Identification of flanking evolutionarily conserved sequences (FECS) and their role in the chromosomal-dependent expression ofjunB

The proto-oncogene junB is an immediate early gene which is induced by diverse mitogens. The product of this gene is a bZip protein which belongs to the AP-1 family. JunB promotes both cell proliferation and differentiation in different cell types. Due to its multiple functions in cell growth and differentiation, a better understanding of junB regulation may help to illuminate the mechanisms of gene expressions. We study the junB regulation from three aspects, which are: regulatory elements, transcription factors and chromatin structure. In order to identify cis regulatory elements, we compared the DNA sequences between human and mouse junB loci. There are nine regions within 5$\sp\prime$ and 3$\sp\prime$ junB flanking DNA that share 72 to 91% identity between human and mouse. These regions are from 47 bp to 487 bp in length. We named these conserved blocks as Flanking Evolutionarily Conserved Sequences or FECS. Some of the FECS are located within DNAse I hypersensitive regions and most of the regulatory elements in junB locus are also in the FECS. In FECS II and FECS III, we have identified specific protein binding sites and regulatory sequences. Some of these cis elements can affect junB induction in stable and transient expression assays. These regulatory elements include a SRE site (SRE III), two Ets sites (Ets II and Ets III) and two novel Ets-linked motifs (ELMs) (ELM II and ELM III). In general, mutation in any single element significantly impairs junB induction in stable functional assay. Furthermore, mutations in SRE III and ELM III also alter the structure of junB 5$\sp\prime$ flanking DNA within chromatin. In the sequence of ELM III, which is CTGGTCCCACC, we find that CCACC is critical for protein binding and transcriptional regulation. This CCACC motif mediates negative transcriptional regulation in transient assay and induces DNA bending through the binding of Sp1. Therefore, Sp1 is a transcription factor with the property of architectural protein in junB regulation. Studies in this dissertation suggest that FECS are critical components not only in junB transcriptional regulation but also important in other genes. Our data also suggest that transcription activity requires multiple regulatory factors which include cis-elements, transcription factors, architectural proteins and protein-protein interactions to form functional three-dimensional nucleoprotein structures