scholarly journals Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy

Circulation ◽  
2020 ◽  
Vol 142 (9) ◽  
pp. 882-898 ◽  
Author(s):  
Jessica Pfleger ◽  
Ryan C. Coleman ◽  
Jessica Ibetti ◽  
Rajika Roy ◽  
Ioannis D. Kyriazis ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Chromatin Immunoprecipitation ◽  
De Novo ◽  
Pressure Overload ◽  
Transverse Aortic Constriction ◽  
Aortic Constriction ◽  
Pol Ii ◽  
Hypertrophic Growth

Background: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. Methods: To address this, we performed FoxO1 chromatin immunoprecipitation–deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg −1 ·mg −1 ), transverse aortic constriction, or vehicle injection/sham surgery. Results: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation–deep sequencing results were aligned with those of pol II chromatin immunoprecipitation–deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction–operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1– and, in vivo, pressure overload–induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. Conclusions: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II–regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

scholarly journals Growth hormone-releasing hormone attenuates cardiac hypertrophy and improves heart function in pressure overload-induced heart failure

2017 ◽  
Vol 114 (45) ◽  
pp. 12033-12038 ◽  
Author(s):  
Iacopo Gesmundo ◽  
Michele Miragoli ◽  
Pierluigi Carullo ◽  
Letizia Trovato ◽  
Veronica Larcher ◽  
...  
Keyword(s):  
Heart Failure ◽  
Growth Hormone ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Therapeutic Potential ◽  
Growth Hormone Releasing Hormone ◽  
Transverse Aortic Constriction ◽  
Aortic Constriction

It has been shown that growth hormone-releasing hormone (GHRH) reduces cardiomyocyte (CM) apoptosis, prevents ischemia/reperfusion injury, and improves cardiac function in ischemic rat hearts. However, it is still not known whether GHRH would be beneficial for life-threatening pathological conditions, like cardiac hypertrophy and heart failure (HF). Thus, we tested the myocardial therapeutic potential of GHRH stimulation in vitro and in vivo, using GHRH or its agonistic analog MR-409. We show that in vitro, GHRH(1-44)NH2 attenuates phenylephrine-induced hypertrophy in H9c2 cardiac cells, adult rat ventricular myocytes, and human induced pluripotent stem cell-derived CMs, decreasing expression of hypertrophic genes and regulating hypertrophic pathways. Underlying mechanisms included blockade of Gq signaling and its downstream components phospholipase Cβ, protein kinase Cε, calcineurin, and phospholamban. The receptor-dependent effects of GHRH also involved activation of Gαs and cAMP/PKA, and inhibition of increase in exchange protein directly activated by cAMP1 (Epac1). In vivo, MR-409 mitigated cardiac hypertrophy in mice subjected to transverse aortic constriction and improved cardiac function. Moreover, CMs isolated from transverse aortic constriction mice treated with MR-409 showed improved contractility and reversal of sarcolemmal structure. Overall, these results identify GHRH as an antihypertrophic regulator, underlying its therapeutic potential for HF, and suggest possible beneficial use of its analogs for treatment of pathological cardiac hypertrophy.

scholarly journals Regulation of Calcineurin through Transcriptional Induction of the calcineurin Aβ Promoter In Vitro and In Vivo

2005 ◽  
Vol 25 (15) ◽  
pp. 6649-6659 ◽  
Author(s):  
Toru Oka ◽  
Yan-Shan Dai ◽  
Jeffery D. Molkentin
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Pressure Overload ◽  
Growth Stimulation ◽  
Feedback Mechanism ◽  
Activated T Cells ◽  
The Central Nervous System ◽  
Positive Feedback Mechanism ◽  
Transcriptional Induction

ABSTRACT The calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway has been shown to be of critical importance in regulating the growth response of cardiac myocytes. We have previously demonstrated that calcineurin Aβ (CnAβ) mRNA and protein are increased in response to growth stimulation, although the precise regulatory mechanism underlying CnAβ upregulation is not clear. Here, we isolated the mouse CnAβ promoter and characterized its responsiveness to growth stimuli in vitro and in vivo. A 2.3-kb promoter fragment was strongly activated by phenylephrine and endothelin-1 stimulation and by cotransfection with constitutively active CnA, NFATc4, and GATA4. Using chromatin immunoprecipitation, sequence regions were identified within the 2.3-kb promoter that associated with NFAT and GATA4, as well as with acetylated histone H3, following agonist stimulation. Consistent with the chromatin immunoprecipitation experiments, deletion of the distal half of the CnAβ promoter severely reduced NFAT, GATA4, and hypertrophic agonist-mediated activation. To investigate in vivo activity, we generated β-galactosidase (LacZ) containing transgenic mice under the control of the CnAβ 2.3-kb promoter. CnAβ-LacZ mice showed expression in the heart that was cyclosporine sensitive, as well as expression in the central nervous system and skeletal muscle from early embryonic stages through adulthood. CnAβ-LacZ mice were subjected to cardiac pressure overload stimulation and crossbreeding with mice containing cardiac-specific transgenes for activated calcineurin and NFATc4, which revealed inducible expression in the heart. These results indicate that the CnAβ 2.3-kb promoter is specifically activated by hypertrophic stimuli through a positive feedback mechanism involving NFAT and GATA4 transcription factors, suggesting transcriptional induction of CnAβ expression as an additional means of regulating calcineurin activity in the heart.

scholarly journals Methylation-Mediated Proviral Silencing Is Associated with MeCP2 Recruitment and Localized Histone H3 Deacetylation

2001 ◽  
Vol 21 (23) ◽  
pp. 7913-7922 ◽  
Author(s):  
Matthew C. Lorincz ◽  
Dirk Schübeler ◽  
Mark Groudine
Keyword(s):  
Chromatin Immunoprecipitation ◽  
De Novo ◽  
Histone H3 ◽  
Transcriptional Silencing ◽  
Dependent Manner ◽  
Histone H4 ◽  
Acetylation Status ◽  
Murine Leukemia

ABSTRACT The majority of 5-methylcytosine in mammalian DNA resides in endogenous transposable elements and is associated with the transcriptional silencing of these parasitic elements. Methylation also plays an important role in the silencing of exogenous retroviruses. One of the difficulties inherent in the study of proviral silencing is that the sites in which proviruses randomly integrate influence the probability of de novo methylation and expression. In order to compare methylated and unmethylated proviruses at the same genomic site, we used a recombinase-based targeting approach to introduce an in vitro methylated or unmethylated Moloney murine leukemia-based provirus in MEL cells. The methylated and unmethylated states are maintained in vivo, with the exception of the initially methylated proviral enhancer, which becomes demethylated in vivo. Although the enhancer is unmethylated and remodeled, the methylated provirus is transcriptionally silent. To further analyze the repressed state, histone acetylation status was determined by chromatin immunoprecipitation (ChIP) analyses, which revealed that localized histone H3 but not histone H4 hyperacetylation is inversely correlated with proviral methylation density. Since members of the methyl-CpG binding domain (MBD) family of proteins recruit histone deacetylase activity, these proteins may play a role in proviral repression. Interestingly, only MBD3 and MeCP2 are expressed in MEL cells. ChIPs with antibodies specific for these proteins revealed that only MeCP2 associates with the provirus in a methylation-dependent manner. Taken together, our results suggest that MeCP2 recruitment to a methylated provirus is sufficient for transcriptional silencing, despite the presence of a remodeled enhancer.

Abstract 355: In Vivo Targeting Of GTF2B Selectively Inhibits Transcription Of Cardiomyopathy-related Genes And Partially Blunts Development Of Cardiac Hypertrophy

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Danish Sayed ◽  
Zhi Yang ◽  
Minzhen He ◽  
Maha Abdellatif
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Splicing ◽  
De Novo ◽  
Transcriptional Profiling ◽  
Essential Genes ◽  
Pol Ii ◽  
Expression Of Genes ◽  
Essential Components

Transcriptional profiling of cardiac genome during hypertrophy identified two categories of genes with distinct modes of regulation. The first set of genes involved in the cells essential functions (e.g. RNA splicing) and whose transcription is expected to be incremental and contribute to the increasing cardiac mass is regulated by promoter clearance of RNA polymerase II (pol II). On the other hand, the second set that include genes with specialized function and show a robust increase in expression upon growth stimulus (cytoskeletal, extracellular matrix) are regulated by de novo pol II recruitment to promoters. Our goal was to identify the transcriptional mechanisms that distinguish these two sets of genes and then to selectively inhibit those that participate in contractile dysfunction, while preserving the expression of genes necessary for essential functions. General Transcription factor IIB (GTF2B), is one of the essential components of transcription machinery and is required for pol II recruitment. Thus, we hypothesized that inhibition of GTF2B would result in inhibition of only the specialized genes, sparing the essential genes. Our in vitro results with shRNA mediated inhibition of GTF2B in hypertrophying neonatal myocytes showed decreased expression of genes that required de novo pol II recruitment for transcription (eg. ACTA1), while no change was observed in the genes regulated by promoter clearance of pol II (Vdac1). Similarly, preliminary results with in vivo knockdown of GTF2B (~80% reduction in mRNA and ~36% in protein) via intravenous injection of modified antisense oligo in mice subjected to transaortic coarctation (TAC) showed inhibition of only cardiomyopathy-related genes that require pol II recruitment (ANF), while expression of essential genes (Vdac1) remained unchanged. Inhibition of GTF2B restricted increase in TAC-induced heart wt to 9%, compared to 29% in TAC hearts with control oligo. Echocardiography showed partial normalization of ejection fraction with GTF2B inhibitor during TAC from 61.5% to 66.4% compared to sham hearts with 71%. Thus, we conclude that by targeting GTF2B we can selectively restrict the expression of detrimental genes during hypertrophy, thereby delaying the onset of cardiac dysfunction and failure

scholarly journals Platelet TGF-β1 contributions to plasma TGF-β1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1064-1074 ◽  
Author(s):  
Alexander Meyer ◽  
Wei Wang ◽  
Jiaxiang Qu ◽  
Lori Croft ◽  
Jay L. Degen ◽  
...  
Keyword(s):  
Cardiac Fibrosis ◽  
Systolic Dysfunction ◽  
In Vitro Release ◽  
Pressure Overload ◽  
Aortic Constriction ◽  
Blood Drawing ◽  
High Concentrations ◽  
Tgf Β1

Abstract Circulating platelets contain high concentrations of TGF-β1 in their α-granules and release it on platelet adhesion/activation. We hypothesized that uncontrolled in vitro release of platelet TGF-β1 may confound measurement of plasma TGF-β1 in mice and that in vivo release and activation may contribute to cardiac pathology in response to constriction of the transverse aorta, which produces both high shear and cardiac pressure overload. Plasma TGF-β1 levels in blood collected from C57Bl/6 mice by the standard retro-bulbar technique were much higher than those obtained when prostaglandin E1 was added to inhibit release or when blood was collected percutaneously from the left ventricle under ultrasound guidance. Even with optimal blood drawing, plasma TGF-β1 was lower in mice rendered profoundly thrombocytopenic or mice with selectively low levels of platelet TGF-β1 because of megakaryocytespecific disruption of their TGF-β1 gene (Tgfb1flox). Tgfb1flox mice were also partially protected from developing cardiac hypertrophy, fibrosis, and systolic dysfunction in response to transverse aortic constriction. These studies demonstrate that plasma TGF-β1 levels can be assessed accurately, but it requires special precautions; that platelet TGF-β1 contributes to plasma levels of TGF-β1; and that platelet TGF-β1 contributes to the pathologic cardiac changes that occur in response to aortic constriction.

Ultra-Deep Sequencing of De Novo IGHV Mutations in Activated CLL Cells: Evidence for Activation-Induced Deaminase Function.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2545-2545
Author(s):  
Piers E.M. Patten ◽  
Thomas MacCarthy ◽  
Xiao-Jie Yan ◽  
Jonathan E. Kolitz ◽  
Steven L. Allen ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
De Novo ◽  
Fold Change ◽  
Total Sequence ◽  
Cytogenetic Aberrations ◽  
In Vitro Activation ◽  
Activation Induced Deaminase ◽  
Nsg Mouse

Abstract Abstract 2545 B-cell chronic lymphocytic leukemia (CLL) clones often acquire new mutations and cytogenetic aberrations over time. In other human cancers, including lymphomas and solid tumors, activation-induced deaminase (AID), which normally causes immunoglobulin (Ig) somatic hypermutation (SHM) and isotype class switch recombination (CSR) in germinal center B cells, is expressed and functions abnormally to cause mutations promoting aggressiveness. In CLL, AID mRNA expression in the leukemic cells correlates with increased adverse cytogenetic aberrations and worse clinical outcomes. Furthermore, CLL cells activated by culture with CD32-transfected murine L cells, anti-CD40 and interleukin-4, produce AID protein with associated functions: DNA breaks, Ig CSR, and Ig heavy chain (IGH) variable (IGHV) gene SHM. To evaluate AID-mediated SHM in CLL more accurately, ultra-deep sequencing was performed on CLL clone's IGH cDNA prior to and after in vitro activation in one unmutated CLL (U-CLL) case (CLL1278, 0.0% mutated IGHV3–30) and one mutated CLL (M-CLL) case (CLL1299, 4.9% mutated IGHV3–23). Additionally, to examine activation of CLL IGH mutation in vivo, ultra-deep sequencing was performed on cells from one U-CLL case (CLL1083, 0.0% mutated IGHV4-b) prior to and after adoptive transfer into the NOD/SCID/γcnull (NSG) mouse, a xenograft model of CLL, where upregulation of AID protein occurs in CD5+CD19+ human CLL cells. Libraries were created for ultra-deep sequencing using the 454 FLX system (Roche) by PCR amplification with IGHV family-specific framework1 (Lprimer) and IGH constant region Cμ (IGHM) (Rprimer) primers on cDNA obtained from CLL cells prior to (day 0) or after in vitro culture for 7 (CLL1278) or 14 days (CLL1278; CLL1299) or from NSG spleen CLL cells collected 35 days after transfer (CLL1083). The resulting 461,153 sequence reads were processed to generate separate datasets with fixed sequence block lengths for each primer. The Lprimer sequence blocks included only 5'IGHV sequence, while the Rprimer sequence blocks encompassed 3'IGHV, IGH diversity, and IGH joining genes (IGHVDJ) as well as 5'IGHM sequence. Individual subclone sequences that occurred at least twice were extracted from each of the datasets and the unique de novo subclones not shared between day 0 and activation were analyzed for mutations. All three CLL cases showed increases in 5'IGHV and 3'IGHVDJ subclones with activation. After in vitro activation, for CLL1278, 123,518 total sequence reads produced 68 unique subclones as compared to 33 at day 0; and for CLL1299, 163,358 total sequence reads produced 78 unique subclones as compared to 61 at day 0. Likewise, after in vivo activation in the NSG mouse, for CLL1083, 174,472 total sequence reads produced 91 unique subclones as compared to 56 at day 0. In contrast, all three CLL cases showed decreases in 5'IGHM subclones after activation. After in vitro activation, CLL1278 and CLL1299 decreased from 22 and 20 unique day 0 subclones to 13 and 16 unique subclones. Similarly, CLL1083 showed a decrease from 20 unique day 0 subclones to 11 unique subclones after transfer into the NSG mouse. After normalization for read number and block sequence length, all three CLL cases showed an increase in 5'IGHV mutation with CLL cell activation (fold change relative to 5'IGHM = 3.4, 2.2, and 4.6 for CLL1278, CLL1299, and CLL1083, respectively). This increase in IGHV mutation relative to IGHM following activation is consistent with AID activity. Furthermore, examination of mutation sites in these subclones revealed an increase in mutations in AID hotspot motifs (GYW or WRC) in the 5'IGHV of all three CLL cases with activation (fold change = 2.0, 1.9, and 2.5 for CLL1278, CLL1299, and CLL1083, respectively), which was not observed further downstream in 3'IGHVDJ and 5'IGHM. Thus, by analyzing a very large number of IGH sequences in CLL cells after activation in vitro or in vivo, a pattern of de novo mutations consistent with AID activity is found. Furthermore, since both U-CLL and M-CLL clones exhibited AID activity, these findings indicate that AID-mediated SHM is not limited by CLL IGHV mutation status. Finally, these data support a model of AID-promoted mistargeted mutations, which may lead to adverse cytogenetic aberrations and unfavorable outcomes in CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cardiac Wnt5a and Wnt11 promote fibrosis by the crosstalk of FZD5 and EGFR signaling under pressure overload

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Yan Zou ◽  
Le Pan ◽  
Yi Shen ◽  
Xiang Wang ◽  
Chenxing Huang ◽  
...  
Keyword(s):  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Pressure Overload ◽  
Mechanical Stretch ◽  
Transverse Aortic Constriction ◽  
Egfr Signaling ◽  
Aortic Constriction ◽  
And Function ◽  
Cardiac Microvascular Endothelial Cells

AbstractProgressive cardiac fibrosis accelerates the development of heart failure. Here, we aimed to explore serum Wnt5a and Wnt11 levels in hypertension patients, the roles of Wnt5a and Wnt11 in cardiac fibrosis and potential mechanisms under pressure overload. The pressure overload mouse model was built by transverse aortic constriction (TAC). Cardiac fibrosis was analyzed by Masson’s staining. Serum Wnt5a or Wnt11 was elevated and associated with diastolic dysfunction in hypertension patients. TAC enhanced the expression and secretion of Wnt5a or Wnt11 from cardiomyocytes (CMs), cardiac fibroblasts (CFs), and cardiac microvascular endothelial cells (CMECs). Knockdown of Wnt5a and Wnt11 greatly improved cardiac fibrosis and function at 4 weeks after TAC. In vitro, shWnt5a or shWnt11 lentivirus transfection inhibited pro-fibrotic effects in CFs under mechanical stretch (MS). Similarly, conditional medium from stretched-CMs transfected with shWnt5a or shWnt11 lentivirus significantly suppressed the pro-fibrotic effects induced by conditional medium from stretched-CMs. These data suggested that CMs- or CFs-derived Wnt5a or Wnt11 showed a pro-fibrotic effect under pressure overload. In vitro, exogenous Wnt5a or Wnt11 activated ERK and p38 (fibrotic-related signaling) pathway, promoted the phosphorylation of EGFR, and increased the expression of Frizzled 5 (FZD5) in CFs. Inhibition or knockdown of EGFR greatly attenuated the increased FZD5, p-p38, and p-ERK levels, and the pro-fibrotic effect induced by Wnt5a or Wnt11 in CFs. Si-FZD5 transfection suppressed the increased p-EGFR level, and the fibrotic-related effects in CFs treated with Wnt5a or Wnt11. In conclusion, pressure overload enhances the secretion of Wnt5a or Wnt11 from CMs and CFs which promotes cardiac fibrosis by activation the crosstalk of FZD5 and EGFR. Thus, Wnt5a or Wnt11 may be a novel therapeutic target for the prevention of cardiac fibrosis under pressure overload.

scholarly journals The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saikat Bhattacharya ◽  
Michaella J. Levy ◽  
Ning Zhang ◽  
Hua Li ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mrna Processing ◽  
Key Factors ◽  
Pol Ii ◽  
Mrna Transcripts ◽  
Hnrnp Protein ◽  
Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

Computational study for suppression of CD25/IL-2 interaction

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Moein Dehbashi ◽  
Zohreh Hojati ◽  
Majid Motovali-bashi ◽  
Mazdak Ganjalikhani-Hakemi ◽  
Akihiro Shimosaka ◽  
...  
Keyword(s):  
Small Molecules ◽  
Cancer Progression ◽  
Immune Escape ◽  
De Novo ◽  
Computational Study ◽  
Treg Cells ◽  
Homology Search ◽  
Small Interfering Rnas

AbstractCancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

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