DNA sequence encoded repression of rRNA gene transcription in chromatin

Eukaryotic genomes are packaged into nucleosomes that occlude DNA from interacting with most DNA-binding proteins. Nucleosome positioning and chromatin organization is critical for gene regulation. We have investigated the mechanism by which nucleosomes are positioned at the promoters of active and silent rRNA genes (rDNA). The reconstitution of nucleosomes on rDNA results in sequence-dependent nucleosome positioning at the rDNA promoter that mimics the chromatin structure of silent rRNA genes in vivo, suggesting that active mechanisms are required to reorganize chromatin structure upon gene activation. Nucleosomes are excluded from positions observed at active rRNA genes, resulting in transcriptional repression on chromatin. We suggest that the repressed state is the default chromatin organization of the rDNA and gene activation requires ATP-dependent chromatin remodelling activities that move the promoter-bound nucleosome about 22-bp upstream. We suggest that nucleosome remodelling precedes promoter-dependent transcriptional activation as specific inhibition of ATP-dependent chromatin remodelling suppresses the initiation of RNA Polymerase I transcription in vitro. Once initiated, RNA Polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes. The results reveal the functional cooperation of DNA sequence and chromatin remodelling complexes in nucleosome positioning and in establishing the epigenetic active or silent state of rRNA genes

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo

DNA methylation in chromatin - complexes and mechanisms

The DNA in the eukaryotic genome is packaged into chromatin whose basic repeating unit is the nucleosome consisting of 147bp of DNA wrapped around an octameric histone core. The histone octamer is composed of two copies of each of the four histone proteins H2A, H2B, H3 and H4. DNA methylation is an epigenetic mechanism that is involved in various important processes in the cell such as differentiation and proliferation, transcriptional regulation, genomic imprinting, X-chromosome inactivation, silencing of repetitive elements, maintenance of genomic stability and DNA repair. In the mammalian genome, DNA methylation occurs almost exclusively in the context of CpG di-nucleotides and is brought about by three DNA cytosine-5-methyltransferases that use S�adenosyl-methionine (SAM) as methyl-group donor. Due to Dnmt1�s preferential activity towards hemi-methylated DNA and the fact that it restores methylation patterns on the newly synthesized daughter strand during replication, it is referred to as the maintenance DNA methyltransferase, whereas the de novo DNA methyltransferases Dnmt3a and Dnmt3b introduce new methylation marks in the genome. Dnmt3L is structurally related but catalytically inactive and serves as a cofactor for Dnmt3a and Dnmt3b. Importantly, DNA methylation is indispensible for mammalian embryogenesis and aberrant DNA methylation is often found concomitant with cancer. Dnmt1 was shown to be implicated in the formation of tumors, however the underlying mechanisms especially the question of Dnmt1 targeting remain largely unknown. The goal of this thesis was to identify new Dnmt1 complexes from native tissues that would allow comparison of complex composition in tumors. ICBP90 (UHRF1) and USP7 were identified as interacting proteins from co-immunoprecipitation experiments. During the course of this study, UHRF1 was described not only to interact with Dnmt1 but to recruit Dnmt1 to replication foci during late S-phase. In vivo and in vitro immunoprecipitations revealed different possible complexes, namely Dnmt1/ICBP90, Dnmt1/USP7 and ICBP90/USP7. Furthermore, a possible trimeric complex of USP7 binding with two different domains to both Dnmt1 and ICBP90 was established. Notably, chromatin immunoprecipitation demonstrated the existence of different Dnmt1/ICBP90/USP7 complexes at four different loci in vivo, however the function in chromatin related processes awaits further investigation. Interestingly, ICBP90 and USP7 are endowed with antagonistic enzymatic activities. ICBP90 exhibits autoubiquitinylation and ubiquitinylation activity towards histone H3, and USP7 in vitro. On the contrary, USP7 was able to target ICBP90 and histones H2A, H2B and H3 for deubiquitination in vitro whereas global levels of ubiquitinylated H2A and H2B were not changed upon knockdown or over-expression of USP7. Binding of ICBP90 and Dnmt1 to USP7 did not influence the in vitro activity of USP7. Moreover, Dnmt1 was ubiquitinylated by ICBP90 in vitro, but Dnmt1 protein or ubiquitinylation levels were not affected by USP7 over-expression or knockdown in vivo. Future research will focus on the role of histone ubiquitinylation/deubiquitination in transcriptional repression and silencing of repetitive elements in heterochromatin. In another project, the properties of Dnmt binding to DNA and mono-nucleosomes and the principle mechanisms of DNA methylation in chromatin by the de novo DNA methyltransferases were in focus. It could be shown that Dnmt3a and Dnmt3b2 stably associated with DNA >35bp in length, albeit longer DNA fragments were preferred indicative of a cooperative binding process. Furthermore, binding to DNA or mono-nucleosomes was highly dynamic and the interaction of mono-nucleosomes with the de novo DNA methyltransferases did not disrupt mono-nucleosomes. Dnmt3a generally bound comparably well to DNA and mono-nucleosomes with different DNA linker length whereas Dnmt3b2 preferentially bound to free DNA and mono-nucleosomes with long linker DNA. In vitro DNA methylation assays, either performed with radioactive SAM or non-radioactive one, but following single-molecule analysis with bisulfite treatment clearly demonstrated that Dnmt3a and Dnmt3b2 could not methylate DNA within the nucleosome but only linker DNA. This indicated that the DNA strand facing opposite the histone octamer did not represent a target for methylation and that nucleosomes constitute a major restriction for DNA methylation. Further experiments will address the role of chromatin remodeling enzymes in this process. Dnmt3L, a stimulatory factor for Dnmt3a and Dnmt3b, was shown to bind to non-methylated H3K4. Therefore, the effect of Dnmt3L on binding to DNA and nucleosomes by Dnmt3a and Dnmt3b was analyzed. Dnmt3L itself neither bound to DNA nor to mono-nucleosomes in EMSA experiments. Addition of Dnmt3L to Dnmt3a and Dnmt3b enhanced DNA binding and modified the binding behavior towards nucleosomes. Interestingly, recombinant Dnmt3L was tightly associated with nucleosomes when purified from Sf21 insect cells. Sucrose density gradient analysis confirmed this observation as Dnmt3L was distributed over the whole gradient with nucleosomal species of different weight. However, when endogenous nucleosomes were substituted for nucleosomal templates of various sizes or �naked� DNA Dnmt3L entered the gradient by 1/3rd, although the peak fractions migrated at higher densities. To unravel the reasons for the stable association of Dnmt3L with endogenous nucleosomes, future work will concentrate on the identification of possible loading factors, specific posttranslational histone modifications and nucleosomal architecture

Overexpression of UHRF1 promotes silencing of tumor suppressor genes and predicts outcome in hepatoblastoma

Background: Hepatoblastoma (HB) is the most common liver tumor of childhood and occurs predominantly within the first 3 years of life. In accordance to its early manifestation, HB has been described to display an extremely low mutation rate. As substitute, epigenetic modifiers seem to play an exceptional role in its tumorigenesis, which holds promise to develop targeted therapies and establish biomarkers for patient risk stratification. Results: We examined the role of a newly described protein complex consisting of three epigenetic regulators, namely E3 ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), ubiquitin-specific-processing protease 7 (USP7), and DNA methyltransferase 1 (DNMT1), in HB. We found the complex to be located on the promoter regions of the pivotal HB-associated tumor suppressor genes (TSGs) HHIP, IGFBP3, and SFRP1 in HB cells, thereby leading to strong repression through DNA methylation and histone modifications. Consequently, knockdown of UHRF1 led to DNA demethylation and loss of the repressive H3K9me2 histone mark at the TSG loci with their subsequent transcriptional reactivation. The observed growth impairment of HB cells upon UHRF1 knockdown could be attributed to reduced expression of genes involved in cell cycle progression, negative regulation of cell death, LIN28B signaling, and the adverse 16-gene signature, as revealed by global RNA sequencing. Clinically, overexpression of UHRF1 in primary tumor tissues was significantly associated with poor survival and the prognostic high-risk 16-gene signature. Conclusion: These findings suggest that UHRF1 is critical for aberrant TSG silencing and sustained growth signaling in HB and that UHRF1 overexpression levels might serve as a prognostic biomarker and potential molecular target for HB patients

Additional file 1: of Overexpression of UHRF1 promotes silencing of tumor suppressor genes and predicts outcome in hepatoblastoma

Figure S1. Relative RNA expression levels of indicated genes in HUH6 cells 24 h after UHRF1 knockdown compared to control-transfected cells. Data were normalized to the expression level of the housekeeping gene TBP. The average of two independent knockdown experiments is shown. Statistical significance of all experiments was calculated using t test (p < 0.05). (PNG 110 kb