1: Biochem J. 2005 Sep 1;390(Pt 2):521-8. 

Cloning and characterization of the human glutathione synthetase 5'-flanking
region.

Lee TD, Yang H, Whang J, Lu SC.

Division of Gastroenterology and Liver Diseases, USC Research Center for Liver
Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases,
Department of Medicine, Keck School of Medicine USC, 2011 Zonal Ave., Los
Angeles, CA 90033, USA.

GSH synthesis occurs through a two-step enzymatic reaction driven by GCL
(glutamate-cysteine ligase; made up of catalytic and modifying subunits) and GSS
(glutathione synthetase). In humans, oxidative stress regulates GCL expression
in an antioxidant response element-dependent manner via Nrf2 [NFE (nuclear
factor erythroid)-related factor 2]. In the rat, GSS and GCL are regulated
co-ordinately by oxidative stress, and induction of GSS further increases GSH
synthetic capacity. Transcriptional regulation of the human GSS has not been
examined. To address this, we have cloned and characterized a 2.2 kb 5'-flanking
region of the human GSS. The transcriptional start site is located 80 nt
upstream of the translation start site. The human GSS promoter efficiently drove
luciferase expression in Chang cells. Overexpression of either Nrf1 or Nrf2
induced the GSS promoter activity by 130 and 168% respectively. Two regions
homologous to the NFE2 motif are demonstrated to be important for basal
expression of human GSS, as mutation of these sites reduced the promoter
activity by 66%. Nrf1, Nrf2 and c-Jun binding to these NFE2 sites under basal
conditions was demonstrated using chromatin immunoprecipitation assays. In
summary, two NFE2 sites in the human GSS promoter play important roles in the
basal expression of GSS and, similar to the GCL subunits, the human GSS gene
expression is also regulated by Nrf2.

PMID: 15890065 [PubMed - in process]


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2: Blood Cells Mol Dis. 2002 Sep-Oct;29(2):145-58. 

Cloning MafF by recognition site screening with the NFE2 tandem repeat of HS2:
analysis of its role in globin and GCSl genes regulation.

Marini MG, Asunis I, Chan K, Chan JY, Kan YW, Porcu L, Cao A, Moi P.

Istituto di Neurogenetica e Neurofarmacologia, CNR, Cagliari 09121, Italy.

The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the
human beta-globin locus control region is required for high level globin gene
expression. We used an oligonucleotide of the NF-E2 tandem repeat, within HS2,
as recognition site probe to screen a K562 cDNA library for interacting
transcription factors. A 2.3 kb full length cDNA encoding the b-zip
transcription factor MafF was isolated. MafF can form both homodimers and high
affinity heterodimers with Nrf1, Nrf2 and Nf-E2, three members of the CNC-bZip
family. Despite obvious structural similarities with the other small Maf
proteins, MafF differs in its tissue distribution and its inability to repress
transcription when overexpressed as homodimer. In fact, in different cell lines
and on different promoters (gamma-globin, beta-globin and glutamylcysteine
synthetase genes) the MafF homodimers do not appreciably affect transcription of
target promoters, whereas MafF/CNC member heterodimers act as weak
transcriptional activators. Even though MafF was cloned using probes derived
from the globin LCR, it is in the context of the GCSl promoter and in
combination with Jun that MafF shows a rather distinct and specific regulatory
role. These observations suggest that a complex network of small Maf and CNC-AP1
protein interactions might be involved in regulating transcription in diverse
tissues or developmental stages.

PMID: 12490281 [PubMed - indexed for MEDLINE]


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