Dynamic binding of RBPJ is determined by Notch signaling status.

Notch signaling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighboring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and, together with the transcription factor moiety of Notch (NICD), regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole-genome binding profiles were generated for RBPJ; its coactivator, p300; NICD; and the histone H3 modifications H3 Lys 4 trimethylation (H3K4me3), H3 Lys 4 monomethylation (H3K4me1), and histone H3 Lys 27 acetylation (H3K27ac) in myogenic cells under active or inhibitory Notch signaling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300 and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signaling mediate their activities and consequently impact on cell fate decisions.

Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk.

Cross-talk between DNA methylation and histone modifications drives the establishment of composite epigenetic signatures and is traditionally studied using correlative rather than direct approaches. Here, we present sequential ChIP-bisulfite-sequencing (ChIP-BS-seq) as an approach to quantitatively assess DNA methylation patterns associated with chromatin modifications or chromatin-associated factors directly. A chromatin-immunoprecipitation (ChIP)-capturing step is used to obtain a restricted representation of the genome occupied by the epigenetic feature of interest, for which a single-base resolution DNA methylation map is then generated. When applied to H3 lysine 27 trimethylation (H3K27me3), we found that H3K27me3 and DNA methylation are compatible throughout most of the genome, except for CpG islands, where these two marks are mutually exclusive. Further ChIP-BS-seq-based analysis in Dnmt triple-knockout (TKO) embryonic stem cells revealed that total loss of CpG methylation is associated with alteration of H3K27me3 levels throughout the genome: H3K27me3 in localized peaks is decreased while broad local enrichments (BLOCs) of H3K27me3 are formed. At an even broader scale, these BLOCs correspond to regions of high DNA methylation in wild-type ES cells, suggesting that DNA methylation prevents H3K27me3 deposition locally and at a megabase scale. Our strategy provides a unique way of investigating global interdependencies between DNA methylation and other chromatin features.

Characterization of genome-wide p53-binding sites upon stress response.

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research.

MK3 modulation affects BMI1-dependent and independent cell cycle check-points.

Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent.

Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.

MBD2 is a subunit of the NuRD complex that is postulated to mediate gene repression via recruitment of the complex to methylated DNA. In this study we adopted an MBD2 tagging-approach to study its genome wide binding characteristics. We show that in vivo MBD2 is mainly recruited to CpG island promoters that are highly methylated. Interestingly, MBD2 binds around 1 kb downstream of the transcription start site of a subset of ∼ 400 CpG island promoters that are characterized by the presence of active histone marks, RNA polymerase II (Pol2) and low to medium gene expression levels and H3K36me3 deposition. These tagged-MBD2 binding sites in MCF-7 show increased methylation in a cohort of primary breast cancers but not in normal breast samples, suggesting a putative role for MBD2 in breast cancer.

A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.

BACKGROUND: DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs). Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD) family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA. METHODOLOGY/PRINCIPAL FINDINGS: For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC). In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs) showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif. CONCLUSIONS/SIGNIFICANCE: The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

CDK2AP1/DOC-1 is a bona fide subunit of the Mi-2/NuRD complex.

The Mi-2/NuRD (NUcleosome Remodeling and histone Deacetylase) chromatin remodeling complex is a large heterogeneous multiprotein complex associated with transcriptional repression. Here we apply a SILAC based quantitative proteomics approach to show that all known Mi-2/NuRD complex subunits co-purify with Cyclin Dependent Kinase 2 Associated Protein1 (CDK2AP1), also known as Deleted in Oral Cancer 1 (DOC-1). DOC-1 displays in vitro binding affinity for methylated DNA as part of the meCpG binding MBD2/NuRD complex. In luciferase reporter assays, DOC-1 is a potent repressor of transcription. Finally, immunofluorescence experiments reveal co-localization between MBD2 and DOC-1 in mouse NIH-3T3 nuclei. Collectively, these results indicate that DOC-1 is a bona fide subunit of the Mi-2/NuRD chromatin remodeling complex.