SummaryCentromeres are specified by sequence-independent epigenetic mechanisms in most organisms. Rarely, centromere repositioning results in neocentromere formation at ectopic sites. However, the mechanisms governing how and where neocentromeres form are unknown. Here, we established a chromosome-engineering system in chicken DT40 cells that allowed us to efficiently isolate neocentromere-containing chromosomes. Neocentromeres appear to be structurally and functionally equivalent to native centromeres. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis with 18 neocentromeres revealed that the centromere-specific histone H3 variant CENP-A occupies an ∼40 kb region at each neocentromere, which has no preference for specific DNA sequence motifs. Furthermore, we found that neocentromeres were not associated with histone modifications H3K9me3, H3K4me2, and H3K36me3 or with early replication timing. Importantly, low but significant levels of CENP-A are detected around endogenous centromeres, which are capable of seeding neocentromere assembly if the centromere core is removed. In summary, our experimental system provides valuable insights for understanding how neocentromeres form.

Leukemia cells in the bone marrow (BM) must meet the biochemical demands of increased cell proliferation and also survive by continually adapting to fluctuations in nutrient and oxygen availability. Thus, targeting metabolic abnormalities in leukemia cells located in the BM is a novel therapeutic approach. In the present study, we investigated the metabolic role of BM adipocytes in supporting the growth of leukemic blasts. Prevention of nutrient starvation-induced apoptosis of leukemic cells by BM adipocytes, as well as the metabolic and molecular mechanisms involved in this process, were investigated using various analytical techniques. In acute monocytic leukemia (AMoL) cells, the prevention of spontaneous apoptosis by BM adipocytes was associated with an increase in fatty acid β-oxidation (FAO) along with the upregulation of PPARγ, FABP4, CD36, and BCL2 genes. In AMoL cells, BM adipocyte co-culture increased adiponectin receptor gene expression and its downstream target stress response kinase AMPK, p38 MAPK with autophagy activation, and upregulated antiapoptotic chaperone heat shock proteins. Inhibition of FAO disrupted metabolic homeostasis, increased reactive oxygen species production, induced the integrated stress response mediator ATF4, and apoptosis in AMoL cells co-cultured with BM adipocytes. Our results suggest that BM adipocytes support AMoL cell survival by regulating their metabolic energy balance, and that the disruption of FAO in BM adipocytes may be an alternative, novel therapeutic strategy for AMoL therapy.

SummaryIn vertebrate cells, centromeres are specified epigenetically through the deposition of the centromere-specific histone CENP-A. Following CENP-A deposition, additional proteins are assembled on centromeric chromatin. However, it remains unknown whether additional epigenetic features of centromeric chromatin are required for kinetochore assembly. Here, we used ChIP-seq analysis to examine centromere-specific histone modifications at chicken centromeres, which lack highly repetitive sequences. We found that H4K20 monomethylation (H4K20me1) is enriched at centromeres. Immunofluorescence and biochemical analyses revealed that H4K20me1 is present at all centromeres in chicken and human cells. Based on immunoprecipitation data, H4K20me1 occurs primarily on the histone H4 that is assembled as part of the CENP-A nucleosome following deposition of CENP-A into centromeres. Targeting the H4K20me1-specific demethylase PHF8 to centromeres reduces the level of H4K20me1 at centromeres and results in kinetochore assembly defects. We conclude that H4K20me1 modification of CENP-A nucleosomes contributes to functional kinetochore assembly.

Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A–H4–HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48–Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A–H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.