HM Loci Research Articles

The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer.

Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATα cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.

Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1

Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2Δ mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2Δ shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2Δ strains. By strong SIRT1 overexpression in sir2Δ cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2Δ mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.

Dimerization of Sir3 via its C-terminal winged helix domain is essential for yeast heterochromatin formation

Gene silencing in budding yeast relies on the binding of the Silent Information Regulator (Sir) complex to chromatin, which is mediated by extensive interactions between the Sir proteins and nucleosomes. Sir3, a divergent member of the AAA+ ATPase-like family, contacts both the histone H4 tail and the nucleosome core. Here, we present the structure and function of the conserved C-terminal domain of Sir3, comprising 138 amino acids. This module adopts a variant winged helix-turn-helix (wH) architecture that exists as a stable homodimer in solution. Mutagenesis shows that the self-association mediated by this domain is essential for holo-Sir3 dimerization. Its loss impairs Sir3 loading onto nucleosomes in vitro and eliminates silencing at telomeres and HM loci in vivo. Replacing the Sir3 wH domain with an unrelated bacterial dimerization motif restores both HM and telomeric repression in sir3Δ cells. In contrast, related wH domains of archaeal and human members of the Orc1/Sir3 family are monomeric and have DNA binding activity. We speculate that a dimerization function for the wH evolved with Sir3's ability to facilitate heterochromatin formation.

Regulating Repression: Roles for the Sir4 N-Terminus in Linker DNA Protection and Stabilization of Epigenetic States

Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR–mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747–1,358) must be complemented with an N-terminal domain (Sir4N; residues 1–270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR–mediated silencing: it contributes to epigenetic repression by stabilizing the SIR–mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.

Promoter Strength Influences the S Phase Requirement for Establishment of Silencing at the Saccharomyces cerevisiae Silent Mating Type Loci

In Saccharomyces cerevisiae, the two cryptic mating type loci, HML and HMR, are transcriptionally silent. Previous studies on the establishment of silencing at HMR identified a requirement for passage through S phase. However, the underlying mechanism for this requirement is still unknown. In contrast to HMR, we found that substantial silencing of HML could be established without passage through S phase. To understand this difference, we analyzed several chimeric HM loci and found that promoter strength determined the S phase requirement. To silence a locus with a strong promoter such as the a1/a2 promoter required passage through S phase while HM loci with weaker promoters such as the α1/α2 or TRP1 promoter did not show this requirement. Thus, transcriptional activity counteracts the establishment of silencing but can be overcome by passage through S phase.

Design of a minimal silencer for the silent mating-type locus HML of Saccharomyces cerevisiae

The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS ΔI) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS ΔI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing.

Saccharomyces cerevisiae Esc2p Interacts with Sir2p through a Small Ubiquitin-like Modifier (SUMO)-binding Motif and Regulates Transcriptionally Silent Chromatin in a Locus-dependent Manner

Saccharomyces cerevisiae Esc2p is a member of a conserved family of proteins that contain small ubiquitin-like modifier (SUMO)-like domains. It has been implicated in transcriptional silencing and shown to interact with the silencing protein Sir2p in a two-hybrid analysis. However, little is known about how Esc2p regulates the structure of silent chromatin. We demonstrate here that ESC2 differentially regulates silent chromatin at telomeric, rDNA, and HM loci. Specifically, ESC2 is required for efficient telomeric silencing and Sir2p association with telomeric silent chromatin and for silencing and maintenance of silent chromatin structure at rDNA. On the other hand, ESC2 negatively regulates silencing at HML and HMR and destabilizes HML silent chromatin without affecting Sir2p association with chromatin. We present evidence that Esc2p is associated with both transcriptionally silent and active loci in the genome, and the abundance of Esc2p is not correlated with the chromatin state at a particular locus. Using affinity pull-down analyses, we show that Esc2p and Sir2p interact in vivo, and recombinant Esc2p and Sir2p interact directly. Moreover, we dissect Esc2p and identify a putative SUMO-binding motif that is necessary and sufficient for interacting with Sir2p and SUMO and is required for the function of Esc2p in transcriptional silencing.

Histone H3 N-terminus regulates higher order structure of yeast heterochromatin

In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Silencing Information Regulator (Sir) proteins with histones. Binding data demonstrate that both the H3 and H4 N-terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment; however, that of the H3 N-terminal tail has remained unclear. To characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We found that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast, the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure.

Reconstitution of Yeast Silent Chromatin: Multiple Contact Sites and O-AADPR Binding Load SIR Complexes onto Nucleosomes In Vitro

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

Sem1p and Ubp6p orchestrate telomeric silencing by modulating histone H2B ubiquitination and H3 acetylation

Histone covalent modifications and 26S proteasome-mediated proteolysis modulate many regulatory events in eukaryotes. In Saccharomyces cerevisiae, heterochromatin mediates transcriptional silencing at telomeres, HM loci and rDNA array. Here, we show that proteasome-associated Sem1p and its interacting partner, Ubp6p (a deubiquitinating enzyme), are essential to maintain telomeric silencing. Simultaneous deletion of SEM1 and UBP6 induces dramatic silencing defect accompanied by significantly increased level of ubiquitinated-histone H2B and markedly reduced levels of acetylated-lysine 14 and 23 on histone H3 at the telomeres. Further, the loss of Sem1p and Ubp6p triggers relocation of silencing factors (e.g. Sir proteins) from telomere to HM loci and rDNA array. Such relocation of silencing factors enhances gene silencing at HM loci and rDNA array, but diminishes telomeric silencing. Interestingly, both Sem1p and Ubp6p participate in the proteolytic function of the proteasome. However, we find that the telomeric silencing is not influenced by proteolysis. Taken together, our data demonstrate that Sem1p and Ubp6p maintain telomeric heterochromatin structure (and hence silencing) through modulation of histone covalent modifications and association of silencing factors independently of the proteolytic function of the proteasome, thus offering a new regulatory mechanism of telomeric silencing.

The Ku Complex in Silencing the Cryptic Mating-Type Loci ofSaccharomyces cerevisiae

Sir1 establishes transcriptional silencing at the cryptic mating-type loci HMR and HML (HM loci) by recruiting the three other Sir proteins, Sir2, -3, and -4, that function directly in silenced chromatin. However, SIR1-independent mechanisms also contribute to recruiting the Sir2-4 proteins to the HM loci. A screen to elucidate SIR1-independent mechanisms that establish HMR silencing identified a mutation in YKU80. The role for Ku in silencing both HMR and HML was masked by SIR1. Ku's role in silencing the HM loci was distinct from its shared role with the nuclear architecture protein Esc1 in tethering the HM loci and telomeres to the nuclear periphery. The ability of high-copy SIR4 to rescue HMR silencing defects in sir1Delta cells required Ku, and chromatin immunoprecipitation (ChIP) experiments provided evidence that Ku contributed to Sir4's physical association with the HM loci in vivo. Additional ChIP experiments provided evidence that Ku functioned directly at the HM loci. Thus Ku and Sir1 had overlapping roles in silencing the HM loci.

Bypassing Sir2 and O-Acetyl-ADP-Ribose in Transcriptional Silencing

The yeast Sir2/3/4 complex forms a heterochromatin-like structure that represses transcription. The proteins nucleate at silencers and spread distally, utilizing the Sir2 NAD(+)-dependent histone deacetylase activity and the affinity of Sir3/4 for deacetylated histone tails. A by-product of the Sir2 reaction, O-acetyl-ADP-ribose (OAADPr), is thought to aid spreading by binding one of the Sir proteins. We developed a protein chimera approach to reexamine the contributions of Sir2. We show that a Sir3 chimera-bearing Hos3, an unrelated NAD(+)-independent histone deacetylase, substitutes for Sir2 in silencing. Sir3-Hos3 operates within the Sir pathway, spreading while deacetylating histones. Moreover, the chimera represses HM loci in strains lacking all five OAADPr-producing deacetylases, indicating that OAADPr is not necessary for silencing. Repression by a Hos3 hybrid bearing the targeting motifs of Sir2 shows that targeting doesn't require the Sir2 reaction. Together, these data demonstrate that protein deacetylation is the only essential function of Sir2 in creating silenced chromatin.

Structural and Functional Studies of the Rap1 C-Terminus Reveal Novel Separation-of-Function Mutants

The yeast Rap1 protein plays an important role in transcriptional silencing and in telomere length homeostasis. Rap1 mediates silencing at the HM loci and at telomeres by recruiting the Sir3 and Sir4 proteins to chromatin via a Rap1 C-terminal domain, which also recruits the telomere length regulators, Rif1 and Rif2. We report the 1.85 Å resolution crystal structure of the Rap1 C-terminus, which adopts an all-helical fold with no structural homologues. The structure was used to engineer surface mutations in Rap1, and the effects of these mutations on silencing and telomere length regulation were assayed in vivo. Our surprising finding was that there is no overlap between mutations affecting mating-type and telomeric silencing, suggesting that Rap1 plays distinct roles in silencing at the silent mating-type loci and telomeres. We also found novel Rap1 phenotypes and new separation-of-function mutants, which provide new tools for studying Rap1 function. Yeast two-hybrid studies were used to determine how specific mutations affect recruitment of Sir3, Rif1, and Rif2. A comparison of the yeast two-hybrid and functional data reveals patterns of protein interactions that correlate with each Rap1 phenotype. We find that Sir3 interactions are important for telomeric silencing, but not mating type silencing, and that Rif1 and Rif2 interactions are important in different subsets of telomeric length mutants. Our results show that the role of Rap1 in silencing differs between the HM loci and the telomeres and offer insight into the interplay between HM silencing, telomeric silencing, and telomere length regulation. These findings suggest a model in which competition and multiple recruitment events modulate silencing and telomere length regulation.

Epistatic Interaction Between the K-Homology Domain Protein HEK2 and SIR1 at HMR and Telomeres in Yeast

In budding yeast, telomeres, the ribosomal DNA array, and HM loci are transcriptionally silenced by chromatin complexes containing Sir proteins. Hek2, a protein containing three evolutionary conserved RNA-binding K-homology domains, was identified as a suppressor of telomeric silencing [telomeric position effect (TPE)]. To explore the mechanisms of Hek2p action in gene silencing, we examined its relationship with Sir proteins. This search revealed an epistatic interaction between HEK2 and SIR1 at telomeres. Both single mutations, sir1Δ and hek2Δ, enhanced TPE, whereas the effect of double mutation, sir1Δ hek2Δ, did not exceed that of the single mutations. The results of chromatin immunoprecipitation analysis demonstrate that the TPE enhancement observed in these mutants is associated with increased binding of Sir2 protein to telomeres. At the HMR locus, hek2Δ rescues the silencing defect caused by sir1Δ mutation and reverses the loss of Sir2p and Sir3p. These data suggest that the epistatic interaction of HEK2 and SIR1 reflects competition between telomeres and HMR for Sir2/3 factors where HEK2 acts to suppress silencing. Because chromatin immunoprecipitation analysis reveals the presence of Hek2p at a subtelomeric region and HMR, its silencing effects at these loci are likely direct. These observations suggest that HEK2 regulates the composition of Sir complexes at HMR and telomeres.

Isw1 Acts Independently of the Isw1a and Isw1b Complexes in Regulating Transcriptional Silencing at the Ribosomal DNA Locus in Saccharomyces cerevisiae

Transcriptional silencing of Pol II-transcribed genes in Saccharomyces cerevisiae occurs at the HM loci, telomeres and ribosomal DNA (rDNA) locus. Gene silencing at these loci requires histone-modifying enzymes as well as factors that regulate local chromatin structure. Previous work has shown that the ATP-dependent chromatin remodeling protein Isw1 is required for silencing of a marker gene inserted at the HMR locus, but not at telomeres. Here we show that Isw1 is required for transcriptional silencing of Pol II-transcribed genes in the ribosomal DNA locus. Our results indicate that Isw1 associates with the rDNA and that this interaction is not altered in cells lacking other members of the Isw1a and Isw1b chromatin remodeling complexes. Further, the association of Isw1 with the rDNA is not altered in cells lacking the histone deacetylase Sir2 or the histone methyltransferase Set1, two factors that are required for gene silencing at the rDNA. Notably, the loss of transcriptional silencing at the rDNA in cells lacking Isw1 is correlated with a change in rDNA chromatin structure. Together, our data support a model in which Isw1 acts independently of the previously characterized Isw1a and Isw1b complexes to maintain a heterochromatin-like structure at the rDNA that is required for gene silencing.

Isolation and Characterization of Conditional Alleles of the Yeast SIR2 Gene

Sir2 is a protein deacetylase that mediates transcriptional silencing at the HM loci, telomeres, and rDNA repeats in yeast. To identify functionally significant regions of the Sir2 protein, we have characterized two types of mutations. First, we used random mutagenesis to create temperature-sensitive alleles of the SIR2 gene. Mutations conferring conditional silencing can be isolated throughout the SIR2 gene, causing both enzymatic and protein interaction defects. We used external deletions to identify regions essential for silencing in the non-conserved regions of Sir2. Deletions of the Sir2 N-terminal 89 amino acid residues caused a subtle increase in silencing, while deletions encompassing residues 110–146 caused loss of Sir2 interactions with both Sir4 and Net1. This loss of protein interaction correlates with a loss of Sir2-mediated silencing, and is consistent with a model in which Net1 and Sir4 compete for interaction with Sir2. These results indicate that recognition of the binding partners of Sir2 is a key function of non-conserved sequences.

Bypassing the Catalytic Activity of SIR2 for SIR Protein Spreading inSaccharomyces cerevisiae

Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed at the HM loci and telomeres in cells containing a stably expressed, but catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Mating in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2'-O-acetyl-ADP ribose by sir2p and Sir2p's enzymatic function for silencing can be bypassed in a subset of cells in a given population. These results also provide genetic evidence for the existence of additional critical substrates of Sir2p for silencing in vivo.

Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

Silencing at the rDNA, HM loci, and telomeres in Saccharomyces cerevisiae requires histone-modifying enzymes to create chromatin domains that are refractory to recombination and RNA polymerase II transcription machineries. To explore how the silencing factor Sir2 regulates the composition and function of chromatin at the rDNA, the association of histones and RNA polymerase II with the rDNA was measured by chromatin immunoprecipitation. We found that Sir2 regulates not only the levels of K4-methylated histone H3 at the rDNA but also the levels of total histone H3 and RNA polymerase II. Furthermore, our results demonstrate that the ability of Sir2 to limit methylated histones at the rDNA requires its deacetylase activity. In sir2Delta cells, high levels of K4-trimethylated H3 at the rDNA nontranscribed spacer are associated with the expression of transcription units in the nontranscribed spacer by RNA polymerase II and with previously undetected alterations in chromatin structure. Together, these data suggest a model where the deacetylase activity of Sir2 prevents euchromatinization of the rDNA and silences naturally occurring intergenic transcription units whose expression has been associated with disruption of cohesion complexes and repeat amplification at the rDNA.

The Requirements for COMPASS and Paf1 in Transcriptional Silencing and Methylation of Histone H3 in Saccharomyces cerevisiae

The Set1-containing complex, COMPASS, methylates histone H3 on lysine 4 (K4) in Saccharomyces cerevisiae. Despite the preferential association of K4-trimethylated H3 with regions of the genome that are transcribed by RNA polymerase II, transcriptional silencing is one of the few cases in S. cerevisiae where histone-methylation defects have a clear effect on gene expression. To better understand the role of COMPASS in transcriptional silencing, we have determined which members of COMPASS are required for silencing at the ribosomal DNA locus (rDNA), a telomere, and the silent mating loci (HM) using Northern analyses. Our findings indicate that most members of COMPASS are required for silencing at the rDNA and telomere, while none are required for silencing of endogenous genes at the HM loci. To complement gene-expression analysis, quantitative Western blot experiments were performed to determine the members of COMPASS that are required for methylation of histone H3. While most are required for trimethylation, cells lacking certain COMPASS proteins maintain reduced levels of K4 mono- and dimethylated H3, suggesting that some COMPASS members have redundant function. Finally, we show Paf1 is required for silencing and K4-methylated H3 at the rDNA, suggesting a possible direct role for K4-methylated H3 in gene silencing.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus

The INK4/ARF locus encodes three tumour suppressors (p15(INK4b), ARF and p16(INK4a)) and is among the most frequently inactivated loci in human cancer. However, little is known about the mechanisms that govern the expression of this locus. Here we have identified a putative DNA replication origin at the INK4/ARF locus that assembles a multiprotein complex containing Cdc6, Orc2 and MCMs, and that coincides with a conserved noncoding DNA element (regulatory domain RD(INK4/ARF)). Targeted and localized RNA-interference-induced heterochromatinization of RD(INK4/ARF) results in transcriptional repression of the locus, revealing that RD(INK4/ARF) is a relevant transcriptional regulatory element. Cdc6 is overexpressed in human cancers, where it might have roles in addition to DNA replication. We have found that high levels of Cdc6 result in RD(INK4/ARF)-dependent transcriptional repression, recruitment of histone deacetylases and heterochromatinization of the INK4/ARF locus, and a concomitant decrease in the expression of the three tumour suppressors encoded by this locus. This mechanism is reminiscent of the silencing of the mating-type HM loci in yeast by replication factors. Consistent with its ability to repress the INK4/ARF locus, Cdc6 has cellular immortalization activity and neoplastic transformation capacity in cooperation with oncogenic Ras. Furthermore, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16(INK4a). We conclude that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RD(INK4/ARF) element.