The Effect Of Transcription Factor CTCF And Enhancer RNA On The Gene Expression Of PCDH Cluster

Protocadherin(Pcdh)cluster can alternatively express more than 50 types of protein.The encoded PCDH proteins play critical roles in neuronal diversity,single cell identity and synaptic connectivity.However,the mechanism of the regulation of gene expression in Pcdh cluster is still not clear.We performed EMSA(Electrophoresis Mobility Shift Assay)experiments and found the regulatory region of Pcdh?? contains CTCF(CCCTC-binding factor)binding sites(CBS),which are conserved between human and mouse.Genome-wide computational analysis revealed many CBS sites close to chromatin marks of regulatory elements,such as enhancer and promoter.We propose that CTCF may mediate specific interactions between enhancers and promoters to form active transcription hub for gene expression.In addition,the enhancers of Pcdh cluster have RNA transcripts,named enhancer RNA(eRNA)that is a type of long noncoding RNA.We mainly studied eRNA transcribed from HS7,HS5-1,HS7 L and HS5-1aL by RT-PCR in mouse cortex and Neuro-2a.The results revealed that all those four sites can transcribe RNA bi-directionally.Interestingly,the eRNA in HS7 has Poly(A)(polyadnelytion)tail,while the eRNA of the remaining three sites do not contain Poly(A)tail.Finally,we carried out shRNA knockdown in Neuron-2a and HEC1-B cell line to study the function of eRNA in HS5-1 site.The results showed that the gene expression of Pcdh? cluster is downregulated when knock down the eRNA in HS5-1 site.Thus,the enhancer RNA may facilitate the gene expression of target gene.In summary,transcription factor CTCF and eRNA play an important role in regulating gene expression,which lay the vitally molecular foundation for the further research of Pcdh cluster.