SARS/YY1 complex compete NFκB1 for binding at vegfa distal -4654/-4623 cis-element to regulate vegfa transcription activity and angiogenesis
Date Issued
2016
Date
2016
Author(s)
Wang, Po-Chun
Abstract
Vascular endothelial growth factor A (VEGFA) is an endothelial cell-specific mitogen which plays a pivotal role in angiogenesis. The expression of vegfa gene is modulated by some transcription factors, including Sp1, AP-2 and STAT-3. These transcription factors bound at proximal regulatory DNA sequences within the 2kb upstream region and thus regulate transcription activity of vegfa. Recent reports have demonstrated that Seryl-tRNA synthetase (SARS) not only has aminoacylation activity, but also localize into nucleus and directly binds to the proximal -62~-36 cis-element to repress transcription of human vegfa (hvegfa). On the other hand, SARS interacts with Yin-Yang1 (YY1) to form a protein complex. Meanwhile, both of them are bound at the distal -4723~-4184 region of hvegfa. Previous studies have focused on transcriptional regulation modulated by cis-element at proximal region of hvegfa gene. However, it is unclear whether SARS control the expression of hvegfa gene through distal upstream DNA sequences beyond 4 kb. First, by DNA sequence analysis, we found a potential YY1 / nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) binding cassette at the -4654~-4623 segment of hvegfa gene. Luciferase assy reveal that -4654~-4623 segment is critical for SARS to regulate the hvegfa transcription activity driven by the -5868~+55 segment. Moreover, we indicate that the transcription activity driven by -4654~-4623 segment were repressed by SARS and YY1, but actived by NFκB1. Furthermore, we prove that SARS control the transcriptional activity of hvegfa by form a complex with YY1. Once the YY1- and NFκB1-specific binding sequences within -4654~-4623 segment are mutated, SARS/YY1 and NFκB1 totally lost their ability to influence the promoter activity. Using western blot assay, we demonstrate that the amount of VEGFA protein produced in cells is dependent on competitive binding between SARS/YY1 complex and NFκB1 at the -4654~-4623 segment in vitro. By in vivo assay of zebrafish embryos from transgenic line Tg(fli1:EGFP)y1, we observe that the blood vessel development is reduced by SARS and YY1 synergistically. By contrast, the blood vessel development is enhanced by NFκB1. Taken together, we conclude that (1) SARS binds tightly to YY1 instead of binding to DNA, in that way SARS/YY1 complex function as a negative effector to regulate expression of human vegfa through -4654~-4623 cis-element. (2) NFκB1 serves as a positive effector through competing with SARS/YY1 binding at the same cis-element. Therefore, SARS/YY1 complex and NFκB1 is a pair of “Yin-Yang” regulator for proper development of a functional vasculature.
Subjects
tanscriptional regulation
angiogenesis
Type
thesis