Abstract

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C. The MARCKS gene is under multiple modes of transcriptional control, including cytokine- and transformation-dependent, cell-specific, and developmental regulation. This study evaluated the transcriptional control of MARCKS gene expression during early development of Xenopus laevis. Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT). The Xenopus MARCKS gene was similar to those of other species, except that a second intron interrupted the 5'- untranslated region. By transiently transfecting XTC-2 cells and microinjecting Xenopus embryos with reporter gene constructs containing serial deletions of 5'-flanking MARCKS sequences, we identified a 124-base pair minimal promoter that was critical for promoter activity. Developmental gel shift assays revealed that a CBF/NF-Y/CP-1-like factor and an Sp1-like factor bound to this region in a manner correlating with the onset of Xenopus MARCKS transcription at MBT. Mutations in the promoter that abolished binding of these two factors also completely inhibited transcriptional activation of the MARCKS gene at MBT. The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might also regulate MARCKS transcription in other species. These studies not only increase our knowledge of the transcriptional regulation of the MARCKS genes but also have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT.

Highlights

  • The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C

  • Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT)

  • Our results indicate that a CBF/NF-Y-like factor and an Sp1like factor bind to the Xenopus MARCKS promoter and that mutations of their binding sites that prevent transcription factor binding inhibit activation of the MARCKS gene at the midblastula transition (MBT)

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Summary

Mechanisms of MARCKS Gene Activation during Xenopus Development*

The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might regulate MARCKS transcription in other species. These studies increase our knowledge of the transcriptional regulation of the MARCKS genes and have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT. It is conceivable that by serial deletion/mutation analysis of the promoter in transgenic mice, it might be possible to identify regulatory elements that are important for developmental control of the MARCKS gene Such experiments would be expensive and time-consuming.

In Vitro Phosphorylation
Cloning of a Xenopus MARCKS cDNA and Genomic Clones
Protein Extracts
Microinjection of Embryos and CAT Assay
Gel Shift and DNase I Protection Analysis
RESULTS
DISCUSSION
Full Text
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