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     

Methylation of p15INK4b and Expression of ANRIL on Chromosome 9p21 Are Associated with Coronary Artery Disease

Background

Genome-wide association studies have identified that multiple single nucleiotide polymorphisms on chromosome 9p21 are tightly associated with coronary artery disease (CAD). However, the mechanism linking this risk locus to CAD remains unclear.

Methodology/Principal Findings

The methylation status of six candidate genes (BAX, BCL-2, TIMP3, p14^ARF, p15^INK4b and p16^INK4a) in 205 patients and controls who underwent coronary angiography were analyzed by quantitative MethyLight assay. Rs10757274 was genotyped and expression of INK4/ARF and antisense non-coding RNA in the INK4 locus (ANRIL) was determined by real-time RT-PCR. Compared with controls, DNA methylation levels at p15^INK4b significantly increased in CAD patients (p = 0.006). To validate and dissect the methylation percentage of each target CpG site at p15^INK4b, pyrosequencing was performed, finding CpG +314 and +332 remarkably hypermethylated in CAD patients. Further investigation determined that p15^INK4b hypermethylation prevalently emerged in lymphocytes of CAD patients (p = 0.013). The rs10757274 genotype was significantly associated with CAD (p = 0.003) and GG genotype carriers had a higher level of ANRIL exon 1–5 expression compared among three genotypes (p = 0.009). There was a stepwise increase in p15^INK4b and p16^INK4a methylation as ANRIL exon 1–5 expression elevated (r = 0.23, p = 0.001 and r = 0.24, p = 0.001, respectively), although neither of two loci methylation was directly linked to rs10757274 genotype.

Conclusions/Significance

p15^INK4b methylation is associated with CAD and ANRIL expression. The epigenetic changes in p15^INK4b methylation and ANRIL expression may involve in the mechanisms of chromosome 9p21 on CAD development.     

Co-regulation of senescence-associated genes by oncogenic homeobox proteins and polycomb repressive complexes

Cellular senescence is a stable cell cycle arrest that can be induced by stresses such as telomere shortening, oncogene activation or DNA damage. Senescence is a potent anticancer barrier that needs to be circumvented during tumorigenesis. The cell cycle regulator p16^INK4a is a key effector upregulated during senescence. Polycomb repressive complexes (PRCs) play a crucial role in silencing the INK4/ARF locus, which encodes for p16^INK4a, but the mechanisms by which PRCs are recruited to this locus as well as to other targets remain poorly understood. Recently we discovered the ability of the homeobox proteins HLX1 (H2.0-like homeobox 1) and HOXA9 (Homeobox A9) to bypass senescence. We showed that HLX1 and HOXA9 recruit PRCs to repress INK4a, which constitutes a key mechanism explaining their effects on senescence. Here we provide evidence for the regulation of additional senescence-associated PRC target genes by HLX1 and HOXA9. As both HLX1 and HOXA9 are oncogenes implicated in leukemogenesis, we discuss the implications that the collaboration between Homeobox proteins and PRCs has for senescence and cancer.     

An AC-repeat adjacent to mouse Cdkn2B allows the detection of specific allelic losses in the p15 INK4b and p16 INK4a tumor suppressor genes

The cyclin-dependent kinase inhibitors p15 ^INK4b and p16 ^INK4a are involved in the development of a wide range of human and murine tumors. These tumor suppressor genes are inactivated by deletions frequently associated to point mutations in the coding regions or hypermethylation of their promoters. In this work, we describe a simple-sequence length polymorphism located in mouse Chromosome (Chr) 4, between the Cdkn2B (p15 ^INK4b ) and Cdkn2A (p16 ^INK4a ) genes, only 700 bp downstream of the Cdkn2B locus. This DNA region was analyzed in different inbred strains showing a variable AC-repetitive DNA sequence. We used this microsatellite to detect loss of heterozygosity of the Cdkn2A and Cdkn2B loci in γ-irradiation-induced thymic lymphomas of C57BL/6J × RF/J F₁ hybrids. Using this specific marker, we were able to locate additional allelic losses not detected by other microsatellites. Since the allelic losses can be detected by a simple PCR amplification, this AC-repetitive sequence is specially useful as a genetic marker for these Cdkn2 genes and specifically for the p15 ^INK4b cell cycle inhibitor. Received: 27 August 1997 / Accepted: 13 November 1997     

SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus

Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16(INK4a) induction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15(INK4b) and p16(INK4a), but not of p14(ARF). Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control.     

Methylation profile of INK4B-ARF-INK4A locus in atherosclerosis

Single-nucleotide polymorphisms (SNPs) in the 9p21.3 locus have recently been demonstrated to be strongly associated with atherosclerosis. However, the pathophysiology of this locus is insufficiently studied. Here, the methylation profile of the nearest mapped genes for cyclin-dependent kinase inhibitors CDKN2A (p16^INK4a, p14^ARF) and CDKN2B (p15^INK4b) in the tissues of the carotid artery in patients with atherosclerosis was evaluated for the first time. Aberrant DNA methylation of the analyzed loci was not established in either the atherosclerotic plaques or in the tissues from the macroscopically intact vascular wall in the same patients.     

ARF——一种新的抑癌因子的研究进展

INK4a/ARF基因位于人染色体9p21,是人类肿瘤中最常见的基因失活位点之一.INK4a/ARF基因有两套各自独立的启动子,通过可变阅读框,能够编码两种蛋白质：p16^INK4a和p14^ARF（ARF在鼠细胞中为p19^ARF）.p16作为CDK4/6的抑制因子,能够阻断pRb磷酸化,将细胞周期阻断在G1期;而ARF可结合原癌蛋白MDM2,稳定p53,将细胞周期阻断在G1期和G2/M转换期,或诱导细胞凋亡.因此ARF蛋白和p16一样也是一种肿瘤抑制因子.     

Expression of p15INK4b and p57KIP2 and Relationship with Clinicopathological Features and Prognosis in Patients with Vulvar Squamous Cell Carcinoma

Background

The cyclin-dependent kinase inhibitors p15^INK4b and p57^KIP2 are important regulators of the cell cycle, and their abnormal expression has been detected in various tumors. However, little is known about the role of p15^INK4b and p57^KIP2 in the pathogenesis of vulvar carcinoma, and the prognostic impact is still unknown. In our current study, we examined the expression of p15^INK4b and p57^KIP2 in a large series of vulvar squamous cell carcinomas to elucidate the prognostic impact.

Methods

Expression of p15^INK4b and p57^KIP2 were examined in 297 vulvar squamous cell carcinomas using immunohistochemistry. Both uni- and multivariate analysis of prognostic factors were performed, and correlations with clinicopathologic parameters were examined.

Results

Compared to the high levels of p15^INK4b and p57^KIP2 in normal vulvar squamous epithelium, low levels of p15^INK4b and p57^KIP2 were found in 82% and 44% of vulvar carcinomas, respectively. Low levels of p15^INK4b and p57^KIP2 correlated significantly with malignant features, including large tumor diameter (p = 0.03 and p = 0.001, respectively) and increased invasiveness (p = 0.003 and p = 0.04, respectively). Although p15^INK4b and p57^KIP2 levels could not be identified as prognostic markers, combined analysis of p14^ARF/p15^INK4b/p16^INK4a showed that patients whose tumors expressed low levels of two or three of these INK4 proteins had a worse prognosis than those with only low levels of one or no protein (univariate analysis p = 0.02). The independent prognostic significance of these INK4 proteins was confirmed by multivariate analysis (p = 0.008).

Conclusions

We show for the first time that p15^INK4b and p57^KIP2 may be involved in the progression of vulvar carcinomas and the combined p14^ARF/p15^INK4b/p16^INK4a status was a statistically independent prognostic factor.     

Bone marrow p16INK4a-deficiency does not modulate obesity, glucose homeostasis or atherosclerosis development

Objective

A genomic region near the CDKN2A locus, encoding p16^INK4a, has been associated to type 2 diabetes and atherosclerotic vascular disease, conditions in which inflammation plays an important role. Recently, we found that deficiency of p16^INK4a results in decreased inflammatory signaling in murine macrophages and that p16^INK4a influences the phenotype of human adipose tissue macrophages. Therefore, we investigated the influence of immune cell p16^INK4a on glucose tolerance and atherosclerosis in mice.

Methods and Results

Bone marrow p16^INK4a-deficiency in C57Bl6 mice did not influence high fat diet-induced obesity nor plasma glucose and lipid levels. Glucose tolerance tests showed no alterations in high fat diet-induced glucose intolerance. While bone marrow p16^INK4a-deficiency did not affect the gene expression profile of adipose tissue, hepatic expression of the alternative markers Chi3l3, Mgl2 and IL10 was increased and the induction of pro-inflammatory Nos2 was restrained on the high fat diet. Bone marrow p16^INK4a-deficiency in low density lipoprotein receptor-deficient mice did not affect western diet-induced atherosclerotic plaque size or morphology. In line, plasma lipid levels remained unaffected and p16^INK4a-deficient macrophages displayed equal cholesterol uptake and efflux compared to wild type macrophages.

Conclusion

Bone marrow p16^INK4a-deficiency does not affect plasma lipids, obesity, glucose tolerance or atherosclerosis in mice.     

p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

Introduction

Recent evidence suggests that tissue accumulation of senescent p16^INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16^INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16^INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).

Methods

We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16^INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16^INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.

Results

p16^INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16^INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16^INK4a. Accordingly, p16^INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.

Conclusions

We disclosed herein a new role of the senescence marker p16^INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.     

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.     

How Disruption of Cell Cycle Regulators Might Predispose to Sun-Induced Skin Cancer

The Ink4a/Arf ( CDKN2a) locus encodes two proteins that regulate distinct important tumor suppressor pathways represented by p53 and Rb. Loss of either p16^INK4a or p19^ARF was recently reported to reduce the ability of mouse cells to repair UV-induced DNA damage and to induce a UV-mutator phenotype. This observation was independent of cell cycle effects incurred by either p16INK4a and/or p19ARF loss, as it was demonstrable in unirradiated cells using UV-treated DNA. We suggest that this might explain why germ line mutations of INK4a/ARF predispose mainly to malignant melanoma, a UV-induced skin cancer, and provides a molecular explanation for the link between melanoma-genesis and impaired DNA repair. It also further demonstrates that regulation of cell cycle check points and DNA repair in response to genomic insults, such as ultraviolet irradiation are intricately interwoven processes. Differences in the apoptotic response to ultraviolet light between melanocytes and keratinocytes might explain why INK4a/ARF mutations predispose to malignant melanoma, but not to keratinocyte-derived skin cancers.     

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 p16^INK4a. 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 p14^ARF expression in normal human cells it activates p14^ARF in cells containing deregulated E2F. Moreover, oncogenic Ras and E2F1 synergize in activating p14^ARF expression. Activation of p14^ARF 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.