Novel insights into cardiac regeneration based on differential fetal and adult ovine heart transcriptomic analysis

2020 ◽  
Vol 318 (4) ◽  
pp. H994-H1007
Author(s):  
Paola Locatelli ◽  
Mariano N. Belaich ◽  
Ayelén E. López ◽  
Fernanda D. Olea ◽  
Martín Uranga Vega ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Differentially Expressed Gene ◽  
Cardiac Regeneration ◽  
Differentially Expressed ◽  
Cell Cycle Regulators ◽  
Large Mammal ◽  
Adult Sheep ◽  
Laboratory Rodents ◽  
Cycle Regulation

The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies. NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration.

The MEIS1 Interactome in Megakaryocytes Reveals a Role in Cell Cycle Regulation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3380-3380
Author(s):  
Vishal A Salunkhe ◽  
Iain Macaulay ◽  
Sylvia Nuernberg ◽  
Cathal McCarthy ◽  
Willem Hendrik Ouwehand ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Cyclin D3 ◽  
Haematopoietic Stem Cell ◽  
Nuclear Fraction ◽  
Cell Cycle Regulators ◽  
Cycle Progression ◽  
Cycle Regulation

Abstract Abstract 3380 Haematopoiesis is highly coordinated process of fate determination at branch points that is regulated by transcription factors and their cofactors. Our comprehensive catalogue of transcripts in the eight main mature blood cell elements, including erythroblasts and megakaryocytes (MKs) showed that the transcription factor MEIS1 is uniquely transcribed in MKs and the CD34+ haematopoietic stem cell. Gene silencing studies in mice and zebrafish has shown a pivotal role for MEIS1 in haematopoiesis, megakaryopoiesis and vasculogenesis, although its precise hierarchical position and function remain unknown. To gain further insight in the role of MEIS1 in megakaryopoiesis, we used a proteomics approach to search for its nuclear interaction partners. Co-immunoprecipitation was used to isolate MEIS1 interacting proteins from the nuclear fraction of the MK cell line, CHRF 288–11 and resulting eluates were subjected to proteomics analysis using one-dimensional electrophoresis and liquid chromatography (LC) coupled to tandem mass spectrometry (MS) or GeLC-MS/MS. In total 70 proteins were identified to co-immunoprecipitate with MEIS1 from 3 replicate MS analyses. These included the previously validated MEIS1 interactors PBX1 and HOXB9, as well as numerous novel interactors such as ARID3B and DHX9. Network analysis of our MEIS1 interactome dataset revealed a strong association with cell cycle regulation. In fact, we had identified a myriad of cell cycle regulators including CDK1, CDK2, CDK9, CUL3, PCNA, CDC5L, ARID3B and MDC1. These interactions are consistent with recent microarray studies in promyelocytic leukemic cell lines that link MEIS1 with cell cycle entry and its regulation of genes such as CDK2, CDK6, CDKN3, CDC7 and Cyclin D3 among others. To confirm the novel interaction of MK MEIS1 with cell cycle regulators we performed reverse immuno-precipitation/immunoblot analysis in CHRF cells and purified MEIS1 containing multiprotein complexes from L8057 murine megakaryoblastic cells. Using a cell cycle specific PCR array, we demonstrate that MEIS1 overexpression in L8057 cells regulates numerous cell cycle regulatory genes. Preliminary analysis using flow cytometry demonstrated that MEIS1 overexpression resulted in an altered cell cycle progression. Furthermore, genome wide ChIP-Seq analysis in CHRF cells for MEIS1 revealed binding sites in Cyclin D3 and CDK6, two known key regulators of the cell cycle and megakaryopoiesis. Taken together this study provides evidence linking MEIS1 to the cell cycle control of MKs and will help elucidate the role of MEIS1 in cell cycle progression, megakaryopoiesis and associated disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dexamethasone Accelerates the Transition of Human BFU-E to CFU-E and Enhances CFU-E Proliferation through Cell Cycle Regulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3620-3620
Author(s):  
Ryan Ashley ◽  
Hongxia Yan ◽  
Brian Dulmovits ◽  
Nan Wang ◽  
Julien Papoin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Cell Cycle Regulation ◽  
Bone Marrow Failure ◽  
Cell Cycle Regulators ◽  
Erythroid Progenitors ◽  
Cd34 Cells ◽  
Cycle Regulation

Abstract Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is frequently managed with glucocorticoids to increase red cell mass. Despite sustained effective clinical use for many decades in DBA and other anemias, we still do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. In order to improve the clinical management of patients with DBA, we studied the mechanisms of action of dexamethasone (Dex) on erythroid progenitors. To study the effects of Dex on erythroid progenitor biology, we employed a serum-free erythroid culture system to differentiate primary human CD34+ cells isolated from the peripheral blood of adults or DBA patients as well as cord blood samples. We found that Dex increases the total proliferation of CD34+ cells from adult peripheral blood but not in cord blood over 14 days in culture. Dex treatment also led to an acceleration of the BFU-E to CFU-E transition in peripheral blood with a minimal effect in cord blood. In methylcellulose colony forming assays, peripheral blood derived CFU-E treated with Dex for 24hrs produced more colonies or larger colonies than the untreated controls while a lesser effect was seen in cord blood derived CFU-E. Both assays revealed that Dex treatment enhanced self-renewal of peripheral blood derived CFU-E. We also noted that BFU-E and CFU-E differentially express the glucocorticoid receptor (GR) isoforms, GRα and GRβ. BFU-E expressed comparable levels of GRα and the dominant-negative GRβ, whereas only the GRα transcript was identified in CFU-E. We observed similar expression patterns for GR between peripheral and cord blood derived progenitors. Importantly, following Dex treatment, GRα was shown to translocate to the nucleus of erythroid progenitors derived from peripheral blood derived CD34+ cells but not in the cord blood derived progenitors. When we examined the cell cycle progression of sorted human erythroid progenitors, CFU-E and BFU-E, we observed a G0/G1 arrest of peripheral blood CFU-E treated with Dex compared to untreated CFU-E. In marked contrast, no such change was noted for either Dex treated BFU-E from peripheral blood or for CFU-E or BFU-E from cord blood. In addition, we found an increase in the expression of cell cycle regulators p57Kip2 and p27Kip1 in Dex treated peripheral blood CFU-E but no such increased expression was seen in cord blood CFU-E. When peripheral blood derived erythroid progenitors were treated with olomoucine (Olo), a small molecule CDK inhibitor, we observed an acceleration of the BFU-E to CFU-E transition similar to that induced by Dex. Upon co-treatment with Dex and Olo, there was a synergistic effect in the accelerated formation of CFU-E population from BFU-Es. To validate this finding of the dependence of CFU-E on cell cycle regulators, we transduced peripheral blood derived CD34+ cells with a lentiviral shRNA construct to knockdown p57 expression. Following successful knockdown of the expression levels of p57, the numbers of CFU-E indeed decreased implying a role for cell cycle in defective transition of BFU-E to CFU-E. Erythroid progenitors derived from cultures of primary CD34+ cells from DBA patients were also studied. Importantly, we noted increased baseline expression of p57Kip2 and p27Kip1 in erythroid progenitors from steroid resistant DBA patients and in contrast to controls, their expression did not increase following Dex treatment. Our findings provide novel mechanistic insights into the regulation of human erythropoiesis by Dex. We have shown that alterations in cell cycle regulation of erythroid progenitors plays a key role in the effects of glucocorticoids on red blood cells. Furthermore, this work has direct relevance for the development of improved therapeutic treatment strategies for patients with bone marrow failure syndromes such as DBA. Disclosures No relevant conflicts of interest to declare.

scholarly journals Developmental Control of the Cell Cycle: Insights from Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 211 (3) ◽  
pp. 797-829 ◽  
Author(s):  
Edward T. Kipreos ◽  
Sander van den Heuvel
Keyword(s):  
Cell Cycle ◽  
Caenorhabditis Elegans ◽  
Cell Cycle Regulation ◽  
Genetic Model ◽  
Somatic Cells ◽  
Model Systems ◽  
Cell Cycle Regulators ◽  
Animal Development ◽  
C Elegans ◽  
Cycle Regulation

During animal development, a single fertilized egg forms a complete organism with tens to trillions of cells that encompass a large variety of cell types. Cell cycle regulation is therefore at the center of development and needs to be carried out in close coordination with cell differentiation, migration, and death, as well as tissue formation, morphogenesis, and homeostasis. The timing and frequency of cell divisions are controlled by complex combinations of external and cell-intrinsic signals that vary throughout development. Insight into how such controls determine in vivo cell division patterns has come from studies in various genetic model systems. The nematode Caenorhabditis elegans has only about 1000 somatic cells and approximately twice as many germ cells in the adult hermaphrodite. Despite the relatively small number of cells, C. elegans has diverse tissues, including intestine, nerves, striated and smooth muscle, and skin. C. elegans is unique as a model organism for studies of the cell cycle because the somatic cell lineage is invariant. Somatic cells divide at set times during development to produce daughter cells that adopt reproducible developmental fates. Studies in C. elegans have allowed the identification of conserved cell cycle regulators and provided insights into how cell cycle regulation varies between tissues. In this review, we focus on the regulation of the cell cycle in the context of C. elegans development, with reference to other systems, with the goal of better understanding how cell cycle regulation is linked to animal development in general.

scholarly journals Gradual evolution of cell cycle regulation by cyclin-dependent kinases during the transition to animal multicellularity

2019 ◽  
Author(s):  
Alberto Perez-Posada ◽  
Omaya Dudin ◽  
Eduard Ocaña-Pallarès ◽  
Iñaki Ruiz-Trillo ◽  
Andrej Ondracka
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Temporal Order ◽  
Human Cells ◽  
Cell Cycle Regulators ◽  
Transcriptional Program ◽  
Cyclin Dependent Kinases ◽  
Cycle Regulation ◽  
Over Time

AbstractProgression through the cell cycle in eukaryotes is regulated on multiple levels. The main driver of the cell cycle progression is the periodic activity of cyclin-dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle is regulated by a transcriptional program that ensures the just-in-time gene expression. Many core cell cycle regulators are present in all eukaryotes, among them cyclins and CDKs; however, periodic transcriptional programs are divergent between distantly related species. In addition, many otherwise conserved cell cycle regulators have been lost and independently evolved in yeast, a widely used model organism for cell cycle research. To gain insight into the cell cycle regulation in a more representative opisthokont, we investigated the cell cycle regulation at the transcriptional level of Capsaspora owczarzaki, a species closely related to animals. We developed a protocol for cell cycle synchronization in Capsaspora cultures and assessed gene expression over time across the entire cell cycle. We identified a set of 801 periodic genes that grouped into five clusters of expression over time. Comparison with datasets from other eukaryotes revealed that the periodic transcriptional program of Capsaspora is most similar to that of animal cells. We found that orthologues of cyclin A, B and E are expressed at the same cell cycle stages as in human cells and in the same temporal order. However, in contrast to human cells where these cyclins interact with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, our results demonstrate that Capsaspora could be a useful unicellular model system for animal cell cycle regulation.Author’s summaryWhen cells reproduce, proper duplication and splitting of the genetic material is ensured by cell cycle control systems. Many of the regulators in these systems are present across all eukaryotes, such as cyclin and cyclin-dependent kinases (CDK), or the E2F-Rb transcriptional network. Opisthokonts, the group comprising animals, yeasts and their unicellular relatives, represent a puzzling scenario: in contrast to animals, where the cell cycle core machinery seems to be conserved, studies in yeasts have shown that some of these regulators have been lost and independently evolved. For a better understanding of the evolution of the cell cycle regulation in opisthokonts, and ultimately in the lineage leading to animals, we have studied cell cycle regulation in Capsaspora owczarzaki, a unicellular amoeba more closely related to animals than fungi that retains the ancestral cell cycle toolkit. Our findings suggest that, in the ancestor of Capsaspora and animals, cyclins oscillate in the same temporal order as in animals, and that expansion of CDKs occurred later in the lineage that led to animals.

scholarly journals Differential Gene Expression in Auristatin PHE-Treated Cryptococcus neoformans

2004 ◽  
Vol 48 (2) ◽  
pp. 561-567 ◽  
Author(s):  
Tanja Woyke ◽  
Michael E. Berens ◽  
Dominique B. Hoelzinger ◽  
George R. Pettit ◽  
Günther Winkelmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cryptococcus Neoformans ◽  
Cellular Response ◽  
Cell Cycle Regulation ◽  
Efflux Pump ◽  
Schizophyllum Commune ◽  
Nucleotide Metabolism ◽  
Differentially Expressed ◽  
Rt Pcr ◽  
Cycle Regulation

ABSTRACT The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 μM) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

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