Principal Investigator/Program Director (Last, first, middle): Woodcock, Christopher, L. C.L.?Woodcock?????MeCP2?as?a?Chromatin?Architectural?Protein.? ? PROJECT?SUMMARY? ? Mutations?of?the?human?MeCP2?protein?lead?to?the?severe?neurodevelopmental?disorder?Rett? Syndrome?(RTT).??Misregulation?of?MeCP2?is?also?seen?in?many?of?the?Autism?Spectrum?Disorders.??MeCP2?is? a?unique?chromatin?architectural?protein?that?transmits?the?epigenetic?information?encoded?in?DNA? methylation,?and?evidence?suggests?that?in?the?context?of?chromatin?binding?it?can?cause?local?transcriptional? repression?both?by?its?recruitment?of?histone?deacetylase?(HDAC)?complexes?and?by?its?innate?ability?to? compact?chromatin.?This?project?is?aimed?at?understanding?these?mechanisms?and?the?root?causes?of?the? pathological?effects?that?are?seen?with?MeCP2?mutations.??Recent?work?by?the?P.I.?and?collaborators?has?led?to? specific?hypotheses?regarding?the?domain?organization?of?MeCP2,?the?functional?associations?between? domains,?its?interactions?with?other?proteins,?and?the?molecular?events?leading?to?chromatin?compaction.??This? proposal?describes?experiments?designed?to?test?these?hypotheses.??? In?Aim?1,?spectroscopic?techniques?are?proposed?to?compare?the?amount?and?type?of?secondary? structure?present?in?the?wildtype?domains?of?MeCP2?with?domains?carrying?RTT?causing?mutations.??Aim?2? employs?similar?strategies?to?complexes?between?MeCP2?and?DNA?to?test?the?hypothesis?that?DNA?binding? leads?to?changes?in?secondary?structure.??Aim?3?is?to?test?the?hypothesis?that?phosphorylation?of?MeCP2?results? in?weakening?of?MeCP2?DNA?interactions,?and?that?this?event?plays?a?major?role?in?its?regulation?in?vivo.??This? will?be?achieved?by?examining?the?interaction?with?DNA?and?chromatin?of?MeCP2?mutations?that?mimic? phosphorylation?as?well?as?MeCP2?phosphorylated?at?selected?sites.??Recent?work?shows?that?histone?H1?and? MeCP2?share?overlapping?binding?sites?on?nucleosomes,?suggesting?that?competition?for?binding?sites?may? play?an?important?regulatory?role.??Aim?4?includes?experiments?to?quantitate?the?competition?between?these? two?chromatin?architectural?proteins?and?examine?the?structural?consequences?on?chromatin?conformation?of? nucleosomal?arrays?which?have?both?MeCP2?and?H1?bound.??The?co?repressor?protein?mSin3A?is?a?major? binding?partner?of?MeCP2,?and?also?recruits?HDACs.??However,?little?is?known?about?the?interactions?with? MeCP2?and?MeCP2?mutants.??Aim?5?proposes?an?examination?of?these?interactions,?the?structures?of?the? protein?complexes,?and?their?impact?on?chromatin?conformation.??? For?common?MeCP2?mutations,?there?is?a?correlation?between?the?severity?of?the?disease,?and?the? magnitude?of?effects?seen?with?the?assays?used?by?the?P.I.??This?suggests?that?the?proposed?in?vitro?experiments? will?be?relevant?for?understanding?RTT.??Although?this?proposal?is?primarily?aimed?at?understanding?the? defects?in?RTT?causing?mutations?of?MeCP2,?it?is?hoped?that?the?information?we?obtain?will?be?used?to?select? and?screen?for?molecules?that?restore?normal?function,?a?strategy?that?is?being?successfully?pursued?with?other? DNA?binding?proteins.??The?work?will?also?contribute?significantly?to?understanding?a?fundamental?aspect?of? epigenetic?control?and?chromatin?remodeling.?? Project Description Page 6
