1: Cardiovasc Res. 2004 Dec 1;64(3):402-11. 

Transcriptional regulation of the murine Connexin40 promoter by cardiac factors
Nkx2-5, GATA4 and Tbx5.

Linhares VL, Almeida NA, Menezes DC, Elliott DA, Lai D, Beyer EC, Campos de
Carvalho AC, Costa MW.

Laboratorio de Cardiologia Celular e Molecular-Instituto de Biofisica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 20941-900,
Brazil.

OBJECTIVE: Connexin40 (Cx40) is a gap junction protein expressed specifically in
developing and mature atrial myocytes and cells of the conduction system. In
this report, we identify cis-acting elements within the mouse Cx40 promoter and
unravel part of the complex pathways involved in the cardiac expression of this
gene. METHODS: To identify the factors involved in the cardiac expression of
Cx40, we used transient transfections in mammalian cells coupled with
electrophoretic mobility shift assays (EMSA) and RT-PCR. RESULTS: Within the
promoter region, we identified the minimal elements required for transcriptional
activity within 150 base pairs (bp) upstream of the transcriptional start site.
Several putative regulatory sites for transcription factors were predicted
within this region by computer analysis, and we demonstrated that the nuclear
factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements
present in the minimal promoter region of the Cx40. Furthermore, co-transfection
experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal
Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis
of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased
activity in rat smooth muscle cell line A7r5. Consistent with this, mouse
embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression. CONCLUSION:
In this work, we cloned the promoter region of the Cx40 and demonstrated that
the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5
and GATA4 acting together with ubiquitous Sp1.

PMID: 15537493 [PubMed - indexed for MEDLINE]


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2: Gene. 2003 Dec 11;322:123-36. 

Analysis of the rat connexin 43 proximal promoter in neonatal cardiomyocytes.

Teunissen BE, Jansen AT, van Amersfoorth SC, O'Brien TX, Jongsma HJ, Bierhuizen
MF.

Department of Medical Physiology, University Medical Center Utrecht, P.O. Box
85060, 3508 AB Utrecht, The Netherlands. B.E.J.Teunissen@med.uu.nl

Altered transcriptional control is likely to contribute to the down-regulation
of connexin 43 (Cx43) expression observed in many forms of heart disease.
However, little is known about the factors regulating Cx43 transcription in the
heart under (patho)physiological conditions. Therefore, a systematic study of
rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal
ventricular cardiomyocytes (NCM) and, for comparison, different cell types was
initiated. Luciferase assays revealed that, in NCM, the proximal promoter is
preserved in a conserved region extending from 148 nucleotides upstream towards
281 nucleotides downstream relative to the transcription initiation site (TIS).
Further deletional analysis suggested the involvement of four putative Sp- and
two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding
elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic
mobility shift assays (EMSA). Promoter-luciferase assays using the natural rCx43
proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed
that all sites contribute to basal promoter activity. Trans-activation of the
Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2)
cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43
promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In
addition, we studied the effect of the cardiac transcription factor Nkx2.5 on
Cx43 regulation. NCM were infected with adenovirus encoding either
beta-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly
decreased after Nkx2.5 infection as shown by Western and Northern blot analyses.
Promoter-reporter assays demonstrated that the rCx43 promoter was down-regulated
approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5
appears to play a role in the regulation of Cx43 expression.

PMID: 14644504 [PubMed - indexed for MEDLINE]


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3: Gene. 2002 Jul 10;294(1-2):259-68. 

Structural and functional characterization of the human PAX7 5'-flanking
regulatory region.

Syagailo YV, Okladnova O, Reimer E, Grassle M, Mossner R, Gattenlohner S, Marx
A, Meyer J, Lesch KP.

Department of Psychiatry and Psychotherapy, University of Wurzburg,
Fuchsleinstrasse 15, 97080, Wurzburg, Germany.

The human PAX7 gene is a member of the paired box containing gene family of
transcription factors implicated in development of the skeletal muscle of the
trunk and limbs as well as elements of the central nervous system. To understand
the molecular mechanisms involved in its expression, we have localized the
transcription start sites in adult skeletal muscle and functionally
characterized the 5'-flanking regulatory region responsible for PAX7 expression
in this tissue. The major transcription start was identified 664 bp upstream
from the ATG codon using primer extension and 5'-rapid amplification of cDNA
ends (5'-RACE). Analysis of the 5'-flanking sequence revealed the absence of a
TATA-box and the presence of an inverted CCAAT-box. Several consensus sites for
common transcriptional regulators including Oct-1, NF1, AP2, AP4, CREB, Sp1,
Nkx2.5, and MyoD are present in the promoter region. To determine the sites
critical for the function of the PAX7 promoter, a series of deletion fragments
of the 5'-flanking region were cloned adjacent to luciferase reporter gene and
expressed in RD, Cos-7 and JAR cell lines. The maximal promoter activity was
achieved by a fragment extending from the position -403 to +373. No strong
positive or negative regulatory elements were discovered by adding of further
sequences (up to 2.97 kb). A polymorphic (CCT)(n) repeat sequence was found 107
bp upstream of the transcription initiation site. PCR-based systematic screening
for length variations in 227 unrelated individuals of a Caucasian population
showed a bimodal distribution of three alleles containing 8, 10 or 11 repeat
units. When different variants of this PAX7 gene-linked polymorphic region
(PAX7-LPR) were fused to a luciferase reporter gene and transfected into RD
cells, the variant with 11 repeat units revealed higher transcriptional
efficiency compared to the 8 or 10 repeat alleles.

PMID: 12234688 [PubMed - indexed for MEDLINE]


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4: Biochim Biophys Acta. 2002 Jun 7;1576(1-2):183-90. 

Genomic structure and promoter characterization of the gene encoding the ErbB
ligand betacellulin.

Lawson J, Wheldrake JF, Dunbar AJ.

Cooperative Research Centre for Tissue Growth and Repair, School of Biological
Sciences, Faculty of Science and Engineering, Flinders University of South
Australia, GPO Box 2100, Adelaide 5001, Australia.

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of
peptide ligands that are characterized by a six-cysteine consensus motif
(EGF-motif) that forms three intra-molecular disulfide bonds, crucial for
binding the ErbB receptor family. A variety of in vitro studies have identified
BTC as an important factor in the growth and/or differentiation of pancreatic
islet cells. The molecular mechanisms that regulate the transcription of the BTC
gene however have not been delineated. As an initial step, we have characterized
the genomic structure of the mouse BTC (mBTC) gene. mBTC cDNA was used as a
probe to screen a mouse 129/SVJ genomic bacterial artificial chromosome (BAC)
library. Three positive clones containing the entire gene were isolated. DNA
sequence analysis identified six exons (1-6) and five introns (A-E); a structure
conserved among the EGF family. PCR analysis showed that introns A-E are
approximately 7.8, 8.9, 3.8, 1.4 and 1.4 kb in length, respectively. The
EGF-motif is encoded by exons 3 and 4 with an intron (intron C) disrupting the
coding sequence between the second and third disulfide loops. All exon-intron
boundaries are consistent with the "gt-ag" rule. Multiple transcription start
sites and one poly(A) site, located 18 bp downstream of a polyadenylation signal
sequence, were identified by 5'- and 3'-RACE, respectively. Approximately 2.6 kb
of 5'-flanking region was sequenced and was shown to lack consensus TATA and
CCAAT boxes, but was found to contain several putative cis-acting regulatory
elements. These included consensus binding sites for transcription factors HNF3
beta, USF, Nkx2-5, AP-4, and Sp1. Functional promoter analysis of the
5'-flanking region in COS-7 cells, using 5'-deletion fragments (-168/+335;
-635/+335; -732/+335; -1175/+335; -1698/+335) cloned into a promoterless firefly
luciferase reporter vector, identified basal promoter activity and both positive
and negative cis-acting elements.

PMID: 12031500 [PubMed - indexed for MEDLINE]


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5: Gene. 2000 Dec 30;260(1-2):103-12. 

Genomic organization, 5'flanking region and tissue-specific expression of mouse
phosphofructokinase C gene.

Gunasekera D, Kemp RG.

Department of Biochemistry and Molecular Biology, Chicago Medical School, 3333
Green Bay Road, North Chicago, IL 60064, USA.

Using a combination of mouse bacterial artificial chromosome (BAC) genomic
library screening, long-range polymerase chain reaction (PCR) amplification,
genomic walking and DNA sequencing, we have characterized the intron/exon
boundaries, the sizes of each intron and 5' flanking region of the mouse PFK-C
gene. The gene spans approximately 55 kb and comprises 22 exons separated by 21
introns. All intron/exon splice junctions conform to the GT/AG rule. The mouse
PFK-C gene organization is similar to that of the human and rabbit PFK-A and
human and mouse PFK-B genes. However, PFK-C has much larger intronic sequences
throughout the gene. Anchored PCR was performed to amplify about 1.0 kb of
genomic DNA upstream of the translational start site. Sequence analysis of the
PFK-C 5' flanking region revealed that it is devoid of TATA and CAAT boxes at
the usual positions, but it contained several putative binding sites for
transcription factors AP1, GATA1, NKX2.5 and STAT. The 5' flanking region was
not enriched in GC dinucleotides and lacked CpG islands and putative binding
sites for SP1. Four transcription initiation sites have been identified by
full-length RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE)
between -61 and -32 bp from the translation initiation codon.Reverse
transcription-PCR analysis revealed that PFK-A, PFK-B and PFK-C genes were
expressed, in all mouse tissues tested, at varying levels. PFK-A mRNA was more
abundantly expressed in all tissues than were the PFK-B and PFK-C genes. Based
on the mouse PFK-C signal normalized to 18S rRNA, the PFK-C mRNA was expressed
at the highest levels in the brain, heart, thymus and testicles, whereas low
levels were observed in the kidney, liver, muscle, and lung.

PMID: 11137296 [PubMed - indexed for MEDLINE]


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6: J Biol Chem. 2001 Jan 12;276(2):1026-33. 

GATA-4 and serum response factor regulate transcription of the muscle-specific
carnitine palmitoyltransferase I beta in rat heart.

Moore ML, Wang GL, Belaguli NS, Schwartz RJ, McMillin JB.

Department of Pathology and Laboratory Medicine, Medical School, University of
Texas-Houston Health Science Center, Baylor College of Medicine, Houston, Texas
77030, USA.

Transcriptional regulation of nuclear encoded mitochondrial proteins is
dependent on nuclear transcription factors that act on genes encoding key
components of mitochondrial transcription, replication, and heme biosynthetic
machinery. Cellular factors that target expression of proteins to the heart have
been well characterized with respect to excitation-contraction coupling. No
information currently exists that examines whether parallel transcriptional
mechanisms regulate nuclear encoded expression of heart-specific mitochondrial
isoforms. The muscle CPT-Ibeta isoform in heart is a TATA-less gene that uses
Sp-1 proteins to support basal expression. The rat cardiac fatty acid response
element (-301/-289), previously characterized in the human gene, is responsive
to oleic acid following serum deprivation. Deletion and mutational analysis of
the 5'-flanking sequence of the carnitine palmitoyltransferase Ibeta (CPT-Ibeta)
gene defines regulatory regions in the -391/+80 promoter luciferase construct.
When deleted or mutated constructs were individually transfected into cardiac
myocytes, CPT-I/luciferase reporter gene expression was significantly depressed
at sites involving a putative MEF2 sequence downstream from the fatty acid
response element and a cluster of heart-specific regulatory regions flanked by
two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and
competition specificity (or supershifts) with oligonucleotides and antibodies.
Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4
enhanced CPT-I reporter gene expression 4-36-fold in CV-1 cells. Although
cotransfection of Nkx and SRF produced additive luciferase expression, the
combination of SRF and GATA-4 cotransfection resulted in synergistic activation
of CPT-Ibeta. The results demonstrate that SRF and the tissue-restricted
isoform, GATA-4, drive robust gene transcription of a mitochondrial protein
highly expressed in heart.

PMID: 11038368 [PubMed - indexed for MEDLINE]


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7: Am J Physiol Heart Circ Physiol. 2000 Mar;278(3):H796-805. 

Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in
Egr-1-deficient mice.

Saadane N, Alpert L, Chalifour LE.

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General
Hospital, Montreal H3T 1E2, Quebec, Canada H3A 1A3.

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar
heart-to-body weight ratios but express lower amounts of atrial natriuretic
factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal actin, NGF1-A binding
protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice.
alpha-MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and
phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF,
beta-MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction
in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1,
GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+
mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased
Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump
removal caused the agonist-treated hearts and gene expression to regress to
control or near-control levels. Thus Egr-1 deficiency caused a blunted
catecholamine-induced hypertrophy response and increased sensitivity to stress.

PMID: 10710348 [PubMed - indexed for MEDLINE]


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