Quantitative assessment of higher-order chromatin structure of the INK4/ARF locus in human senescent cells

Somatic cells can be reset to oncogene-induced senescent (OIS) cells or induced pluripotent stem (iPS) cells by expressing specified factors. The INK4/ARF locus encodes p15(INK4b) , ARF, and p16(INK4a) genes in human chromosome 9p21, the products of which are known as common key reprogramming regulators. Compared with growing fibroblasts, the CCCTC-binding factor CTCF is remarkably up-regulated in iPS cells with silencing of the three genes in the locus and is reversely down-regulated in OIS cells with high expression of p15(INK4b) and p16(INK4a) genes. There are at least three CTCF-enriched sites in the INK4/ARF locus, which possess chromatin loop-forming activities. These CTCF-enriched sites and the p16(INK4a) promoter associate to form compact chromatin loops in growing fibroblasts, while CTCF depletion disrupts the loop structure. Interestingly, the loose chromatin structure is found in OIS cells. In addition, the INK4/ARF locus has an intermediate type of chromatin compaction in iPS cells. These results suggest that senescent cells have distinct higher-order chromatin signature in the INK4/ARF locus.     

Mutations in the INK4a/ARF melanoma susceptibility locus functionally impair p14ARF

The INK4a/ARF locus encodes two cell cycle regulatory proteins, the cyclin-dependent kinase inhibitor, p16(INK4a), and the p53 activator, p14(ARF). Germline mutations in this locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Many of these mutations specifically impair p16(INK4a), whereas mutations uniquely targeting p14(ARF) are rare. Nevertheless, the importance of p14(ARF) has not been excluded because more than 40% of INK4a/ARF alterations affect p16(INK4a) and p14(ARF). We now report that p14(ARF) is functionally impaired in melanoma kindreds carrying INK4a/ARF mutations. Of the seven INK4a/ARF mutations tested, three altered the subcellular distribution of p14(ARF) and diminished the ability of p14(ARF) to activate the p53 pathway. This work establishes the importance of p14(ARF) in melanoma predisposition.     

p16INK4A and p19ARF act in overlapping pathways in cellular immortalization

The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.     

The regulation of INK4/ARF in cancer and aging

Loss of the INK4a/ARF/INK4b locus on chromosome 9p21 is among the most frequent cytogenetic events in human cancer. The products of the locus--p15(INK4b), p16(INK4a), and ARF--play widespread and independent roles in tumor suppression. Recent data also suggest that expression of p16(INK4a) induces an age-dependent decrease in the proliferative capacity of certain tissue-specific stem cells and unipotent progenitors. Here, we discuss the regulation and role of p16(INK4a), ARF, and p15(INK4b) in cancer and aging.     

Epigenetic regulation of the INK4b-ARF-INK4a locus: In sickness and in health

The INK4b-ARF-INK4a locus encodes for two cyclin-dependent kinase inhibitors, p15^INK4b and p16^INK4a, and a regulator of the p53 pathway, ARF. In addition ANRIL , a non-coding RNA, is also transcribed from the locus. ARF, p15^INK4b and p16^INK4a are well-established tumor suppressors which function is frequently disabled in human cancers. Recent studies showed that single nucleotide polymorphisms mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including cardiovascular disease, ischemic stroke, type 2 diabetes, frailty and Alzheimer disease. The INK4b-ARF-INK4a locus is regulated by Polycomb repressive complexes (PRCs) and its expression can be invoked by activating signals. Other epigenetic modifiers such as the histone demethylases JMJD3 and JHDM1B, the SWI/SNF chromatin remodeling complex and DNA methyltransferases regulate the locus interplaying with PRCs. In view of the intimate involvement of the INK4b-ARF-INK4a locus on disease, to understand its regulation is the first step for manipulate it to therapeutic benefit.Key words: senescence, p16^INK4a, ARF, p15^INK4b, ANRIL, polycomb, histone demethylases, DNA methylation     

Modeling INK4/ARF tumor suppression in the mouse

The INK4/ARF locus encodes the p15(INK4B), p16(INK4A) and p14(ARF) tumor suppressor proteins whose loss of function is associated with the pathogenesis of many human cancers. Dissecting the relative contribution of these genes to growth control in vivo is complicated by their physical contiguity and the frequency of homozygous deletions that inactivate all three components of this locus. While genetically engineered mouse models provide a rigorous system for elucidating cancer gene function, there is some evidence to suggest there are cross-species differences in regulating tumor biology. Given the prevalence of mouse models in cancer research and the potential contribution of such models to preclinical studies, it is important determine to what degree the function of these critical tumor suppressors is conserved between organisms. In this review, we assess the relative biological roles of INK4A, INK4B and ARF in mice and humans with the aim of determining the faithfulness of mouse models and also of obtaining insights into the pattern of specific tumor types that are associated with germline and somatic mutations at components of this locus. We will discuss 1) the contribution of INK4A, INK4B and ARF to growth control in vitro in a series of cell types, 2) the in vivo phenotypes associated with germline loss of function of this locus and 3) the study of Ink4a and Arf in different cancer-specific mouse models.     

The ARF-p53 senescence pathway in mouse and human cells

Mouse and human cells have most frequently been used for studies that have led to the elucidation of various molecular pathways involved in senescence. The ARF-p53 pathway has been assigned as one of the major protagonists in these phenomena. ARF is an alternative reading frame protein encoded along with p16INK4A by the INK4a locus on human chromosome 9p21 and the corresponding locus on mouse chromosome 4. Whereas the mouse ARF (p19ARF) consists of 169 amino acids, the human ARF (p14ARF) consists of 132 amino acids, truncated at the C-terminus. Molecular studies on the regulation of ARF activity by its binding partners have revealed that mouse ARF protein, but not human ARF protein, interacts with a cytoplasmic protein, Pex19p. This interaction of mouse ARF with Pex19p results in its milder p53 activation function in mouse cells as compared to human cells and thus accounts, at least in part, for the weaker tumor surveillance and frequent immortalization of mouse cells.     

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.     

Epigenetic regulation of the INK4b-ARF-INK4a locus

The INK4b-ARF-INK4a locus encodes for two cyclin-dependent kinase inhibitors, p15^INK4b and p16^INK4a and a regulator of the p53 pathway, ARF. In addition ANRIL, a non-coding RNA, is also transcribed from the locus. ARF, p15^INK4b and p16^INK4a are well-established tumor suppressors which function is frequently disabled in human cancers. Recent studies showed that single nucleotide polymorphisms mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including cardiovascular disease, ischemic stroke, type 2 diabetes, frailty and Alzheimer’s disease. The INK4b-ARF-INK4a locus is regulated by Polycomb repressive complexes (PRCs), and its expression can be invoked by activating signals. Other epigenetic modifiers such as the histone demethylases JMJD3 and JHDM1B, the SWI/SNF chromatin remodeling complex and DNA methyltransferases regulate the locus interplaying with PRCs. In view of the intimate involvement of the INK4b-ARF-INK4a locus on disease, to understand its regulation is the first step for manipulate it to therapeutic benefit.     

INK4a/ARF: a multifunctional tumor suppressor locus

The INK4a/ARF locus encodes two physically linked tumor suppressor proteins, p16(INK4a) and ARF, which regulate the RB and p53 pathways, respectively. The unusual genomic relationship of the open reading frames of these proteins initially fueled speculation that only one of the two was the true tumor suppressor, and loss of the other merely coincidental in cancer. Recent human and mouse genetic data, however, have firmly established that both proteins possess significant in vivo tumor suppressor activity, although there appear to be species- and cell-type specific differences between the two. For example, ARF plays a clear role in preventing Myc-induced lymphomagenesis in mice, whereas the role for p16(INK4a) is human carcinomas is more firmly established. In this review, I discuss the evolutionary history of the locus, the relative importance of these tumor suppressor genes in human cancer, and recent information suggesting novel biochemical and physiologic functions of these proteins in vivo.     

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.     

Regulation of the INK4a/ARF locus by histone deacetylase inhibitors

Despite the importance of the INK4a/ARF locus in tumor suppression, its modulation by histone deacetylase inhibitors (HDACis) remains to be characterized. Here, we have shown that the levels of p16INK4a are decreased in human and murine fibroblasts upon exposure to relatively high concentrations of trichostatin A and sodium butyrate. Interestingly, the levels of p19ARF are strongly upregulated in murine cells even at low concentrations of HDACis. Using ARF-deficient cells, we have demonstrated that p19ARF plays an active role in HDACi-triggered cytostasis and the contribution of p19ARF to this arrest is of higher magnitude than that of the well established HDACi target p21Waf1/Cip. Moreover, chemically induced fibrosarcomas in ARF-null mice are more resistant to the therapeutic effect of HDACis than similar tumors in wild type or p21Waf1/Cip-null mice. Together, our results have established the tumor suppressor ARF as a relevant target for HDACi chemotherapy.     

E2F and Ras synergize in transcriptionally activating p14ARF expression

The INK4a/ARF locus, which is frequently inactivated in human tumors, encodes two distinct tumor suppressive proteins, ARF and p16INK4a. ARF stabilizes and activates p53 by negating the effects of mdm2 on p53. Furthermore, its function is not restricted to the p53 pathway and it also inhibits cell proliferation in cells lacking p53. Expression of ARF is up-regulated in response to a number of oncogenic stimuli including E2F1. We show here that while oncogenic Ras does not significantly affect p1(4AR)F expression in normal human cells it activates p1(4AR)F in cells containing deregulated E2F. Moreover, oncogenic Ras and E2F1 synergize in activating p1(4AR)F expression. Activation of p1(4AR)F promoter by E2F1 persists in the absence of the consensus E2F-binding sites in this promoter, indicating that this activation also occurs through non- canonical binding sites. The activation by oncogenic Ras requires both E2F and Sp-1 activity, demonstrating the complex regulation of p14(ARF) in response to oncogenic stimuli.     

INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.