Insights into transcriptional regulation of the receptor guanylyl cyclase C gene : role of HNF4, coup-TFs and GATA-6 transcription factors

Abstract

Transcriptional regulation in eukaryotes is a multi-step process involving the ordered assembly of multiprotein complexes on regulatory regions of DNA. DNA-bound specific transcription factors trigger the recruitment of general transcription factors at the core promoter, leading to gene expression.

This thesis investigates the role of specific transcription factors in regulating the expression of Guanylyl Cyclase C (GC-C), the receptor for heat-stable enterotoxins (ST) and the guanylin family of peptide hormones.

Background GC-C is a membrane-bound glycoprotein receptor activated upon ligand binding, resulting in the formation of cGMP.

cGMP activates protein kinase G, which phosphorylates and activates CFTR, leading to ion and water secretion into the intestinal lumen.

GC-C is robustly expressed in the intestine and other tissues (liver, lung, kidney, reproductive organs).

GC-C knockout mice are viable but resistant to ST-mediated diarrhea.

GC-C also plays roles in cell cycle regulation, apoptosis, and serves as a marker for colorectal cancer metastasis.

Tissue-specific expression suggests transcriptional regulation is crucial for its physiological functions.

Chapter Summaries Chapter 1

Overview of eukaryotic transcription, RNA polymerase II core promoter, basal transcription machinery, and transcription cycle.

Discussion of nuclear receptor family, focusing on HNF4 and COUP-TF, and their mechanisms of action.

Review of guanylyl cyclases and transcriptional regulation of GC-C.

Chapter 3

Characterization of the 5 upstream sequence of GC-C revealed binding sites for HNF4, Cdx2, and GATA factors.

In HepG2 cells, dexamethasone upregulated GC-C expression by enhancing HNF4 recruitment to the proximal promoter.

Chapter 4

Cloning and characterization of the rat GC-C promoter showed high sequence identity with mouse (93%) and human (78%).

Subtle differences included loss of a GATA binding site in the rat promoter and altered HNF4 binding affinity (~10-fold higher in rat).

Suggests differential regulation across species.

Chapter 5

Studies on GATA-6 and its interplay with HNF4.

GATA-6 regulates GC-C promoter activity; mutation of the GATA site reduces activity by 50%.

Synergistic activation observed with HNF4 and GATA-6 in human promoter; synergy persists in rat promoter despite lacking GATA site.

Co-immunoprecipitation and ChIP assays confirmed simultaneous binding of HNF4 and GATA-6.

Chapter 6

Expression and regulation of GC-C in rat epididymis.

Established epididymal epithelial primary cultures.

Demonstrated COUP-TFII expression in epididymis.

EMSA and ChIP assays showed COUP-TFII binding to GC-C promoter in epididymal tissue, unlike HNF4 binding in intestinal cells.

Chapter 7

Mechanism of PKC-mediated downregulation of GC-C transcription in T84 cells.

PMA treatment reduced HNF4 binding to the proximal promoter.

PKC/MAPK pathway implicated in transcriptional repression.

Cell-specific phenomenon, as HepG2 and COS-7 cells showed no change.

Conclusion The work emphasizes the combinatorial control of gene expression in eukaryotes.

Interplay between transcription factors (HNF4, COUP-TF, GATA-6) leads to cell-specific regulation.

Rat vs. human promoter differences highlight species-specific transcriptional mechanisms.

Signal transduction pathways (PKC/MAPK) modulate transcription factors post-translationally, contributing to cell-specific regulation of GC-C.