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Transcriptional control of CC chemokine receptor 3

Title
Transcriptional control of CC chemokine receptor 3
Other Titles
CC chemokine receptor 3의 전사조절
Author
김병수
Alternative Author(s)
김병수
Advisor(s)
정일엽
Issue Date
2011-08
Publisher
한양대학교
Degree
Doctor
Abstract
A combinatorial interaction of transcriptiona factors, including GATA-1, AML-1, PU.1, and C/EBP, plays a major role in controling eosinophil development and differentiation. The almost identical set of the transcription factors also regulates expression of eosinophil-specific genes. Thus, analysis of the transcription factors that regulate eosinophil-specific genes may offer insights into mechanisms behind the commitment and differentiation to the eosinophil lineage. In the present study, I have investigated how these factors individually and/or combinatorially regulate transcription of CC chemokine receptor 3 (CCR3), one of eosinophil specific genes, at the molecular level. The regulatory region of the CCR3 gene includes at least the binding elements for all these transcription factors. In the first series of experiments I focused on CCR3 transcription regulation by GATA-1. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1 of CCR3. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all the seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene. In the second series of experiments, I have investigated how AML-1, PU.1, and C/EBPα regulate CCR3 gene transcription. There are two AML-1 binding sites in the regulatory region of CCR3. A mutant in which either AML-1 binding site was disrupted had reduced transcription activity severely to moderately, suggesting that AML-1 is involved in CCR3 transcription. However, when the two AML-1 binding sites were disrupted along with the functional GATA element (the first GATA site), one had an additive effect while the other had elevated transcription activity. Thus, the mode of action of AML-1 appears to be position-dependent. On the other hand, there were also two PU.1 binding sites in tandem in the regulatory region of CCR3. A construct in which both two PU.1 sites were mutated had a reduced transcription activity. A double mutant in which both PU.1 and functional GATA sites were disrupted had a further reduced activity in an additive manner. EMSA analysis, which was carried out with nuclear extract from K562 cells that expressed both AML-1a and PU.1 at low levels, showed that both AML-1a and PU.1 appeared to bind their respective sites, although the binding specificity was confirmed for AML-1 by a supershift. Introduction of AML-1 and PU.1 siRNAs resulted in a decrease in reporter activity in Jurkat cells that do not express GATA-1 but do express AML-1 and PU.1. Thus, these results suggest that both AML-1 and PU.1 play an active role in CCR3 transcription. Enforced expression of C/EBPa in K562 cells (which do not express C/EBPa) enhanced the transactivation of the Ex-In1 construct that do not include the C/EBP binding sequence suggesting that C/EBPa may participate in CCR3 transcription through a physical interaction with other transcription factors. Taken together, not only GATA-1 but also AML-1, PU.1, and C/EBPa regulate CCR3 transcription individually or in combination. As such, the combination of these transcription factors that regulate eosinophil development/differentiation is used exactly for transcriptional control of the eosinophil-specific gene, CCR3.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/138853http://hanyang.dcollection.net/common/orgView/200000417334
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Theses (Ph.D.)
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