| Summary: | 博士 === 國立臺灣大學 === 生化科學研究所 === 88 === Abstract
CSB (Conserved Sequence Block), a block of 125 nucleotide in length, is located between Jα3 and Jα4, approximately 5 kb to the 5'' end of Cα exon l, and its sequence is highly conserved between human and mouse. There are only six differences over 125 nucleotide positions (95% similar). This degree of conservation is much higher than the ~71% overall sequence similarity found between human and mouse in the non-coding region of the TCR Cα/Cδ loci, and is also higher than that obtained when TCR α enhancer region sequences are compared. In addition, no transcript was shown to hybridize to the CSB region in different mouse tissues and zoo blot analysis using the CSB as a probe suggests that this sequence may be conserved in other vertebrates. Given these observations, it seems plausible that this conserved sequence may play a role relevant to the evolutionarily conserved coordinate regulation of TCR gene expression/rearrangement.
In this study, a series of detailed analyses were performed to explore the function of CSB. Transient transfection results showed that the CSB-containing element in conjunction with the TCR α enhancer up-regulated the α enhancer activity, whereas no enhancer activity was detected when CSB alone was assayed. In vitro occupancy analyses of CSB by nuclear factors revealed the existence of an unexpectedly intricate network of CSB-protein and protein-protein interactions. Lymphoid-specific as well as T lineage-specific nuclear factors are involved to differentially form CSB-bound complexes in extracts of various tissues and cell lines. In addition, certain CSB-binding factors were shown to be expressed in a developmental stage-specific fashion. These factors were easily detected in adult thymic extracts but barely in fetal thymic extracts, or vise versa. Similarly, certain factors involved in forming CSB-bound complexes were expressed in CD4+CD8+ immature T cell as well as CD4+ and CD8+ mature lymphocytes, but were barely detectable in immature CD4-CD8- thymocytes. Furthermore, CSB - bound complexes were formed in extracts of mouse fetal brain and liver tissues; however they were absent in extracts of adult''s brain and liver tissues. The results obtained from these studies also showed that transcription factor Oct-1 as well as lymphoid-specific DNA-binding factors Oct-2 and GATA-3 were indeed involved in interacting with CSB both in vivo as demonstrated by a chromatin immunoprecipitation assay and in vitro as evidenced by electrophoretic mobility shift assays in the mouse thymus. Finally, nuclear extracts from chicken tissues were shown to contain the activity of forming tissue-specific CSB-bound complexes similar to that observed in mouse, and the transcription factors GATA-3 and Oct-2 that have been highly conserved during vertebrate evolution were shown to be involved in forming in vitro CSB-bound complexes in chicken. These findings strongly suggest that CSB is a cis-regulatory element which may play a role of regulating the chromatin structure in the TCR Jα region flanking CSB, so that the V to Jα rearrangement in this region could be temporally regulated during T cell development. These results also suggest that further identification of these CSB-interacting factors would help understand the molecular mechanism involved in regulating the rearrangement and expression of genes in TCR α/δ locus during T cell development.
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