Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes.

Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9). Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe. The chromodomain of mouse HP1 (and Swi6 in S. pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly. We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin. In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length. Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome. These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.     

Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin.

Post-translational modifications of histone amino termini are an important regulatory mechanism that induce transitions in chromatin structure, thereby contributing to epigenetic gene control and the assembly of specialized chromosomal subdomains. Methylation of histone H3 at lysine 9 (H3-Lys9) by site-specific histone methyltransferases (Suv39h HMTases) marks constitutive heterochromatin. Here, we show that H3-Lys9 methylation also occurs in facultative heterochromatin of the inactive X chromosome (Xi) in female mammals. H3-Lys9 methylation is retained through mitosis, indicating that it might provide an epigenetic imprint for the maintenance of the inactive state. Disruption of the two mouse Suv39h HMTases abolishes H3-Lys9 methylation of constitutive heterochromatin but not that of the Xi. In addition, HP1 proteins, which normally associate with heterochromatin, do not accumulate with the Xi. These observations suggest the existence of an Suv39h-HP1-independent pathway regulating H3-Lys9 methylation of facultative heterochromatin.     

TGF-beta signaling in tumor suppression and cancer progression

Epithelial and hematopoietic cells have a high turnover and their progenitor cells divide continuously, making them prime targets for genetic and epigenetic changes that lead to cell transformation and tumorigenesis. The consequent changes in cell behavior and responsiveness result not only from genetic alterations such as activation of oncogenes or inactivation of tumor suppressor genes, but also from altered production of, or responsiveness to, stimulatory or inhibitory growth and differentiation factors. Among these, transforming growth factor beta (TGF-beta) and its signaling effectors act as key determinants of carcinoma cell behavior. The autocrine and paracrine effects of TGF-beta on tumor cells and the tumor micro-environment exert both positive and negative influences on cancer development. Accordingly, the TGF-beta signaling pathway has been considered as both a tumor suppressor pathway and a promoter of tumor progression and invasion. Here we evaluate the role of TGF-beta in tumor development and attempt to reconcile the positive and negative effects of TGF-beta in carcinogenesis.     

Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases

Telomeres are capping structures at the ends of eukaryotic chromosomes composed of TTAGGG repeats bound to an array of specialized proteins. Telomeres are heterochromatic regions. Yeast and flies with defects in activities that modify the state of chromatin also have abnormal telomere function, but the putative role of chromatin-modifying activities in regulating telomeres in mammals is unknown. Here we report on telomere length and function in mice null with respect to both the histone methyltransferases (HMTases) Suv39h1 and Suv39h2 (called SUV39DN mice). Suv39h1 and Suv39h2 govern methylation of histone H3 Lys9 (H3-Lys9) in heterochromatic regions. We show that primary cells derived from SUV39DN mice have abnormally long telomeres relative to wild-type controls. Using chromatin immunoprecipitation (ChIP) analysis, we found that telomeres were enriched in di- and trimethylated H3-Lys9 but that telomeres of SUV39DN cells had less dimethylated and trimethylated H3-Lys9 but more monomethylated H3-Lys9. Concomitant with the decrease in H3-Lys9 methylation, telomeres in SUV39DN cells had reduced binding of the chromobox proteins Cbx1, Cbx3 and Cbx5, homologs of Drosophila melanogaster heterochromatin protein 1 (HP1). These findings indicate substantial changes in the state of telomeric heterochromatin in SUV39DN cells, which are associated with abnormal telomere elongation. Taken together, the results indicate epigenetic regulation of telomere length in mammals by Suv39h1 and Suv39h2.     

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.     

Heterochromatin protein 1 modifies mammalian PEV in a dose- and chromosomal-context-dependent manner

Locus control regions (LCRs) are gene regulatory elements in mammals that can overcome the highly repressive effects normally associated with heterochromatic transgene locations (for example the centromere) in mice. Deletion of essential LCR sequences renders the cognate gene susceptible to this form of repression, so a proportion of the cells from transgenic mice that would normally express the transgene are silenced-a phenomenon known as position effect variegation (PEV). We show here that PEV can also occur when the transgene is non-centromeric and that the extent of variegation can be developmentally regulated. Furthermore, by overexpressing a mammalian homologue (M31) of Drosophila melanogaster heterochromatin protein 1 (HP1; refs 7,8) in transgenic mouse lines that exhibit PEV, it is possible to modify the proportion of cells that silence the transgene in a dose-dependent manner. Thus, we show M31 overexpression to have two contrasting effects which are dependent on chromosomal context: (i) it enhanced PEV in those lines with centromeric or pericentromeric transgene locations; and (ii) it suppressed PEV when the transgene was non-centromeric. Our results indicate that components or modifiers of heterochromatin may have a chromosomal-context-dependent role in gene silencing and activation decisions in mammals.     

The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression

Most preneoplastic lesions are quiescent and do not progress to form overt tumors. It has been proposed that oncogenic stress activates the DNA damage response and the key tumor suppressor p53, which prohibits tumor growth. However, the molecular pathways by which cells sense a premalignant state in vivo are largely unknown. Here we report that tissue-specific inactivation of the stress signaling kinase MKK7 in KRas(G12D)-driven lung carcinomas and NeuT-driven mammary tumors markedly accelerates tumor onset and reduces overall survival. Mechanistically, MKK7 acts through the kinases JNK1 and JNK2, and this signaling pathway directly couples oncogenic and genotoxic stress to the stability of p53, which is required for cell cycle arrest and suppression of epithelial cancers. These results show that MKK7 functions as a major tumor suppressor in lung and mammary cancer in mouse and identify MKK7 as a vital molecular sensor to set a cellular anti-cancer barrier.     

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding beta-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free beta-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.     

Telomere length regulates the epigenetic status of mammalian telomeres and subtelomeres

Mammalian telomeres have epigenetic marks of constitutive heterochromatin. Here, we study the impact of telomere length on the maintenance of heterochromatin domains at telomeres. Telomerase-deficient Terc(-/-) mice with short telomeres show decreased trimethylation of histone 3 at Lys9 (H3K9) and histone 4 at Lys20 (H4K20) in telomeric and subtelomeric chromatin as well as decreased CBX3 binding accompanied by increased H3 and H4 acetylation at these regions. Subtelomeric DNA methylation is also decreased in conjunction with telomere shortening in Terc(-/-) mice. In contrast, telomere repeat factors 1 and 2 show normal binding to telomeres independent of telomere length. These results indicate that loss of telomeric repeats leads to a change in the architecture of telomeric and subtelomeric chromatin consisting of loss of heterochromatic features leading to a more 'open' chromatin state. These observations highlight the importance of telomere repeats in the establishment of constitutive heterochromatin at mammalian telomeres and subtelomeres and point to histone modifications as important in counting telomere repeats.     

Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice

We have used large-scale insertional mutagenesis to identify functional landmarks relevant to cancer in the recently completed mouse genome sequence. We infected Cdkn2a(-/-) mice with Moloney murine leukemia virus (MoMuLV) to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a-encoded tumor suppressors p16INK4a and p19ARF. Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. We isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer-related, we identified a total of 37 new common insertion sites (CISs), of which 8 encode components of signaling pathways that are involved in cancer. The effectiveness of large-scale insertional mutagenesis in a sensitized genetic background is demonstrated by the preference for activation of MAP kinase signaling, collaborating with Cdkn2a loss in generating the lymphoid and myeloid tumors. Collectively, our results show that large-scale retroviral insertional mutagenesis in genetically predisposed mice is useful both as a system for identifying genes underlying cancer and as a genetic framework for the assignment of such genes to specific oncogenic pathways.     

Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.     

Epigenetic remodeling in colorectal cancer results in coordinate gene suppression across an entire chromosome band

We report a new mechanism in carcinogenesis involving coordinate long-range epigenetic gene silencing. Epigenetic silencing in cancer has always been envisaged as a local event silencing discrete genes. However, in this study of silencing in colorectal cancer, we found common repression of the entire 4-Mb band of chromosome 2q.14.2, associated with global methylation of histone H3 Lys9. DNA hypermethylation within the repressed genomic neighborhood was localized to three separate enriched CpG island 'suburbs', with the largest hypermethylated suburb spanning 1 Mb. These data change our understanding of epigenetic gene silencing in cancer cells: namely, epigenetic silencing can span large regions of the chromosome, and both DNA-methylated and neighboring unmethylated genes can be coordinately suppressed by global changes in histone modification. We propose that loss of gene expression can occur through long-range epigenetic silencing, with similar implications as loss of heterozygosity in cancer.     

Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon

Kras is commonly mutated in colon cancers, but mutations in Nras are rare. We have used genetically engineered mice to determine whether and how these related oncogenes regulate homeostasis and tumorigenesis in the colon. Expression of K-Ras(G12D) in the colonic epithelium stimulated hyperproliferation in a Mek-dependent manner. N-Ras(G12D) did not alter the growth properties of the epithelium, but was able to confer resistance to apoptosis. In the context of an Apc-mutant colonic tumor, activation of K-Ras led to defects in terminal differentiation and expansion of putative stem cells within the tumor epithelium. This K-Ras tumor phenotype was associated with attenuated signaling through the MAPK pathway, and human colon cancer cells expressing mutant K-Ras were hypersensitive to inhibition of Raf, but not Mek. These studies demonstrate clear phenotypic differences between mutant Kras and Nras, and suggest that the oncogenic phenotype of mutant K-Ras might be mediated by noncanonical signaling through Ras effector pathways.