Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.DOI: http://dx.doi.org/10.7554/eLife.13722.001

The cell proliferation antigen Ki-67 is widely used in cancer histopathology, but estimations of Ki-67 expression levels are inconsistent and understanding of its regulation is limited. Here we show that cell-cycle regulation underlies variable Ki-67 expression in all situations analyzed, including nontransformed human cells, normal mouse intestinal epithelia and adenomas, human cancer cell lines with or without drug treatments, and human breast and colon cancers. In normal cells, Ki-67 was a late marker of cell-cycle entry; Ki-67 mRNA oscillated with highest levels in G while protein levels increased throughout the cell cycle, peaking in mitosis. Inhibition of CDK4/CDK6 revealed proteasome-mediated Ki-67 degradation in G After cell-cycle exit, low-level Ki-67 expression persisted but was undetectable in fully quiescent differentiated cells or senescent cells. CDK4/CDK6 inhibition and in tumors in mice caused G cell-cycle arrest and eliminated Ki-67 mRNA in RB1-positive cells but had no effect in RB1-negative cells, which continued to proliferate and express Ki-67. Thus, Ki-67 expression varies due to cell-cycle regulation, but it remains a reliable readout for effects of CDK4/CDK6 inhibitors on cell proliferation. .

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56bright, NK cells. Most NK cells in healthy human donors are CD45RA+CD45RO-. The CD45RA-RO+ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RAdimRO- (CD45RAdim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RAdim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RAdim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells.

Summary Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible.

Ki-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial–mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses.

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22–producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ–producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ–expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22–expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22–inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.