Authors:

Amira Bailey

Date Created:

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About Paper:

The heart is the first organ that forms in mammalian development, which is mainly composed of progenitor cells derived from the mesoderm. While the Wnt signaling pathway plays a key role in mesoderm development, the direct transcriptional regulation of genes involved in the pathway is still unclear. The VEZF1 protein has been speculated to be involved in the regulation of Wnt signaling genes due to the impairment of mesoderm formation and reduced expression of Wnt signaling genes when VEZF1 is absent. We hypothesize that VEZF1 operates by binding and inducing the expression of Wnt signaling genes during mesoderm differentiation. To test our hypothesis, we grew and differentiated wild-type (WT), knockout (KO), and knockdown (KD) embryonic stem cells into mesoderm. We then used the differentiated cells to perform Chromatin Immunoprecipitation with the use of VEZF1 antibodies conjugated to magnetic beads to identify VEZF1 binding sites on DNA. Primers specific to Wnt signaling genes were designed using SnapGene and NCBI primer blast for putative VEZF1 binding sites. The primers were then utilized to perform quantitative-PCR to identify VEZF1 binding to Wnt signaling genes. The Dnmt3b gene was used as a positive control to verify the Chromatin Immunoprecipitation experiment. Our results show a significant binding of VEZF1 to the Dnmt3b promoter in WT cells, and reduced binding in both KO and KD cells, confirming the success of the ChIP experiment. Further investigation with the use of qPCR is currently being conducted with genes specific to the Wnt signaling pathway to determine the binding of VEZF1.

Source:

Purdue University / 2023

Topics:

No topics listed

Co-authors:

Amira Bailey