1: Biochim Biophys Acta. 2005 Sep 13; [Epub ahead of print] 

Transcriptional regulation of the human reduced folate carrier A1/A2 promoter:
Identification of critical roles for the USF and GATA families of transcription
factors.

Payton SG, Liu M, Ge Y, Matherly LH.

Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, and Wayne
State University School of Medicine, Detroit, MI, USA.

The human reduced folate carrier (hRFC) gene has a complex regulation involving
6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and
E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript
with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC
protein with altered transport function. In this report, we characterize the
hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells
with 5' and 3' deletion constructs, a transcriptionally important 270 bp region
was identified. Gel shift assays identified transcription factor binding to
three E-box elements and one GATA site within this region. These elements were
verified by transfections of mutant constructs into HepG2 cells. Cotransfections
in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A
physical association between USF1 and GATA1 was demonstrated by their
co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells,
USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our
results demonstrate a transcriptionally important region in the hRFC-A1/A2
promoter including E-box and GATA elements, and a transactivation by USF1 and
GATA1 proteins. Our results further establish the complexity of hRFC regulation,
as a means of ensuring adequate folate cofactor transport for cell
proliferation.

PMID: 16225938 [PubMed - as supplied by publisher]


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2: J Biol Chem. 2003 Jan 24;278(4):2571-80. Epub 2002 Nov 8. 

Regulation of expression of the phospholipid hydroperoxide/sperm nucleus
glutathione peroxidase gene. Tissue-specific expression pattern and
identification of functional cis- and trans-regulatory elements.

Borchert A, Savaskan NE, Kuhn H.

Institute of Biochemistry, Humboldt University Medical School Charite,
Monbijoustrasse 2, 10117 Berlin, Germany.

A sperm nucleus glutathione peroxidase (snGPx), which is closely related to the
phospholipid hydroperoxide glutathione peroxidase (phGPx), was recently
discovered in late spermatids. Both GPx isoforms originate from a joint ph/snGPx
gene, but their N-terminal peptides are encoded by alternative first exons. The
expression of the two enzymes is differentially regulated in various cells, but
little is known about the regulatory mechanisms. To explore the tissue-specific
regulation of expression of the two isoenzymes, we first investigated their
tissue distribution. Whereas phGPx is expressed at low levels in many organs,
snGPx was only detected in testis, kidney, and in the human embryonic kidney
cell line HEK293. Subcellular fractionation studies and immunoelectron
microscopy revealed a cytosolic localization. To explore the mechanistic reasons
for the differential expression pattern, we first tested the activity of the
putative phGPx and snGPx promoters. The 5'-flanking region of the joint ph/snGPx
gene exhibits strong promoter activity. In contrast, the putative snGPx
promoter, which comprises 334 bp of intronic sequences, lacks major promoter
activity. However, it strongly suppresses the activity of the ph/snGPx promoter.
These data suggest negative regulatory elements in the first intron of the
ph/snGPx gene, and DNase protection assays revealed the existence of several
protein-binding sites. The corresponding trans-regulatory proteins (SP1, ERG1,
GATA1, SREBP1, USF1, and CREBP1) were identified, and in vivo binding of EGR1
and SREBP1 was shown by chromatin immunoprecipitation. These data indicate for
the first time somatic expression of the snGPx and provide evidence for the
existence of intronic negative cis-regulatory elements in the ph/snGPx gene. Our
failure to detect an alternative snGPx promoter suggests that transcription of
the ph/snGPx gene may be regulated by a joint basic promoter. The decision,
which GPx isoform is expressed in a given cell, appears to be made by
alternative splicing of a joint primary transcript.

PMID: 12427732 [PubMed - indexed for MEDLINE]


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