MECP2 LIMITS DNA ACCESSIBILITY IN MONONUCLEOSOMES AND HIGHER ORDER CHROMATIN ARRAYS

Within the cell all DNA templated processes (transcription, replication, etc.) must act on the DNA double helix within the context of chromatin, the DNA and protein complex responsible for the organized compaction of the genome. Chromatin architectural proteins (CAPs) bind the entry / exit DNA of nucleosomes, the basic, repeating unit of chromatin, to form chromatosomes. CAPs are expressed in specific patterns based on tissue type and development stage in order to maintain the transcriptional integrity of cells. By binding nearly every nucleosome within the cell, CAPs establish distinct transcriptional profiles through the formation of specific higher chromatin structures that lead to active or repressed regions of the genome. Using FRET (Förster Resonance Energy Transfer) on mononucleosomes and REDA (restriction enzyme digest analysis) on 17-nucleosome chromatin arrays we studied how MeCP2 (methyl CpG binding protein 2), a CAP essential for mature neuron function, binds to nucleosomes without epigenetic modifications and alters DNA target site accessibility. Loss of function mutations in MeCP2 lead to Rett Syndrome, a severe neurodevelopment disorder, yet there are conflicting reports on whether MeCP2 acts as an activator or repressor throughout the genome. Therefore, in order to determine if MeCP2 acts as an activating or repressive CAP, we performed the same experiments with the repressive CAP linker histone H1 and the activating CAP HMGD1 (high mobility group D 1) for comparison. We found that MeCP2 has a slightly lower affinity for chromatosome formation than histone H1. However MeCP2 chromatosomes limit DNA target site accessibility to the same extent as histone H1 chromatosomes. Our results suggest that without epigenetic signals MeCP2 behaves as a repressive CAP. Furthermore we show that our mononucleosome FRET system, which can be modified to incorporate epigenetic marks and disease causing mutations, is able to provide key details needed to characterize CAP binding and function.