CArG box gene transcriptions

Editor-In-Chief: Henry A. Hoff

File:Smooth muscle cell CArG.jpeg

The diagram shows a model for epigenetic regulation of SRF binding to CArG box chromatin. Credit: Oliver G. McDonald, Brian R. Wamhoff, Mark H. Hoofnagle, and Gary K. Owens.

CArG boxes are present in the promoters of smooth muscle cell genes. Template:TOCright

Boxes

Main article: Box gene transcriptions

Def. "a repeating sequence of nucleotides that forms a transcription or a regulatory signal" is called a box.

Consensus sequences

Main article: Consensus sequences

"CArG box [CC(A/T)₆GG] DNA [consensus] sequences present within the promoters of SMC genes play a pivotal role in controlling their transcription".

The consensus sequence of CC(A/T)₆GG is confirmed.

"MADS-box proteins bind to a consensus sequence, the CArG box, that has the core motif CC(A/T)₆GG (15)."

"Of the [Flowering Locus C] FLC binding sites, 69% contained at least one CArG-box motif with the core consensus sequence CCAAAAAT(G/A)G and an AAA extension at the 3′ end [...]."

Three "other MADS-box flowering-time regulators, SOC1, SVP, and AGAMOUS-LIKE 24 (AGL24), bind to two different CArG-box motifs at 502 bp (CTAAATATGG) and 287 bp (CAATAATTGG) upstream of the translation start in the SEP3 gene (24), consistent with different specificities for the different MADS-box proteins." These together with the core motif CC(A/T)₆GG (15) suggest a more general CArG-box motif of (C(C/A/T)(A/T)₆(A/G)G).

Smooth muscle cells

"Serum response factor (SRF) controls [ smooth muscle cell ] SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression."

"SMC genes examined in this study display SMC-specific histone modifications at the 5′-CArG boxes."

"The SRF-CArG association is required for transcriptional activation of SMC genes [...] the SMC genes examined in this study display SMC-specific histone modifications at the 5′-CArG boxes. [...] enrichment of H4 and H3 acetylation [...] were relatively low from positions –2,800 to –1,600 in the 5′ region. However, at position –1,600 to –1,200, there was a sharp rise in these modifications, which was increased even further at +400 in the coding region. We observed similar patterns for H3K4dMe and H3 Lys79 di-methylation [...]. SRF, TFIID, and RNA polymerase II displayed enrichments that were consistent with the positions of the CArG boxes, TATA box, and coding region, respectively".

The CArG boxes occur between -400 and -200 nts, between the E boxes and the TC elements.

"Functionally important CArG boxes have been identified in transcriptional regulatory elements controlling expression of sets of myogenic contractile and cytoskeletal proteins (reviewed elsewhere^8,25). Of note, in cardiac and skeletal muscle cells, functionally important CArG boxes have been identified in transcriptional regulatory element controlling a relatively limited subset of myofibrillar proteins.²⁶"

"In the nucleus, MRTFs physically associate with SRF, facilitating the binding of SRF to single or dual CArG boxes, activating transcription of genes encoding cytoskeletal and myogenic proteins [...].^{39,40,53,55,56}"

"The binding of SRF to SMC CArG boxes is associated with specific alterations in chromatin structure including the methylation and acetylation of histones.^76,79"

"Both PDGF-BB and KLF-4 inhibit SRF binding to CArG boxes downregulating transcription of SMC contractile genes.⁹²"

Gene transcriptions

Main article: Gene transcriptions

"SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues."

"Ca²⁺􏰀/calmodulin-dependent protein kinase IV activates cysteine-rich protein 1 through adjacent CRE and CArG elements."

"Smooth muscle-specific transcription is controlled by a multitude of transcriptional regulators that cooperate to drive expression in a temporospatial manner. Previous analysis of the cysteine-rich protein 1 (CRP1/Csrp) gene revealed an intronic enhancer that is sufficient for expression in arterial smooth muscle cells and requires a serum response factor-binding CArG element for activity. The presence of a CArG box in smooth muscle regulatory regions is practically invariant; however, it stands to reason that additional elements contribute to the modulation of transcription in concert with the CArG."

A "conserved cAMP response element (CRE) [...] binds the cAMP response element-binding protein (CREB) and is activated by Ca²⁺􏰀/calmodulin-dependent protein kinase IV (CaMKIV), but not by CaMKII."

"CaMKIV stimulates CRP1 expression not only through the CRE but also through the CArG box."

A "conserved cyclic AMP-response element (CRE) within the CRP1 gene is critical for enhancer activity. The CRE is an 8-bp motif with the consensus sequence TGACGTCA (34). [...] CRE serves as a transcriptional conduit for cyclic AMP-stimulated processes, but it also responds to a variety of other stimuli, including intracellular Ca²⁺􏰀 through the activation of Ca²⁺􏰀/calmodulin-dependent protein kinases (30, 31, 49). The primary factors that bind CRE are the cAMP element-binding protein (CREB) and the related proteins activating transcription factor (ATF)-1 and CRE modulator (CREM). The activity of CREB on the CRE is dependent largely on phosphorylation of a Ser133 residue. This phosphorylation event transforms CREB into a potent transcriptional activator and facilitates interactions with additional regulators, namely, CREB-binding protein (CBP) (31, 49). With respect to function, CREB has been implicated in governing a host of cellular processes and adaptive responses, including differentiation, metabolic changes, cell survival, and proliferation (3, 18, 19, 28, 37, 44, 53)."

The "utilization of two conserved binding sites within the CRP1-5.0 enhancer: a newly identified CRE and the CArG box [...] might serve to amplify a response. Given that these two elements are separated by only 14 bp, CREB and SRF could cooperate by assisting in the recruitment of CBP, both of which bind to CBP’s NH₂ terminus."

A1BG samplings

Testing the more general 3'-C(C/A/T)(A/T)₆(A/G)G-5':

1. negative strand in the negative direction (from ZSCAN22 to A1BG) is SuccessablesCArG--.bas, looking for 3'-C(C/A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G)G-5', 0,
2. negative strand in the positive direction (from ZNF497 to A1BG) is SuccessablesCArG-+.bas, looking for 3'-C(C/A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G)G-5', 0,
3. positive strand in the negative direction is SuccessablesCArG+-.bas, looking for 3'-C(C/A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G)G-5', 2, 3'-CAAAAAAAAG-5', 1399, 3'-CATTAAAAGG-5', 3441,
4. positive strand in the positive direction is SuccessablesCArG++.bas, looking for 3'-C(C/A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G)G-5', 0,
5. complement, negative strand, negative direction is SuccessablesCArGc--.bas, looking for 3'-G(A/G/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/T)C-5', 2, 3'-GTTTTTTTTC-5', 1399, 3'-GTAATTTTCC-5', 3441,
6. complement, negative strand, positive direction is SuccessablesCArGc-+.bas, looking for 3'-G(A/G/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/T)C-5', 0,
7. complement, positive strand, negative direction is SuccessablesCArGc+-.bas, looking for 3'-G(A/G/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/T)C-5', 0,
8. complement, positive strand, positive direction is SuccessablesCArGc++.bas, looking for 3'-G(A/G/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/T)C-5', 0,
9. inverse complement, negative strand, negative direction is SuccessablesCArGci--.bas, looking for 3'-C(C/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G/T)G-5', 0,
10. inverse complement, negative strand, positive direction is SuccessablesCArGci-+.bas, looking for 3'-C(C/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G/T)G-5', 0,
11. inverse complement, positive strand, negative direction is SuccessablesCArGci+-.bas, looking for 3'-C(C/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G/T)G-5', 0,
12. inverse complement, positive strand, positive direction is SuccessablesCArGci++.bas, looking for 3'-C(C/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(A/G/T)G-5', 0,
13. inverse, negative strand, negative direction, is SuccessablesCArGi--.bas, looking for 3'-G(A/G)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/A/T)C-5', 0,
14. inverse, negative strand, positive direction, is SuccessablesCArGi-+.bas, looking for 3'-G(A/G)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/A/T)C-5', 0,
15. inverse, positive strand, negative direction, is SuccessablesCArGi+-.bas, looking for 3'-G(A/G)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/A/T)C-5', 0,
16. inverse, positive strand, positive direction, is SuccessablesCArGi++.bas, looking for 3'-G(A/G)(A/T)(A/T)(A/T)(A/T)(A/T)(A/T)(C/A/T)C-5', 0.

Actins

"Positively acting, rate-limiting regulatory factors that influence tissue-specific expression of the human cardiac α-actin gene in a mouse muscle cell line are shown by in vivo competition and gel mobility-shift assays to bind to upstream regions of its promoter but to neither vector DNA nor a β-globin promoter. Although the two binding regions are distinctly separated, each corresponds to a cis region required for muscle-specific transcriptional stimulation, and each contains a core CC(A+T-rich)₆GG sequence (designated CArG box), which is found in the promoter regions of several muscle-associated genes. Each site has an apparently different binding affinity for trans-acting factors, which may explain the different transcriptional stimulation activities of the two cis regions. [The] two CArG box regions are responsible for muscle-specific transcriptional activity of the cardiac α-actin gene through a mechanism that involves their binding of a positive trans-acting factor in muscle cells."

"SRF binds to an A/T-rich sequence (CCWWWWWWGG) that has been designated as the CArG box.^10–12 CArG boxes were originally identified in transcriptional regulatory elements controlling expression of a set of growth- or serum-responsive genes including c-fos and egr-1.^13,14 Subsequently, CArG boxes were identified in transcriptional regulatory elements controlling expression of a subset of genes encoding myogenic contractile and cytoskeletal proteins including α-cardiac actin, smooth muscle (SM)-α-actin, α-skeletal actin, and SM22α.^15–19"

Early growth responses

"Exposure of human HL-525 cells to x-rays was associated with increases in EGR1 mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation of EGR1 gene transcription. Sequences responsive to ionizing radiation-induced signals were determined by deletion analysis of the EGR1 promoter. The results demonstrate that x-ray inducibility of the EGR1 gene is conferred by a region containing six serum response or CC(A+T-rich)₆GG (CArG) motifs. Further analysis confirmed that the region encompassing the three distal or upstream CArG elements is functional in the x-ray response. Moreover, this region conferred x-ray inducibility to a minimal thymidine kinase gene promoter. Taken together, these results indicate that ionizing radiation induces EGR1 transcription through CArG elements."

Myocardins

The "promyogenic SRF [SRF GeneID: 6722] coactivator myocardin [MYOCD GeneID: 93649] increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. [...] myocardin/SRF complexes physically interact with H3K4dMe and that the interaction of SRF with CArG box chromatin and H3K4dMe is sensitive to expression levels of myocardin."

Kruppel-like factor 4

The "myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. [...] deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. [...] KLF4 can bind to evolutionarily conserved TGF-β [control element] (TCE) DNA sequences adjacent to CArG boxes of SM gene promoters"

Epigenomes

Main article: Epigenomes

"SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo."

Histone modifications in SMCs include H3K4dMe, H3 Lys79 di-methylation, H3 Lys9 acetylation, H4Ac, and SRF binding.

MADS boxes

Main articles: MADS box gene transcriptions and MADS boxes

"RIN [Ripening Inhibitor] binds to DNA sequences known as the CA/T-rich-G (CArG) box, which is the general target of MADS box proteins (Ito et al., 2008)."

Human genes

An "interaction between serum response factor (SRF)¹ and the CArG box has been identified as a core machinery in the transcription of several muscle-specific genes, including the skeletal 􏰀𝛂-actin (8), caldesmon (9), cardiac 􏰀𝛂-actin (10), 􏰀𝛂₁ integrin (11), SM22􏰀𝛂 (12), smooth muscle myosin heavy chain (13), smooth muscle 𝛂􏰀-actin (14), calponin (15), atrial natriuretic factor (16), and 𝛃􏰁-tropomyosin (17) genes."

Actin genes

Gene ID: 58 is ACTA1 actin alpha 1, skeletal muscle. "The product encoded by this gene belongs to the actin family of proteins, which are highly conserved proteins that play a role in cell motility, structure and integrity. Alpha, beta and gamma actin isoforms have been identified, with alpha actins being a major constituent of the contractile apparatus, while beta and gamma actins are involved in the regulation of cell motility. This actin is an alpha actin that is found in skeletal muscle. Mutations in this gene cause a variety of myopathies, including nemaline myopathy, congenital myopathy with excess of thin myofilaments, congenital myopathy with cores, and congenital myopathy with fiber-type disproportion, diseases that lead to muscle fiber defects with manifestations such as hypotonia."

Gene ID: 70 is ACTC1 actin alpha cardiac muscle 1. "Actins are highly conserved proteins that are involved in various types of cell motility. Polymerization of globular actin (G-actin) leads to a structural filament (F-actin) in the form of a two-stranded helix. Each actin can bind to four others. The protein encoded by this gene belongs to the actin family which is comprised of three main groups of actin isoforms, alpha, beta, and gamma. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. Defects in this gene have been associated with idiopathic dilated cardiomyopathy (IDC) and familial hypertrophic cardiomyopathy (FHC)."

Gene ID: 800 is CALD1 caldesmon 1. "This gene encodes a calmodulin- and actin-binding protein that plays an essential role in the regulation of smooth muscle and nonmuscle contraction. The conserved domain of this protein possesses the binding activities to Ca(2+)-calmodulin, actin, tropomyosin, myosin, and phospholipids. This protein is a potent inhibitor of the actin-tropomyosin activated myosin MgATPase, and serves as a mediating factor for Ca(2+)-dependent inhibition of smooth muscle contraction. Alternative splicing of this gene results in multiple transcript variants encoding distinct isoforms."

1. NP_004333.1 caldesmon isoform 2: Transcript Variant: This variant (2) uses an alternate in-frame splice site and lacks an alternate in-frame exon in the central coding region, compared to variant 1. It is mainly expressed in non-muscle tissues or cells. The resulting isoform (2, also known as WI-38 l-CaD II) lacks an internal region, compared to isoform 1. pfam02029 Location:267 → 525, Caldesmon; Caldesmon.
2. NP_149129.2 caldesmon isoform 1: Transcript Variant: This variant (1) encodes the longest isoform (1, also known as aorta h-CaD). It is predominantly expressed in smooth muscle tissues. pfam02029, Location:517 → 780, Caldesmon; Caldesmon.
3. NP_149130.1 caldesmon isoform 4: Transcript Variant: This variant (4) differs in the 5' UTR and 5' coding region, and uses an alternate in-frame splice site in the central coding region, compared to variant 1. It is mainly expressed in non-muscle tissues or cells. The resulting isoform (4, also known as HeLa l-CaD I) contains a distinct N-terminus and is shorter than isoform 1. pfam02029 Location:282 → 545, Caldesmon; Caldesmon.
4. NP_149131.1 caldesmon isoform 5: Transcript Variant: This variant (5) differs in the 5' UTR and 5' coding region, and uses an alternate in-frame splice site and lacks an alternate in-frame exon in the central coding region, compared to variant 1. It is mainly expressed in non-muscle tissues or cells. The resulting isoform (5, also known as HeLa l-CaD II) has a distinct N-terminus and is shorter than isoform 1. pfam02029 Location:256 → 519, Caldesmon; Caldesmon.
5. NP_149347.2 caldesmon isoform 3: Transcript Variant: This variant (3) uses two alternate in-frame splice sites in the central coding region, compared to variant 1. It is mainly expressed in non-muscle tissues or cells. The resulting isoform (3, also known as WI-38 l-CaD I) is shorter than isoform 1. pfam02029 Location:288 → 550, Caldesmon; Caldesmon.
6. XP_016868139.1 caldesmon isoform X1: pfam02029 Location:517 → 779, Caldesmon; Caldesmon.
7. XP_024302710.1 caldesmon isoform X2: pfam02029 Location:293 → 551, Caldesmon; Caldesmon.
8. XP_024302711.1 caldesmon isoform X4: pfam02029 Location:267 → 524, Caldesmon; Caldesmon.
9. XP_016868141.1 caldesmon isoform X3: pfam02029 Location:267 → 525, Caldesmon; Caldesmon.
10. XP_016868143.1 caldesmon isoform X5.
11. XR_002956488.1 RNA Sequence.
12. XR_001744877.2 RNA Sequence.
13. XR_002956489.1 RNA Sequence.
14. XR_002956490.1 RNA Sequence.
15. XR_001744880.2 RNA Sequence.
16. XR_927535.2 RNA Sequence.
17. XR_927541.2 RNA Sequence.
18. XR_927537.3 RNA Sequence.
19. XR_001744879.2 RNA Sequence.
20. XR_927542.3 RNA Sequence.
21. XR_001744881.2 RNA Sequence.

Fos genes

Gene ID: 2353 is FOS Fos proto-oncogene, AP-1 transcription factor subunit. "The Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2. These genes encode leucine zipper proteins that can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. As such, the FOS proteins have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, expression of the FOS gene has also been associated with apoptotic cell death." "Serum response factor and the (CC(A/T)₆GG) (CArG) box interact to promote the transcription of c-fos and muscle genes".

1. NP_005243.1 proto-oncogene c-Fos, cd14721 Location:147 → 200, bZIP_Fos; Basic leucine zipper (bZIP) domain of the oncogene Fos (Fos): a DNA-binding and dimerization domain.

Integrin genes

Gene ID: 3672 is ITGA1 integrin subunit alpha 1, aka VLA1; CD49a. "This gene encodes the alpha 1 subunit of integrin receptors. This protein heterodimerizes with the beta 1 subunit to form a cell-surface receptor for collagen and laminin. The heterodimeric receptor is involved in cell-cell adhesion and may play a role in inflammation and fibrosis. The alpha 1 subunit contains an inserted (I) von Willebrand factor type I domain which is thought to be involved in collagen binding."

1. NP_852478.1 integrin alpha-1 precursor, smart00191 Location:568 → 621, Int_alpha; Integrin alpha (beta-propellor repeats), cd01469 Location:171 → 351, vWA_integrins_alpha_subunit; Integrins are a class of adhesion receptors that link the extracellular matrix to the cytoskeleton and cooperate with growth factor receptors to promote cell survival, cell cycle progression and cell migration. Integrins consist of an alpha and a beta sub-unit. Each sub-unit has a large extracellular portion, a single transmembrane segment and a short cytoplasmic domain. The N-terminal domains of the alpha and beta subunits associate to form the integrin headpiece, which contains the ligand binding site, whereas the C-terminal segments traverse the plasma membrane and mediate interaction with the cytoskeleton and with signalling proteins. The VWA domains present in the alpha subunits of integrins seem to be a chordate specific radiation of the gene family being found only in vertebrates. They mediate protein-protein interactions., pfam08441 Location:664 → 1056, Integrin_alpha2; Integrin alpha."

Hypotheses

Main article: Hypotheses

1. A1BG is not transcribed using a CArG box.
2. A CArG box on either side of A1BG may show that it is actively used to transcribe A1BG.

Results

There is a more general CArG box, 3'-CATTAAAAGG-5', at 3441 from ZSCAN22, or -1019 nts from the TSS of A1BG in the distal promoter.

A second more general CArG box, 3'-CAAAAAAAAG-5', at 1399 from ZSCAN22, or -3061 nts from the A1BG TSS may be a CArG box for ZSCAN22 in the negative direction on the positive strand in the distal promoter.

Acknowledgements

The content on this page was first contributed by: Henry A. Hoff.

Initial content for this page in some instances came from Wikiversity.

See also

• A1BG gene transcriptions
• CAAT box gene transcriptions
• C and D box gene transcriptions
• CArG box gene transcription laboratory
• CGCG box gene transcriptions
• mir-143
• Serum response elements
• Synaptic Activity-Responsive Elements

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