The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site.

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.

Retinoid resistance and multifaceted impairment of retinoic acid synthesis in glioblastoma.

Measuring concentrations of the differentiation-promoting hormone retinoic acid (RA) in glioblastoma tissues would help to understand the reason why RA treatment has been inefficient in clinical trials involving brain tumor patients. Here, we apply a recently established extraction and measurement protocol to screen glioblastoma tissues for the levels of the RA precursor retinol and biologically active RA. Combining this approach with mRNA analyses of 26 tumors and 8 normal brains, we identify a multifaceted disturbance of RA synthesis in glioblastoma, involving multiple aldehyde dehydrogenase 1 family and retinol dehydrogenase enzymes. Through database studies and methylation analyses, we narrow down chromosomal deletions and aberrant promoter hypermethylation as potential mechanisms accounting for these alterations. Employing chromatin immunoprecipitation analyses and cell-culture studies, we further show that chromatin at RA target genes is poised to RA substitution, but most glioblastoma cell cultures are completely resistant to RA treatment. This paradoxical RA response is unrelated to alternative RA signaling through the fatty acid-binding protein 5/peroxisome proliferator-activated receptor delta axis. Our data suggest a multifaceted disturbance of RA synthesis in glioblastoma and contribute to reconsider current RA treatment strategies.

Reduced promoter methylation and increased expression of CSPG4 negatively influences survival of HNSCC patients.

Proteoglycans are often overexpressed in tumors and can be found on several normal and neoplastic stem cells. In this study, we analyzed in-depth the role of CSPG4 in head and neck squamous cell carcinomas (HNSCC). Analysis of CSPG4 in a homogeneous study sample of HPV-negative stage IVa HNSCCs revealed overexpression of protein and mRNA levels in a subgroup of HNSCC tumors and a significant association of high CSPG4 protein levels with poor survival. This could be validated in three publicly available microarray datasets. As a potential cause for upregulated CSPG4 expression, we identified DNA hypomethylation in a CpG-island of the promoter region. Accordingly, we found an inverse correlation of methylation and patient outcome. Finally, CSPG4 re-expression was achieved by demethylating treatment of highly methylated HNSCC cell lines establishing a direct link between methylation and CSPG4 expression. In conclusion, we identified CSPG4 as a novel biomarker in HNSCC on several biological levels and established a causative link between DNA methylation and CSPG4 protein and mRNA expression.