting a hypertrophic regenerative response, without increasing apoptosis. Mdr2-/- mice contain hepatocytes with a notable persistent DNA damage response (γH2AX, 53BP1) due to chronic inflammation. We show that liver regeneration in this microenvironment leads to a striking increase in hepatocytes bearing micronuclei, a marker of genomic instability, which is suppressed by IL6 blockade. CONCLUSION: Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611).

CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly.

Human centromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive alphoid DNA sequences. By inducing rapid, complete degradation of endogenous CENP-A, we now demonstrate that once the first steps of centromere assembly have been completed in G1/S, continued CENP-A binding is not required for maintaining kinetochore attachment to centromeres or for centromere function in the next mitosis. Degradation of CENP-A prior to kinetochore assembly is found to block deposition of CENP-C and CENP-N, but not CENP-T, thereby producing defective kinetochores and failure of chromosome segregation. Without the continuing presence of CENP-A, CENP-B binding to alphoid DNA sequences becomes essential to preserve anchoring of CENP-C and the kinetochore to each centromere. Thus, there is a reciprocal interdependency of CENP-A chromatin and the underlying repetitive centromere DNA sequences bound by CENP-B in the maintenance of human chromosome segregation.

A two-step mechanism for epigenetic specification of centromere identity and function.

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

In vivo evidence suggesting reciprocal renal hypoxia-inducible factor-1 upregulation and signal transducer and activator of transcription 3 activation in response to hypoxic and non-hypoxic stimuli.

In vitro studies suggest that combined activation of hypoxia-inducible factor (HIF) and signal transducer and activator of transcription 3 (STAT3) promotes the hypoxia response. However, their interrelationship in vivo remains poorly defined. The present study investigated the possible relationship between HIF-1 upregulation and STAT3 activation in the rodent kidney in vivo. Activation of HIF-1 and STAT3 was analysed by immunohistochemical staining and western blot analysis in: (i) models of hypoxia-associated kidney injury induced by radiocontrast media or rhabdomyolysis; (ii) following activation of STAT3 by the interleukin (IL)-6-soluble IL-6 receptor complex; or (iii) following HIF-1α stabilization using hypoxic and non-hypoxic stimuli (mimosine, FG-4497, CO, CoCl(2)) and in targeted von Hippel-Lindau-knockout mice. Western blot analysis and immunostaining revealed marked induction of both transcription factors under all conditions tested, suggesting that in vivo STAT3 can trigger HIF and vice versa. Colocalization of HIF-1α and phosphorylated STAT3 was detected in some, but not all, renal cell types, suggesting that in some cells a paracrine mechanism may be responsible for the reciprocal activation of the two transcription factors. Nevertheless, in several cell types spatial concordance was observed under the majority of conditions tested, suggesting that HIF-1 and STAT3 may act as cotranscription factors. These in vivo studies suggest that, in response to renal hypoxic-stress, upregulation of HIF-1 and activation of STAT3 may be both reciprocal and cell type dependent.

Replicating centromeric chromatin: spatial and temporal control of CENP-A assembly.

The centromere is the fundamental unit for insuring chromosome inheritance. This complex region has a distinct type of chromatin in which histone H3 is replaced by a structurally different homologue identified in humans as CENP-A. In metazoans, specific DNA sequences are neither required nor sufficient for centromere identity. Rather, an epigenetic mark comprised of CENP-A containing chromatin is thought to be the major determinant of centromere identity. In this view, CENP-A deposition and chromatin assembly are fundamental processes for the maintenance of centromeric identity across mitotic and meiotic divisions. Several lines of evidence support CENP-A deposition in metazoans occurring at only one time in the cell cycle. Such cell cycle-dependent loading of CENP-A is found in divergent species from human to fission yeast, albeit with differences in the cell cycle point at which CENP-A is assembled. Cell cycle dependent CENP-A deposition requires multiple assembly factors for its deposition and maintenance. This review discusses the regulation of new CENP-A deposition and its relevance to centromere identity and inheritance.

Diabetes blockade of sevoflurane postconditioning is not restored by insulin in the rat heart: phosphorylated signal transducer and activator of transcription 3- and phosphatidylinositol 3-kinase-mediated inhibition.

BACKGROUND: The possibility of restoring sevoflurane postconditioning (sevo-postC) cardioprotection in diabetic animals is uncertain. We hypothesized that attenuation of myocardial injury by sevo-postC might be hindered by inhibition of signal transducer and activator of transcription (STAT) 3-regulated activity of phosphatidylinositol 3-kinase (PI3K) in diabetic animals. To determine whether postC cardioprotection can be restored by normoglycemia, we treated rats with insulin. METHODS: Diabetic or nondiabetic rats were randomly subjected to 30-min ischemia/reperfusion, with ischemic postC or sevo-postC, with and without mitochondrial adenosine triphosphate-dependent potassium channel blocker 5-hydroxy decanoate sodium and PI3K antagonist wortmannin. The infarct area, phosphorylated STAT3, and apoptosis were examined. Studies were repeated after insulin treatment. RESULTS: Ischemic postC and sevo-postC significantly reduced infarct size by 50% in the nondiabetic rats (P < 0.002), a phenomenon completely reversed by 5-hydroxy decanoate sodium and wortmannin. Diabetes mellitus blocked the protective effect of postC, and insulin treatment to achieve normoglycemia did not restore cardioprotection. Phosphorylated STAT3 nuclear retention was significantly increased after ischemia-reperfusion and was further enhanced in response to ischemic postC (P < 0.05) but was significantly reduced in diabetic rats (by 43%; P < 0.01). CONCLUSIONS: The effective reduction in infarct size and apoptosis in the nondiabetic rat heart by postC was completely abrogated in diabetic rats. This inhibition is not relieved by insulin-induced normoglycemia. The PI3K pathway and mitochondrial adenosine triphosphate-dependent potassium channel activation are involved in the mechanism of postC. In diabetic rats, STAT3 activation was strongly reduced, as was postC cardioprotection, suggesting that the inability of insulin to restore postC may be attributed to diabetes-induced STAT3-mediated inhibition of PI3K signaling.

Early hepatocyte DNA synthetic response posthepatectomy is modulated by IL-6 trans-signaling and PI3K/AKT activation.

BACKGROUND & AIMS: Interleukin-6 (IL-6) is a crucial factor in liver regeneration following partial hepatectomy (PH); however, the role of IL-6 and IL-6 trans-signaling in particular, in hepatocyte mitosis remains controversial. IL-6 trans-signaling relies upon the release of the soluble IL-6R (sIL-6R), which binds IL-6 to form an agonistic IL-6/sIL-6R complex. Herein we have examined the hypothesis that IL-6 trans-signaling plays a crucial and distinct role in liver regeneration following PH. METHODS: The specific IL-6/sIL-6R antagonist, sgp130Fc, was expressed in mice and analyzed for its effect on hepatocyte mitosis following PH. Alternatively, we examined the effect of the IL-6/sIL-6R super-agonist, Hyper-IL-6, or IL-6 expressed either alone or in combination with hepatocyte growth factor (HGF) on hepatocyte mitosis in the absence of PH. RESULTS: Following PH, the dramatic rise of circulating IL-6 levels is accompanied by a concurrent ∼2-fold increase in circulating sIL-6R levels. Ectopic expression of sgp130Fc reduced hepatocyte mitosis by about 40% at early times following PH, while substantially reducing AKT, but not STAT3, activation. But, ectopic Hyper-IL-6 expression in mice without PH was not mitogenic to hepatocytes in vivo. Rather, Hyper-IL-6, but not IL-6, markedly increased HGF-induced hepatocyte mitosis. This cooperative effect correlated with greater resistance of HIL-6 than IL-6 to HGF-mediated reduction of AKT activation, rather than changes in STAT3 or MAPK signaling, and was completely blocked by PI3K inhibition. CONCLUSIONS: Following PH, IL-6/sIL-6R cooperates with growth factors, through a PI3K/AKT-dependent mechanism to promote entry of hepatocytes into the cell cycle.

IL-6/IL-6R axis plays a critical role in acute kidney injury.

The response to tissue injury involves the coordination of inflammatory and repair processes. IL-6 expression correlates with the onset and severity of acute kidney injury (AKI), but its contribution to pathogenesis remains unclear. This study established a critical role for IL-6 in both the inflammatory response and the resolution of AKI. IL-6-deficient mice were resistant to HgCl2-induced AKI compared with wild-type mice. The accumulation of peritubular neutrophils was lower in IL-6-deficient mice than in wild-type mice, and neutrophil depletion before HgCl2 administration in wild-type mice significantly reduced AKI; these results demonstrate the critical role of IL-6 signaling in the injurious inflammatory process in AKI. Renal IL-6 expression and STAT3 activation in renal tubular epithelial cells significantly increased during the development of injury, suggesting active IL-6 signaling. Although a lack of renal IL-6 receptors (IL-6R) precludes the activation of classical signaling pathways, IL-6 can stimulate target cells together with a soluble form of the IL-6R (sIL-6R) in a process termed trans-signaling. During injury,serum sIL-6R levels increased three-fold, suggesting a possible role for IL-6 trans-signaling in AKI. Stimulation of IL-6 trans-signaling with an IL-6/sIL-6R fusion protein activated STAT3 in renal tubular epithelium and prevented AKI. IL-6/sIL-6R reduced lipid peroxidation after injury, suggesting that its protective effect may be largely mediated through amelioration of oxidative stress. In summary, IL-6 simultaneously promotes an injurious inflammatory response and, through a mechanism of trans-signaling, protects the kidney from further injury.