Initial Study Of Interaction Mechanism Of CoREST SANT Domain And Non-coding Small DsRNA

Neural restrictive silencer factor （NRSF）, also designated as repressor element 1 （RE1） silencing transcription factor （REST）, is an essential negative transcriptional repressor for neuron-specific genes in non-neuronal cells and neuronal progenitors.NRSF/REST is a Kruppel-type zinc-finger protein that binds specifically to a 21- bp core sequence of neural-restrictive silencer element, NRSE/RE1 in the regulatory regions of many neuron-specific genes. Recent study showed that small, non-coding dsRNA whose sequence is defined by NRSE/RE1 triggers gene expression of neuron-specific genes through interaction with the NRSF/REST transcriptional machinery. However, its detailed mechanism is still unknown.Previous studies indicated that the zinc finger 9 （ZF9） in the NRSF/REST’s C-terminal repressor domain can interact with the CoREST SANT motif to enhance NRSF/REST negative transcriptional repressor. To study how small RE1 dsRNA activate neuron-gene expression, the research in this thesis consists of two following parts:The 1^st part was focused on CoREST SANT1 plasmid construction and expressive condition optimization. Three recombinant plasmids such as pSJ3 SANT1 （including 8×His·tag）、pSMT3 SANT1 （containing SUMOtag and FLAG·tag） and pHGB SANT1 （with GB·tag and His·tag） were contracted, then were transferred into E.coli BL21（DE3） for expression. By optimizing temperature of induction, concentration of IPTG, time of induction, small amount of pHGB CoREST SANT1 was found to express in supernatant.The 2^nd part was to study the interaction mode between CoREST SANT2 and ZF9 in NRSF/REST, NRSE/RE1 dsDNA, NRSE/RE1 dsRNA. 15N labeled CoREST SANT2 was obtained by utilizing the recombinant plasmid pET28a SANT2 in M9 medium. Then a series of 2D 1H-15N HSQC spectra of free SANT2, and bound SANT2 （with ZF9 in NRSF/REST, NRSE/RE1 dsDNA, NRSE/RE1 dsRNA） were acquired. Further analysis revealed that non-coding small dsRNA might activate neuron-gene expression through interfering the interaction between CoREST SANT2 and ZF9 in NRSF/REST.