Synopsis

DNA methyltransferase I (DNMT1) plays a key role in establishing and maintaining patterns of DNA methylation. Dysregulation of DNA methylation is associated with different kinds of cancer. The goal of this study was to identify DNMT1 interacting proteins with a view to understanding how DNMT1 is itself regulated.

We used a novel affinity purification-mass spectrometry (AP-MS) work flow that makes use of endogenously tagged cell-lines (Zhang et al, 2008) to identify DNMT1-interacting proteins. Epitope (FLAG) tagged DNMT1 protein was expressed in colorectal cancer (RKO) cells and DNMT1 protein complexes recovered using anti-FLAG antibody. Gel-free mass spectrometry proteomics was used to identify DNMT1-interacting proteins.

We identified ubiquitin-specific peptidase 7 (USP7) as a DNMT1 interacting protein and subsequently showed that USP7 regulates DNMT1 stability through de-ubiquitination (thus protecting DNMT1 from proteasomal degradation) (Du et al, 2010).

Tasks & Questions

• Search the MS/MS spectra from the AP-MS experiment and its control experiment using Mascot search engine (or Tandem on command line)
• How many proteins are identified?
• Are the DNMT1 and USP7 proteins identified?
• Using the spectral count data, which proteins are likely to be non-specific interactors or contaminants? [open the spectral count file using Scaffold]

• Analyze the identified proteins using STRING
• Are there any DNMT1-connected proteins in the STRING network?

Data	Notes
Raw Data MS/MS spectra from AP-MS experiment (a filtered subset of the spectra are provided) MS/MS spectra from Control experiment Search Results Identified proteins from AP-MS experiment Identified proteins from Control experiment	Taxonomy: Homo sapiens
Spectral count data	Install Scaffold software to open this file

References

Du et al. DNMT1 Stability Is Regulated by Proteins Coordinating Deubiquitination and Acetylation-Driven Ubiquitination. Science Signaling, 2010 (paper|PubMed).

Zhang et al. Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies. Nat Methods, 2008 (PubMed)