The Scc2-Scc4 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions.

The cohesin complex is at the heart of many chromosomal activities, including sister chromatid cohesion and transcriptional regulation. Cohesin loading onto chromosomes depends on the Scc2-Scc4 cohesin loader complex, but the chromatin features that form cohesin loading sites remain poorly understood. Here we show that the RSC chromatin remodeling complex recruits budding yeast Scc2-Scc4 to broad nucleosome-free regions, which the cohesin loader helps to maintain. Consequently, inactivation of either the cohesin loader or the RSC complex has similar effects on nucleosome positioning, gene expression and sister chromatid cohesion. These results show an intimate link between local chromatin structure and higher-order chromosome architecture. Our findings pertain to the similarities between two severe human disorders, Cornelia de Lange syndrome, which is caused by alterations in the human cohesin loader, and Coffin-Siris syndrome, which results from alterations in human RSC complex components. Both syndromes can arise from gene misregulation due to related changes in the nucleosome landscape.

Budding yeast Wapl controls sister chromatid cohesion maintenance and chromosome condensation.

The establishment of stable sister chromatid cohesion during DNA replication requires acetylation of the chromosomal cohesin complex by the replication fork-associated acetyltransferase Eco1. Cohesin acetylation is thought to facilitate replication fork progression by counteracting an as yet ill-defined cohesion "antiestablishment" activity imposed by the Wapl protein. Here, using budding yeast, we find no evidence that cohesin acetylation must overcome Wapl during replication fork progression. Instead, Wapl emerges as a negative regulator of cohesion maintenance in G2, a function that it likely exerts through its role as destabilizer of unacetylated, chromosome-bound cohesin. Our results suggest that acetylation renders cohesin Wapl-resistant from S phase onward until mitosis. In the absence of Wapl, sister chromatid cohesion functions well, suggesting that Wapl partakes in a cohesin function outside of sister chromatid cohesion. We find that Wapl is not required for cohesin's known role in transcriptional regulation. Rather, cells lacking Wapl display increased chromosome condensation in both interphase and mitosis. Thus, as a conserved regulator of cohesin dynamics on chromosomes, Wapl controls cohesion maintenance after its establishment in S phase and adjusts the chromosome condensation status.

Facile synthesis of budding yeast a-factor and its use to synchronize cells of α mating type.

The ease with which populations of the budding yeast Saccharomyces cerevisiae can be synchronized using the mating pheromone α-factor has been invaluable for studies of the cell cycle. The α-factor response pathway has also remained an important model to study the molecular mechanism of G-protein coupled receptor signalling. α-Factor is a 13 amino acids long peptide that is readily available by automated peptide synthesis. However, only cells of the a mating type respond to α-factor. Cells of the opposite α mating type respond to a-factor, a farnesylated and C-terminally methylated 12 amino acids peptide. Because of its more difficult chemical synthesis, a-factor is not readily available and consequently the a-factor response is less well understood. Here we describe an improved strategy for producing a-factor, based on solid-phase peptide synthesis, followed by two simple steps in solution that show favourable characteristics and good yield. We demonstrate the successful use of the resulting a-factor to synchronize cell cycle progression of α cells. Notably, the a-factor concentrations required for cell synchronization are an order of magnitude lower than typically used α-factor concentrations. Despite a similar cell cycle response, shmoo formation was less pronounced compared to α-factor-treated a cells. Our protocol makes a-factor widely accessible, extending the ease of cell cycle synchronization to budding yeast cells of both mating types and facilitating the study of a-factor signalling.

Regulation of SNAIL1 and E-cadherin function by DNMT1 in a DNA methylation-independent context.

Mammalian DNA methyltransferase 1 (DNMT1) is essential for maintaining DNA methylation patterns after cell division. Disruption of DNMT1 catalytic activity results in whole genome cytosine demethylation of CpG dinucleotides, promoting severe dysfunctions in somatic cells and during embryonic development. While these observations indicate that DNMT1-dependent DNA methylation is required for proper cell function, the possibility that DNMT1 has a role independent of its catalytic activity is a matter of controversy. Here, we provide evidence that DNMT1 can support cell functions that do not require the C-terminal catalytic domain. We report that PCNA and DMAP1 domains in the N-terminal region of DNMT1 are sufficient to modulate E-cadherin expression in the absence of noticeable changes in DNA methylation patterns in the gene promoters involved. Changes in E-cadherin expression are directly associated with regulation of ß-catenin-dependent transcription. Present evidence suggests that the DNMT1 acts on E-cadherin expression through its direct interaction with the E-cadherin transcriptional repressor SNAIL1.

Hos1 deacetylates Smc3 to close the cohesin acetylation cycle.

Cohesion between sister chromatids is mediated by the chromosomal cohesin complex. In budding yeast, cohesin is loaded onto chromosomes during the G1 phase of the cell cycle. During S phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to promote the establishment of sister chromatid cohesion. At the time of anaphase, Smc3 loses its acetylation again, but the Smc3 deacetylase and the possible importance of Smc3 deacetylation are unknown. Here, we show that the class I histone deacetylase family member Hos1 is responsible for Smc3 deacetylation. Cohesin is protected from deacetylation while bound to chromosomes but is deacetylated as soon as it dissociates from chromosomes at anaphase onset. Nonacetylated Smc3 is required as a substrate for cohesion establishment in the following cell cycle. Our results complete the description of an Smc3 acetylation cycle and provide unexpected insight into the importance of de novo Smc3 acetylation for cohesion establishment.

New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

Identification of PMF1 methylation in association with bladder cancer progression.

PURPOSE: Polyamines are important regulators of cell growth and death. The polyamine modulated factor-1 (PMF-1) is involved in polyamine homeostasis. After identifying an enriched CpG island encompassing the PMF1 promoter, we aimed at evaluating the clinical relevance of PMF1 methylation in bladder cancer. EXPERIMENTAL DESIGN: The epigenetic silencing of PMF1 by hypermethylation was tested in bladder cancer cells (n = 11) after azacytidine treatment. PMF1 methylation status was evaluated in 507 bladder tumors and 118 urinary specimens of bladder cancer patients and controls. PMF1 protein expression was analyzed by immunohistochemistry on tissue arrays containing bladder tumors for which PMF1 methylation was assessed (n = 218). RESULTS: PMF1 hypermethylation was associated with gene expression loss, being restored in vitro by a demethylating agent. An initial set of 101 primary frozen bladder tumors served to identify PMF1 hypermethylation in 88.1% of the cases. An independent set of 406 paraffin-embedded tumors also revealed a high PMF1 methylation rate (77.6%). PMF1 methylation was significantly associated with increasing stage (P = 0.025). Immunohistochemical analyses revealed that PMF1 methylation was associated with cytoplasmic PMF1 expression loss (P = 0.032). PMF1 protein expression patterns were significantly associated with stage (P < 0.001), grade (P < 0.001), and poor overall survival using univariate (P < 0.001) and multivariate (P = 0.011) analyses. Moreover, PMF1 methylation in urinary specimens distinguished bladder cancer patients from controls (area under the curve = 0.800). CONCLUSION: PMF1 was identified to be epigenetically modified in bladder cancer. The association of PMF1 methylation with tumor progression and its diagnostic ability using urinary specimens support including PMF1 assessment for the clinical management of bladder cancer patients.

Mecp2-null mice provide new neuronal targets for Rett syndrome.

BACKGROUND: Rett syndrome (RTT) is a complex neurological disorder that is one of the most frequent causes of mental retardation in women. A great landmark in research in this field was the discovery of a relationship between the disease and the presence of mutations in the gene that codes for the methyl-CpG binding protein 2 (MeCP2). Currently, MeCP2 is thought to act as a transcriptional repressor that couples DNA methylation and transcriptional silencing. The present study aimed to identify new target genes regulated by Mecp2 in a mouse model of RTT. METHODOLOGY/PRINCIPAL FINDINGS: We have compared the gene expression profiles of wild type (WT) and Mecp2-null (KO) mice in three regions of the brain (cortex, midbrain, and cerebellum) by using cDNA microarrays. The results obtained were confirmed by quantitative real-time PCR. Subsequent chromatin immunoprecipitation assays revealed seven direct target genes of Mecp2 bound in vivo (Fkbp5, Mobp, Plagl1, Ddc, Mllt2h, Eya2, and S100a9), and three overexpressed genes due to an indirect effect of a lack of Mecp2 (Irak1, Prodh and Dlk1). The regions bound by Mecp2 were always methylated, suggesting the involvement of the methyl-CpG binding domain of the protein in the mechanism of interaction. CONCLUSIONS: We identified new genes that are overexpressed in Mecp2-KO mice and are excellent candidate genes for involvement in various features of the neurological disease. Our results demonstrate new targets of MeCP2 and provide us with a better understanding of the underlying mechanisms of RTT.

Epigenetic inactivation of the Groucho homologue gene TLE1 in hematologic malignancies.

An undifferentiated status and the epigenetic inactivation of tumor-suppressor genes are hallmarks of transformed cells. Promoter CpG island hypermethylation of differentiating genes, however, has rarely been reported. The Groucho homologue Transducin-like Enhancer of Split 1 (TLE1) is a multitasked transcriptional corepressor that acts through the acute myelogenous leukemia 1, Wnt, and Notch signaling pathways. We have found that TLE1 undergoes promoter CpG island hypermethylation-associated inactivation in hematologic malignancies, such as diffuse large B-cell lymphoma and AML. We also observed a mutual exclusivity of the epigenetic alteration of TLE1 and the cytogenetic alteration of AML1. TLE1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas TLE1-short hairpin RNA depletion in unmethylated cells enhances tumor growth. We also show that these effects are mediated by TLE1 transcriptional repressor activity on its target genes, such as Cyclin D1, Colony-Stimulating Factor 1 receptor, and Hairy/Enhancer of Split 1. These data suggest that TLE1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting critical differentiation and growth-suppressing pathways.

Combinatorial effects of splice variants modulate function of Aiolos.

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

Epigenetic disruption of ribosomal RNA genes and nucleolar architecture in DNA methyltransferase 1 (Dnmt1) deficient cells.

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.

A profile of methyl-CpG binding domain protein occupancy of hypermethylated promoter CpG islands of tumor suppressor genes in human cancer.

Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.