TY - JOUR
T1 - USF in the Lytechinus sea urchin embryo may act as a transcriptional repressor in non-aboral ectoderm cells for the cell lineage-specific expression of the LpS1 genes
AU - Seid, Christopher A.
AU - George, Jenny M.
AU - Sater, Amy K.
AU - Kozlowski, Mark T.
AU - Lee, Haemin
AU - Govindarajan, Venkatesh
AU - Ramachandran, Ravi K.
AU - Tomlinson, Craig R.
N1 - Funding Information:
We thank Drs William H. Klein, Yolanda Sanchez and Akif Uzman for critical reading of the manuscript. We are most appreciative for the clones donated by Drs William H. Klein and Martin Chalfie and for the cDNA library sent by Dr David McClay. We thank Drs Deborah Kimbrell and Diane Shakes for the use of their microscope, Dr Lisa Meffert for her advice on the statistical analysis, and Richard Falzone for his technical assistance. This work was supported by Texas Advanced Research Program grant 003652-131 to A.K.S. and by the American Heart Association grants 91 G-441 and 93R-441 to C.R.T.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 1996/11/22
Y1 - 1996/11/22
N2 - Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed that a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type LpS1 promoter and the same LpS1 promoter with a mutated USF binding site fused to the bacterial chloramphenicol acetyltransferase reporter gene were tested. The mutation in the USF binding site caused an increase in chloramphenicol acetyltransferse activity. We selected a clone that encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RNA or a DNA construct consisting of the LvUSF cDNA clone fused to the Lytechinus pictus metallothionein promoter coinjected with the wild-type or mutated LpS1 promoter-chloramphenicol acetyltransferase gene construct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 promoter DNA fragments ligated to the green fluorescent protein reporter gene were used to examine spatial expression. The reporter gene constructs containing the mutated USF binding site were expressed inappropriately in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expressed primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in later embryos. The data are consistent with a model depicting Lytechinus USF, as a temporal and spatial regulator by repressing LpS1 gene transcription in non-aboral ectoderm cells.
AB - Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed that a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type LpS1 promoter and the same LpS1 promoter with a mutated USF binding site fused to the bacterial chloramphenicol acetyltransferase reporter gene were tested. The mutation in the USF binding site caused an increase in chloramphenicol acetyltransferse activity. We selected a clone that encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RNA or a DNA construct consisting of the LvUSF cDNA clone fused to the Lytechinus pictus metallothionein promoter coinjected with the wild-type or mutated LpS1 promoter-chloramphenicol acetyltransferase gene construct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 promoter DNA fragments ligated to the green fluorescent protein reporter gene were used to examine spatial expression. The reporter gene constructs containing the mutated USF binding site were expressed inappropriately in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expressed primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in later embryos. The data are consistent with a model depicting Lytechinus USF, as a temporal and spatial regulator by repressing LpS1 gene transcription in non-aboral ectoderm cells.
UR - http://www.scopus.com/inward/record.url?scp=0030598355&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030598355&partnerID=8YFLogxK
U2 - 10.1006/jmbi.1996.0619
DO - 10.1006/jmbi.1996.0619
M3 - Article
C2 - 8950263
AN - SCOPUS:0030598355
VL - 264
SP - 7
EP - 19
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 1
ER -