scholarly journals The cooperation of cis-elements during M-cadherin promoter activation

2021 ◽  
Vol 478 (4) ◽  
pp. 911-926
Author(s):  
Yung-Jui Lin ◽  
Chien-Han Kao ◽  
Sheng-Pin Hsiao ◽  
Shen-Liang Chen
Keyword(s):  
Transcription Initiation ◽  
Myogenic Differentiation ◽  
Transmembrane Protein ◽  
Initiation Site ◽  
Culture Conditions ◽  
Proximal Promoter ◽  
Cis Elements ◽  
Promoter Activation ◽  
Muscle Creatine Kinase ◽  
E Boxes

M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47. Furthermore, MyoD/E47 heterodimer and MyoD ∼ E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM. We also found that GC boxes and CDE positively affected MyoD mediated activation. The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer. Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin. Using ChIP assay, we further found significant reduction in MyoD recruitment to M-cadherin promoter when CDE was deleted. Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.

Myogenic regulatory factors regulate M-cadherin expression by targeting its proximal promoter elements

2010 ◽  
Vol 428 (2) ◽  
pp. 223-233 ◽  
Author(s):  
Sheng Pin Hsiao ◽  
Shen Liang Chen
Keyword(s):  
Cell Adhesion ◽  
Transcription Initiation ◽  
Myogenic Differentiation ◽  
Initiation Site ◽  
Proximal Promoter ◽  
Myogenic Regulatory Factors ◽  
Regulatory Factors ◽  
Cell Cell

M- and N-cadherin are members of the Ca2+-dependent cell–cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell–cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from −2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix–loop–helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.

A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene

1989 ◽  
Vol 9 (2) ◽  
pp. 594-601
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
M E Perry ◽  
E N Olson
Keyword(s):  
Creatine Kinase ◽  
Growth Factors ◽  
Myogenic Differentiation ◽  
Regulatory Elements ◽  
Negative Control ◽  
Proximal Promoter ◽  
Muscle Creatine Kinase ◽  
Kinase Gene ◽  
Muscle Creatine ◽  
Ras Oncogenes

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.

Characterization of the rat NHE3 promoter

1996 ◽  
Vol 271 (3) ◽  
pp. F629-F636 ◽  
Author(s):  
A. Cano
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Initiation ◽  
Transmembrane Protein ◽  
Initiation Site ◽  
Proximal Tubule Cell ◽  
Specific Expression ◽  
Genomic Libraries ◽  
Sequence Elements ◽  
Flanking Region

NHE3, a transmembrane protein involved in transcellular ion transport, is expressed in the apical membrane of renal and gastrointestinal epithelia. Chronic regulation by multiple stimuli, including glucocorticoid-induced transcriptional regulation, has been demonstrated. To study the tissue-specific expression and transcriptional regulation of NHE3, the 5' flanking region of the rat NHE3 gene was cloned. Two genomic libraries were screened with the 5' end of the NHE3 cDNA. The 5' flanking region and first exon were isolated. Primer extension mapped a single transcription start site in stomach, colon and kidney. The NHE3 promoter near the transcription initiation site is characterized by the absence of TATA and CAAT sequences. Two Sp1 sites, one Egr-1 site, and an initiator with the sequence GGGATTAAA mark the area of transcription initiation. Upstream sequences include multiple DNA sequence elements recognized by the glucocorticoid and thyroid receptors, Sp1, atriopeptin-2, and several other transcription factors. Transcriptional regulation by glucocorticoids and chronic acidosis was demonstrated. Promoter activity was present in OKP cells, a renal proximal tubule cell line, but not in fibroblasts. This suggests that the NHE3 promoter contains elements conferring epithelial cell-specific expression.

scholarly journals Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site

2007 ◽  
Vol 407 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Ian Pearse ◽  
Ying X. Zhu ◽  
Eleanor J. Murray ◽  
Pradeep K. Dudeja ◽  
Krishnamurthy Ramaswamy ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Critical Role ◽  
Constitutive Expression ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Proximal Promoter ◽  
Gc Box

We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the −40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, −10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5′ flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at −25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5′-UTR (5′-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5′-UTR may co-operate with the 5′-flanking region to achieve the optimal promoter activity.

Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4078-4083 ◽  
Author(s):  
Li Xue ◽  
Xiaoyong Chen ◽  
Yanjie Chang ◽  
James J. Bieker
Keyword(s):  
Transcription Initiation ◽  
Fetal Liver ◽  
Critical Role ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Proximal Promoter ◽  
Erythroid Cell ◽  
Mammalian Development ◽  
Specific Expression ◽  
Position Effects

Abstract Erythroid Krüppel-like factor (EKLF) plays an essential role in enabling β-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose.

Isolation and primary function detection of Nile tilapia (Oreochromis niloticus) β-actin promoter

2008 ◽  
Vol 5 (2) ◽  
pp. 141-146
Author(s):  
Yu Er-Meng ◽  
Ye Xing ◽  
Wang Hai-Ying ◽  
Bai Jun-Jie ◽  
Xia Shi-Ling ◽  
...  
Keyword(s):  
Oreochromis Niloticus ◽  
Transcription Initiation ◽  
Nile Tilapia ◽  
Gene Promoter ◽  
Pcr Amplification ◽  
Transgenic Fish ◽  
Initiation Site ◽  
Proximal Promoter ◽  
Regulatory Sequence ◽  
Transcription Activity

AbstractA 5′-flanking region and partial open reading frame (ORF) of the β-actin gene (GenBank accession No. EF026001) of Nile tilapia (Oreochromis niloticus) was cloned by polymerase chain reaction (PCR) amplification. The segment included a 1643 bp regulatory sequence and a 90 bp partial ORF which encoded a 30-amino-acid peptide. The regulatory sequence comprised a 108 bp 5′ proximal promoter, the first untranslated exon and the first intron of the β-actin gene. The proximal promoter region contained elements that were critical for transcription activity, including a CCAAT, TATA and CArG box located at –92, –29 and –62 bp upstream of the transcription initiation site, respectively. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector to construct the expressing vector pNA-DsRed. The linearized pNA-DsRed was microinjected into the fertilized eggs of Tanichthys albonubes. The expression of the DsRed2 gene in transgenic fish could be observed under the microfluoroscope and anatomical lens. The results showed that the β-actin gene promoter possessed effective transcription activity.

scholarly journals A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.

1989 ◽  
Vol 9 (2) ◽  
pp. 594-601 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
M E Perry ◽  
E N Olson
Keyword(s):  
Creatine Kinase ◽  
Growth Factors ◽  
Myogenic Differentiation ◽  
Regulatory Elements ◽  
Negative Control ◽  
Proximal Promoter ◽  
Muscle Creatine Kinase ◽  
Kinase Gene ◽  
Muscle Creatine ◽  
Ras Oncogenes

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.

P/CAF rescues the Bhlhe40-mediated repression of MyoD transactivation

2009 ◽  
Vol 422 (2) ◽  
pp. 343-352 ◽  
Author(s):  
Sheng P. Hsiao ◽  
Kai M. Huang ◽  
Hsin Y. Chang ◽  
Shen L. Chen
Keyword(s):  
Transcription Initiation ◽  
Myogenic Differentiation ◽  
Binding Protein ◽  
Core Promoter ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Boxes

Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α) by targeting a short region, from nt −49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1α core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1α and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.

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