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Background: CXXC-type zinc-finger protein CXXC5 has been reported to be associated with the development of cardiovascular disease. Recently, through signaling pathway screening we found that CXXC5 activated Tgfβ and myocardial differentiation signaling pathways simultaneously. Although the physiological and pathological function of CXXC5 in many organs has been well elucidated, its function in heart remains unclear., Methods and Results: Here, we found that zebrafish CXXC5 and SMAD were interacting through ZF-CXXC and MH1 domain. Over-expression of CXXC5 in cardiomyocyte increased the luciferase report activity of Tgfβ signaling pathway. Spatiotemporal expression profile of cxxc5 showed that it consistently expressed during cardiogenesis. Knockdown of cxxc5 in zebrafish displayed looping defects, cardiac dysplasia, pericardial edema, and decreased contraction ability, accompanied with down-regulation of members referring to cardiac looping downstream genes of Tgfβ signaling pathway, such as nkx2.5, hand2, and has2. Co-injection of hand2 mRNA with cxxc5 morpholino rescued the cardiac looping detects., Conclusion: Our study is the first to provide an in vivo evidence for cxxc5 regulating heart development and cardiac looping via Tgfβ related signaling pathway. This finding suggested that CXXC5 may serve as a possible marker that has potential diagnostic and prognostic value in fetus with congenital heart disease., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Previous studies have demonstrated that Smyd1 plays a critical role in cardiomyocyte differentiation, cardiac morphogenesis and myofibril organization. In this study, we uncovered a novel function of Smyd1 in the regulation of endothelial cells (ECs). Our data showed that Smyd1 is expressed in vascular endothelial cells, and knockdown of SMYD1 in endothelial cells impairs EC migration and tube formation. Furthermore, Co-IP and GST pull-down assays demonstrated that SMYD1 is associated with the Serum Response Factor (SRF). EMSA assays further showed that SMYD1 forms a complex with SRF and enhances SRF DNA binding activity. Our studies indicate that SMYD1 serves as an SRF-interacting protein, enhances SRF DNA binding activity, and is required for EC migration and tube formation to regulate angiogenesis.

In this study, we report the identification and characterization of a novel C2H2 zinc finger protein, ZNF552, from a human embryonic heart cDNA library. ZNF552 is composed of three exons and two introns and maps to chromosome 19q13.43. The cDNA of ZNF552 is 2.3 kb, encoding 407 amino acids with an amino-terminal KRAB domain and seven carboxyl-terminal C2H2 zinc finger motifs in the nucleus and cytoplasm. Northern blotting analysis indicated that a 2.3 kb transcript specific for ZNF552 was expressed in liver, lung, spleen, testis and kidney, especially with a higher level in the lung and testis in human adult tissues. Reporter gene assays showed that ZNF552 was a transcriptional repressor, and overexpression of ZNF552 in the COS-7 cells inhibited the transcriptional activities of AP-1 and SRE, which could be relieved through RNAi analysis. Deletion studies showed that the KRAB domain of ZNF552 may be involved in this inhibition.

SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporal-spatial manner. Zinc finger-containing transcription factors have been implicated as critical regulators of multiple cardiac-expressed genes, and are thought to be important for human heart development and diseases. Here, we have identified and characterized a novel zinc finger gene named ZNF418 from a human embryo heart cDNA library. The gene spans 13.5 kb on chromosome 19q13.43 encompassing six exons, and transcribes a 3.7-kb mRNA that encodes a protein with 676 amino acid residues. The predicted protein contains a KRAB-A box and 17 tandem C2H2 type zinc finger motifs. Northern blot analysis indicates that ZNF418 is expressed in multiple fetal and adult tissues, but is expressed at higher levels in the heart. Reporter gene assays show that ZNF418 is a transcriptional repressor, and the KRAB motif of ZNF418 represents the basal repressive domain. Overexpression of ZNF418 in COS-7 cells inhibits the transcriptional activity of SRE and AP-1 which may be silenced by siRNA. These results suggest that ZNF418 is a member of the zincfinger transcription factor family and may act as a negative regulator in MAPK signaling pathway.

We have cloned a novel KRAB-related zinc finger gene, ZNF307, encoding a protein of 545aa. ZNF307 is conserved across species in evolution and is differentially expressed in human adult and fetal tissues. The fusion protein of EGFP-ZNF307 localizes in the nucleus. Transcriptional activity assays show ZNF307 suppresses transcriptional activity of L8G5-luciferase. Overexpressing ZNF307 in different cell lines also inhibits the transcriptional activities of p53 and p21. Moreover, ZNF307 works by reducing the p53 protein level and p53 protein reduction is achieved by increasing transcription of MDM2 and EP300. ZNF307 might suppress p53-p21 pathway through activating MDM2 and EP300 expression and inducing p53 degradation.

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc finger proteins are involved in the regulation of the MAPK signaling pathways. Here we report the identification and characterization of a novel human zinc finger protein, ZNF436. The cDNA of ZNF436 is 3.8 kb, encoding 470 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from rat to human. RT-PCR indicates that ZNF436 is expressed in all the human fetal tissues examined, with a high level in brain and heart. Overexpression of pCMV-tag2A-ZNF436 in the COS-7 cells represses the transcriptional activities of SRE and AP-1. These results suggest that ZNF436 is a member of the zinc finger transcription factor family and may act as a negative regulator in gene transcription mediated by the MAPK signaling pathways.

Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. The zinc-finger-containing transcription factors have been previously revealed to be involved in the regulation of the MAPK signaling pathways. Here, we have identified a novel human zinc-finger transcriptional repressor, ZNF325, that contains a RBaK-like RB-binding domain and 15 tandem repeated C2H2 type zinc fingers. Northern blot analysis indicates that a 2.7 kb transcript specific for ZNF325 is widely expressed in all tissues examined at adult stage and in most of the embryonic tissues. Overexpression of ZNF325 in COS-7 cells inhibits the transcriptional activities of AP-1 and SRE. The deletion and RNAi analysis indicate that the C2H2 zinc finger motifs represent the basal transcriptional repressive activity. These results indicate that the ZNF325 protein may act as a novel transcription repressor in MAPK signaling pathway to mediate cellular functions.

Transcription factors play an essential role in altering gene expression. A great progress about transcription factors has been made towards the understanding of normal physiological processes, embryonic development, and human diseases. Here we report the identification and characterization of a novel KRAB-containing zinc-finger protein, ZNF569, which is isolated from a human embryonic heart cDNA library. ZNF569 encodes a putative protein of 686 amino acids. The protein is conserved across different species during evolution. Expression of ZNF569 was found in most of the examined human adult and embryonic tissues with a higher level in heart and skeletal muscles. The KRAB and ZNF motifs of ZNF569 represent potent repression domains. When ZNF569 is fused to Gal-4 DNA-binding domain and co-transfected with VP-16, ZNF569 protein suppresses transcriptional activity. Overexpression of ZNF569 in COS-7 cells inhibited the transcriptional activities of SRE and AP-1, which may be silenced by siRNA. The results suggest that ZNF569 protein may act as a transcriptional repressor that suppresses MAPK signaling pathway to mediate cellular functions.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C(2)H(2)-type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.

Mitogen-activated protein kinases (MAPKs) are major components of pathways controlling embryogenesis, cell differentiation, cell proliferation, and cell death. One of the most explored functions of MAPK signaling is the regulation of gene expression by direct or indirect phosphorylation and subsequent activation of transcription factors. In this article, we isolated a novel KRAB-related zinc finger gene named ZNF383 from an early embryo heart cDNA library. The cDNA of ZNF383 is 2220bp, encoding a protein of 475 amino acids. The protein is conserved in evolution across different species. Northern blot analysis indicates that a 2.2kb transcript specific for ZNF383 is detected in most of the examined human adult and embryonic tissues with a higher level in skeletal muscle. In COS-7 cells, ZNF383 protein is localized to nucleus and cytoplasm. ZNF383 is a transcription repressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Deletion analysis indicates that the KRAB box of ZNF383 is responsible for the transcriptional repressor activity. Overexpression of ZNF383 in cells inhibits the transcriptional activities of AP-1 and SRE, suggesting that ZNF383 may act as a negative regulator in MAPK-mediated signaling pathways.

The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C2H2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions.

Src homology 3 (SH3) domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Such protein-protein interactions can activate the protein kinase cascade that mediates MAPK signaling pathway. The human hole gene, hhole, is a 319-amino acid six-transmembrane protein with proline-rich C-terminal motifs and N-terminal ERK binding domains (D-domains). The hhole protein is highly conserved in evolution across different species from elegent, mouse to human. Northern blot analysis indicates that hhole is expressed in heart, liver, skeletal muscle, and pancreas at adult stages and in most of the examined embryonic tissues, especially at a higher level in heart. Using a GFP-labeled hhole protein, we demonstrate that hhole is localized in plasma membrane or proximal region of the membrane. Overexpression of hhole in COS-7 cells strongly inhibited the transcriptional activities of AP-1 and SRE while deletion of the C-terminal proline-rich motifs or the N-terminal ERK binding D-domain motifs reduced the repressive activity of the gene. These results suggest that the hhole protein may interact with SH3-domain proteins or ERKs to mediate signaling pathways/networks that lead to the suppression of AP-1 and SRE.

Kruppel-related zinc-finger proteins constitute the largest individual family of transcription factors in mammals [C. Looman, L. Hellman, M. Abrink, A novel Kruppel-associated box identified in a panel of mammalian zinc-finger proteins, Mammalian Genome 15 (1) (2004) 35-40.[1]]. Here we identified and characterized a novel zinc-finger gene named ZNF446. The predicted protein contains a KRAB and three C(2)H(2) zinc fingers. Northern blot analysis shows that ZNF446 is expressed in a variety of human adult tissues with the highest expression level in muscle. ZNF446 is a transcription repressor when fused to GAL4 DNA-binding domain and co-transfected with VP-16. Overexpression of ZNF446 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1, in which the KRAB motif represents the basal transcriptional repressive activity, suggesting that the ZNF446 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions.

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here we report the identification and characterization of a novel human zinc-finger gene named ZNF649. The cDNA of ZNF649 is 3176 bp, encoding a protein of 505 amino acids in the nuclei. Northern blot analysis indicates that ZNF649 is expressed in most of the examined human adult and embryonic tissues. ZNF649 is a transcription suppressor when fused to GAL-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF649 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF649 protein may act as a transcriptional repressor in mitogen-activated protein kinase signaling pathway to mediate cellular functions.

The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted alpha-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway., (Copyright 2004 Elsevier Inc.)

The zinc-finger motif found in many transcription factors is thought to be important for human heart development and diseases. In this study, we have identified and characterized a novel zinc-finger gene named ZNF480 using degenerate primers from an early human embryo heart cDNA library. ZNF480 contains a KRAB-A box and 12 C2H2 zinc fingers. The cDNA sequence contains an open reading frame of 1551 bp, encoding a putative protein of 516 amino acid residues with a predicted molecular mass of 57 kDa. Northern blot analysis indicates that a 4.7kb transcript specific for ZNF480 is expressed only in embryonic heart. In the adult tissues, the expression of ZNF480 is restricted largely to heart, skeletal muscle, pancreas, and placenta. Overexpression of ZNF480 in cells activates the transcriptional activities of AP-1 and SRE. Therefore, our data suggest that ZNF480 may act as a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. The zinc-finger-containing transcription factor has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. The MAPKs function inside the nucleus and target transcription factors that are prebound to DNA. Many transcription factors are probably important MAPK targets. Here, we have cloned a new zinc-finger gene named ZNF411 using degenerate primers from an early embryo heart cDNA library, which mapped to 19p13.11. The ZNF411 gene consists of 2360 nucleotides and encodes a protein of 499 amino acids with an amino-terminal KRAB domain and eleven carboxy-terminal C2H2 zinc-finger units. Northern blot analysis indicates that a 2.4 kb transcript specific for ZNF411 is expressed in heart, skeletal muscle, and placenta at adult stage and is expressed in most of the examined embryonic tissues, especially at a higher level in skeletal muscle, heart, and pancreas. ZNF411 protein distributes evenly in nuclei when overexpressed in the cells. Reporter gene assays show that ZNF411 is a transcriptional repressor and overexpression of ZNF411 in the COS-7 cells inhibits the transcriptional activities of AP-1 and SRE. These results indicate that ZNF411 is a member of the zinc-finger transcription factor family and may be involved in the heart development, and it probably works as a negative regulator in MAPK signaling pathway.

With the aim of identifying genes involved in human heart development and disease, we have isolated a novel KRAB-related zinc-finger gene named ZNF382 from heart cDNA library. The ZNF382 gene has a predicted 548-amino acid open reading frame, encoding a putative 64kDa zinc-finger protein. The N-terminus of the ZNF382 coding region has a well-conserved Krüpple-associated box domain that consists of KRAB boxes A and B, whereas the C-terminus contains a Krüpple-type zinc-finger domain possessing nine C(2)H(2) zinc-finger motifs in tandem arrays. The ZNF382 gene is mapped to chromosome 19q13.13. Northern blot analysis indicates that a 2.9-kb transcript specific for ZNF382 is expressed at very early embryonic stage of human (at least earlier than gestation 34 day) and widely in human embryo tissues. At the adult stage, ZNF382 expression is restricted largely to heart tissue suggesting a potential role in heart development and function.

The C(2)H(2) zinc-finger motif found in many transcription factors is thought to be important for nucleic acid binding and/or dimerization. Here, we have identified and characterized a novel zinc-finger gene named ZNF323 using degenerate primers from an early human embryo heart cDNA library. The predicted protein contains six different C(2)H(2) type zinc fingers and a SCAN box. ZNF323 maps to chromosome 6p22.1-22.3. The expression levels were different during different development stages of human embryo between 15 and 23 weeks. Northern blot analysis shows that a 3.2-kb transcript specific for ZNF323 was expressed at high levels in the lung, liver, and kidney, while weakly expressed in intestine, brain, muscle, cholecyst, heart, and pancreas. In adult tissues, ZNF323 is expressed at high levels in liver and kidney, weakly in lung, pancreas, brain, placenta, muscle, and heart. Taken together, these results indicate that ZNF323 is a member of the zinc-finger transcription factor family and may be involved in the development of multiple embryonic organs.