scholarly journals Progressive methylation of POU5F1 regulatory regions during blastocyst development

Reproduction ◽  
2018 ◽  
Vol 156 (2) ◽  
pp. 145-161 ◽  
Author(s):  
E Canon ◽  
L Jouneau ◽  
T Blachère ◽  
N Peynot ◽  
N Daniel ◽  
...  
Keyword(s):  
De Novo ◽  
Regulatory Region ◽  
Upstream Region ◽  
Distal Enhancer ◽  
Blastocyst Development ◽  
Differentiated Cells ◽  
Rabbit Blastocyst ◽  
Highly Correlated ◽  
And Function

ThePOU5F1gene encodes one of the ‘core’ transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5′ regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere withPOU5F1expression and function inin vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends onde novomethylation of its promoter. However, little is known about the epigenetic regulation ofPOU5F1expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of thePOU5F15′ upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5′ regulatory region and the first exon accompanying differentiation and the gradual repression ofPOU5F1. Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.

Abstract 193: RNA-sequencing of Disease-specific iPSC as a New Filter to Identify Genes Associated With Hypoplastic Left Heart Syndrome

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Almudena Martinez Fernandez ◽  
Xing Li ◽  
Jeanne L Theis ◽  
Andre Terzic ◽  
Timothy M Olson ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Hypoplastic Left Heart Syndrome ◽  
De Novo ◽  
Nuclear Family ◽  
Cardiac Differentiation ◽  
Left Heart ◽  
Hypoplastic Left Heart ◽  
Differentiated Cells ◽  
Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is a complex multifactorial disease for which no definitive genetic causes have been found. Current genetic filtering strategies render lists of genes with unknown relevance in terms of pathogenesis. A complementary filter based on biological evidence would create a new approach to prioritize relevant candidate genes and mutations. In our study, 5 members of a nuclear family including a child with HLHS were evaluated using echocardiography and their genetic information was obtained through whole genome sequencing (WGS). Data filtering including rarity, functional impact and mode of inheritance was implemented, resulting in identification of 34 genes with recessive or de novo variants potentially involved in the pathogenesis of HLHS. Additionally, iPSC were derived from proband and parents and subjected to RNA-sequencing at the undifferentiated state and following spontaneous differentiation. Comparative transcriptional analyses identified genes differentially expressed in proband samples at each stage. These gene sets were used as an additional filter for the previously generated WGS data. This strategy revealed that out of 34 mutated genes originally identified, 10 displayed transcriptional differences in undifferentiated iPSC from the HLHS-affected individual while 16 out of 34 mutated genes showed significantly different expression levels in differentiated cells from proband. Furthermore, expression dynamics were studied during guided cardiac differentiation for the 9 genes fulfilling all applied criteria. Two genes not previously linked to HLHS, ELF4 and HSPG2 were found to behave significantly different in HLHS-iPSC when compared to control counterparts. In summary, filtering WGS data according to a new layer of transcriptional information that leverages iPSC plasticity allows prioritization of genes associated with HLHS in an in vitro model of disease.

scholarly journals Dynamic changes in chromatin accessibility, altered adipogenic gene expression, and total versus de novo fatty acid synthesis in subcutaneous adipose stem cells of normal-weight polycystic ovary syndrome (PCOS) women during adipogenesis: evidence of cellular programming

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Karen L. Leung ◽  
Smriti Sanchita ◽  
Catherine T. Pham ◽  
Brett A. Davis ◽  
Mariam Okhovat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
De Novo ◽  
Polycystic Ovary ◽  
Total Content ◽  
Chromatin Accessibility ◽  
Normal Weight ◽  
Triacylglycerol Synthesis ◽  
And Function

Abstract Background Normal-weight polycystic ovary syndrome (PCOS) women exhibit adipose resistance in vivo accompanied by enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with accelerated lipid accumulation per cell in vitro. The present study examines chromatin accessibility, RNA expression and fatty acid (FA) synthesis during SC abdominal ASC differentiation into adipocytes in vitro of normal-weight PCOS versus age- and body mass index-matched normoandrogenic ovulatory (control) women to study epigenetic/genetic characteristics as well as functional alterations of PCOS and control ASCs during adipogenesis. Results SC abdominal ASCs from PCOS women versus controls exhibited dynamic chromatin accessibility during adipogenesis, from significantly less chromatin accessibility at day 0 to greater chromatin accessibility by day 12, with enrichment of binding motifs for transcription factors (TFs) of the AP-1 subfamily at days 0, 3, and 12. In PCOS versus control cells, expression of genes governing adipocyte differentiation (PPARγ, CEBPα, AGPAT2) and function (ADIPOQ, FABP4, LPL, PLIN1, SLC2A4) was increased two–sixfold at days 3, 7, and 12, while that involving Wnt signaling (FZD1, SFRP1, and WNT10B) was decreased. Differential gene expression in PCOS cells at these time points involved triacylglycerol synthesis, lipid oxidation, free fatty acid beta-oxidation, and oxidative phosphorylation of the TCA cycle, with TGFB1 as a significant upstream regulator. There was a broad correspondence between increased chromatin accessibility and increased RNA expression of those 12 genes involved in adipocyte differentiation and function, Wnt signaling, as well as genes involved in the triacylglycerol synthesis functional group at day 12 of adipogenesis. Total content and de novo synthesis of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and oleic (C18:1) acid increased from day 7 to day 12 in all cells, with total content and de novo synthesis of FAs significantly greater in PCOS than controls cells at day 12. Conclusions In normal-weight PCOS women, dynamic chromatin remodeling of SC abdominal ASCs during adipogenesis may enhance adipogenic gene expression as a programmed mechanism to promote greater fat storage.

Identification and function of phosphorylation in the glucose-regulated transcription factor ChREBP

2008 ◽  
Vol 411 (2) ◽  
pp. 261-270 ◽  
Author(s):  
Nikolas G. Tsatsos ◽  
Michael N. Davies ◽  
Brennon L. O'callaghan ◽  
Howard C. Towle
Keyword(s):  
Transcription Factor ◽  
High Glucose ◽  
Leucine Zipper ◽  
De Novo ◽  
Regulatory Region ◽  
De Novo Lipogenesis ◽  
Nuclear Accumulation ◽  
Helix Loop Helix ◽  
Genes Encoding ◽  
And Function

In the liver, induction of genes encoding enzymes involved in de novo lipogenesis occurs in response to increased glucose metabolism. ChREBP (carbohydrate-response-element-binding protein) is a basic helix–loop–helix/leucine zipper transcription factor that regulates expression of these genes. To evaluate the potential role of ChREBP phosphorylation in its regulation, we used MS to identify modified residues. In the present paper, we report the detection of multiple phosphorylation sites of ChREBP expressed in hepatocytes, several of which are only observed under high-glucose conditions. Mutation of each of these serine/threonine residues of ChREBP did not alter its ability to respond to glucose. However, mutation of five N-terminal phosphoacceptor sites resulted in a major decrease in activity under high-glucose conditions. These phosphorylated residues are located within a region of ChREBP (amino acids 1–197) that is critical for glucose regulation. Mutation of Ser56 within this region to an aspartate residue resulted in increased nuclear accumulation and activity under high-glucose conditions. Together, these data suggest that ChREBP activity is regulated by complex multisite phosphorylation patterns involving its N-terminal regulatory region.

scholarly journals The NtcA-Dependent P1 Promoter Is Utilized for glnA Expression in N2-Fixing Heterocysts of Anabaena sp. Strain PCC 7120

2004 ◽  
Vol 186 (21) ◽  
pp. 7337-7343 ◽  
Author(s):  
Ana Valladares ◽  
Alicia M. Muro-Pastor ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Binding Site ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Gene Encoding ◽  
Differentiated Cells ◽  
Multiple Promoters ◽  
Key Enzyme ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N2-fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNAI originates from a canonical NtcA-dependent promoter (P1) and RNAII originates from a σ70-type promoter (P2), RNAIV is influenced by NtcA but the corresponding promoter (P3) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNAV, which has previously been detected only in in vitro transcription assays and should originate from P4. However, in heterocysts, which are differentiated cells specialized in N2 fixation, RNAI was the almost exclusive glnA transcript. Analysis of P glnA ::lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P1, P2, or P3 and P4 have the ability to promote transcription. Mutation of the NtcA-binding site in P1 eliminated P1-directed transcription and allowed increased use of P2. The NtcA-binding site in the P1 promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

scholarly journals Lethal Respiratory Failure and Mild Primary Hypothyroidism in a Term Girl with a de Novo Heterozygous Mutation in the TITF1/NKX2.1 Gene

2009 ◽  
Vol 94 (1) ◽  
pp. 197-203 ◽  
Author(s):  
Emilie Maquet ◽  
Sabine Costagliola ◽  
Jasmine Parma ◽  
Christiane Christophe-Hobertus ◽  
Luc L. Oligny ◽  
...  
Keyword(s):  
Respiratory Failure ◽  
Respiratory Distress ◽  
De Novo ◽  
Primary Hypothyroidism ◽  
Heterozygous Mutation ◽  
Pulmonary Tissue ◽  
Pulmonary Vasodilators ◽  
Thyroid Transcription Factor 1 ◽  
And Function

Abstract Context: Thyroid transcription factor 1 (TITF1/NKX2.1) is expressed in the thyroid, lung, ventral forebrain, and pituitary. In the lung, TITF1/NKX2.1 activates the expression of genes critical for lung development and function. Titf/Nkx2.1−/− mice have pituitary and thyroid aplasia but also impairment of pulmonary branching. Humans with heterozygous TITF1/NKX2.1 mutations present with various combinations of primary hypothyroidism, respiratory distress, and neurological disorders. Objective: The objective of the study was to report clinical and molecular studies of the first patient with lethal neonatal respiratory distress from a novel heterozygous TITF1/NKX2.1 mutation. Participant: This girl, the first child of healthy nonconsanguineous French-Canadian parents, was born at 41 wk. Birth weight was 3460 g and Apgar scores were normal. Soon after birth, she developed acute respiratory failure with pulmonary hypertension. At neonatal screening on the second day of life, TSH was 31 mU/liter (N <15) and total T4 245 nmol/liter (N = 120–350). Despite mechanical ventilation, thyroxine, surfactant, and pulmonary vasodilators, the patient died on the 40th day. Results: Histopathology revealed pulmonary tissue with low alveolar counts. The thyroid was normal. Sequencing of the patient’s lymphocyte DNA revealed a novel heterozygous TITF1/NKX2.1 mutation (I207F). This mutation was not found in either parent. In vitro, the mutant TITF-1 had reduced DNA binding and transactivation capacity. Conclusion: This is the first reported case of a heterozygous TITF1/NKX2.1 mutation leading to neonatal death from respiratory failure. The association of severe unexplained respiratory distress in a term neonate with mild primary hypothyroidism is the clue that led to the diagnosis.

scholarly journals Motoneuron Excitability and Muscle Spasms Are Regulated by 5-HT2B and 5-HT2C Receptor Activity

2011 ◽  
Vol 105 (2) ◽  
pp. 731-748 ◽  
Author(s):  
Katherine C. Murray ◽  
Marilee J. Stephens ◽  
Edmund W. Ballou ◽  
Charles J. Heckman ◽  
David J. Bennett
Keyword(s):  
Spinal Cord ◽  
Calcium Currents ◽  
Receptor Subtypes ◽  
Constitutive Activity ◽  
Spinal Neurons ◽  
Cord Injury ◽  
Muscle Spasms ◽  
Highly Correlated ◽  
And Function

Immediately after spinal cord injury (SCI), a devastating paralysis results from the loss of brain stem and cortical innervation of spinal neurons that control movement, including a loss of serotonergic (5-HT) innervation of motoneurons. Over time, motoneurons recover from denervation and function autonomously, exhibiting large persistent calcium currents (Ca PICs) that both help with functional recovery and contribute to uncontrolled muscle spasms. Here we systematically evaluated which 5-HT receptor subtypes influence PICs and spasms after injury. Spasms were quantified by recording the long-lasting reflexes (LLRs) on ventral roots in response to dorsal root stimulation, in the chronic spinal rat, in vitro. Ca PICs were quantified by intracellular recording in synaptically isolated motoneurons. Application of agonists selective to 5-HT2B and 5-HT2C receptors (including BW723C86) significantly increased the LLRs and associated Ca PICs, whereas application of agonists to 5-HT1, 5-HT2A, 5-HT3, or 5-HT4/5/6/7 receptors (e.g., 8-OH-DPAT) did not. The 5-HT2 receptor agonist–induced increases in LLRs were dose dependent, with doses for 50% effects (EC50) highly correlated with published doses for agonist receptor binding ( Ki) at 5-HT2B and 5-HT2C receptors. Application of selective antagonists to 5-HT2B (e.g., RS127445) and 5-HT2C (SB242084) receptors inhibited the agonist-induced increase in LLR. However, antagonists that are known to specifically be neutral antagonists at 5-HT2B/C receptors (e.g., RS127445) had no effect when given by themselves, indicating that these receptors were not activated by residual 5-HT in the spinal cord. In contrast, inverse agonists (such as SB206553) that block constitutive activity at 5-HT2B or 5-HT2C receptors markedly reduced the LLRs, indicating the presence of constitutive activity in these receptors. 5-HT2B or 5-HT2C receptors were confirmed to be on motoneurons by immunolabeling. In summary, 5-HT2B and 5-HT2C receptors on motoneurons become constitutively active after injury and ultimately contribute to recovery of motoneuron function and emergence of spasms.

scholarly journals Two Distantly Homologous DnaG Primases from Thermoanaerobacter tengcongensis Exhibit Distinct Initiation Specificities and Priming Activities

2010 ◽  
Vol 192 (11) ◽  
pp. 2670-2681 ◽  
Author(s):  
Jie Li ◽  
Jingfang Liu ◽  
Ligang Zhou ◽  
Huadong Pei ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Amino Acids ◽  
De Novo ◽  
Thermophilic Bacterium ◽  
Structure And Function ◽  
Thermoanaerobacter Tengcongensis ◽  
Wide Range ◽  
Dna Binding Affinity ◽  
Template Specificity ◽  
And Function

ABSTRACT Primase, encoded by dnaG in bacteria, is a specialized DNA-dependent RNA polymerase that synthesizes RNA primers de novo for elongation by DNA polymerase. Genome sequence analysis has revealed two distantly related dnaG genes, TtdnaG and TtdnaG 2, in the thermophilic bacterium Thermoanaerobacter tengcongensis. Both TtDnaG (600 amino acids) and TtDnaG2 (358 amino acids) exhibit primase activities in vitro at a wide range of temperatures. Interestingly, the template recognition specificities of these two primases are quite distinctive. When trinucleotide-specific templates were tested, TtDnaG initiated RNA primer synthesis efficiently only on templates containing the trinucleotide 5′-CCC-3′, not on the other 63 possible trinucleotides. When the 5′-CCC-3′ sequence was flanked by additional cytosines or guanines, the initiation efficiency of TtDnaG increased remarkably. Significantly, TtDnaG could specifically and efficiently initiate RNA primer synthesis on a limited set of tetranucleotides composed entirely of cytosines and guanines, indicating that TtDnaG initiated RNA primer synthesis more preferably on GC-containing tetranucleotides. In contrast, it seemed that TtDnaG2 had no specific initiation nucleotides, as it could efficiently initiate RNA primer synthesis on all templates tested. The DNA binding affinity of TtDnaG2 was usually 10-fold higher than that of TtDnaG, which might correlate with its high activity but low template specificity. These distinct priming activities and specificities of TtDnaG and TtDnaG2 might shed new light on the diversity in the structure and function of the primases.

scholarly journals Regulation of cell–cell adhesion by the cadherin–catenin complex

2008 ◽  
Vol 36 (2) ◽  
pp. 149-155 ◽  
Author(s):  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
De Novo ◽  
Cytoplasmic Proteins ◽  
Cytoskeleton Reorganization ◽  
Dependent Cell ◽  
And Function ◽  
Cell Cell

Ca2+-dependent cell–cell adhesion is regulated by the cadherin family of cell adhesion proteins. Cadherins form trans-interactions on opposing cell surfaces which result in weak cell–cell adhesion. Stronger cell–cell adhesion occurs by clustering of cadherins and through changes in the organization of the actin cytoskeleton. Although cadherins were thought to bind directly to the actin cytoskeleton through cytoplasmic proteins, termed α- and β-catenin, recent studies with purified proteins indicate that the interaction is not direct, and instead an allosteric switch in α-catenin may mediate actin cytoskeleton reorganization. Organization and function of the cadherin–catenin complex are additionally regulated by phosphorylation and endocytosis. Direct studies of cell–cell adhesion has revealed that the cadherin–catenin complex and the underlying actin cytoskeleton undergo a series of reorganizations that are controlled by the Rho GTPases, Rac1 and RhoA, that result in the expansion and completion of cell–cell adhesion. In the present article, in vitro protein assembly studies and live-cell studies of de novo cell–cell adhesion are discussed in the context of how the cadherin–catenin complex and the actin cytoskeleton regulate cell–cell adhesion.

scholarly journals Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription

2020 ◽  
Vol 295 (51) ◽  
pp. 17632-17645
Author(s):  
Yusuke Watanabe ◽  
Daiki Seya ◽  
Dai Ihara ◽  
Shuhei Ishii ◽  
Taiki Uemoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Regulatory Region ◽  
Mouse Embryos ◽  
Great Vessel ◽  
Distal Enhancer ◽  
Enhancer Activity ◽  
Cell Specificity ◽  
Vascular Formation

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

scholarly journals Pancreatic-Duodenal Homeobox 1 Regulates Expression of Liver Receptor Homolog 1 during Pancreas Development

2003 ◽  
Vol 23 (19) ◽  
pp. 6713-6724 ◽  
Author(s):  
Jean-Sébastien Annicotte ◽  
Elisabeth Fayard ◽  
Galvin H. Swift ◽  
Lars Selander ◽  
Helena Edlund ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Regulatory Region ◽  
Mobility Shift ◽  
Pancreatic Development ◽  
Downstream Target ◽  
Regulatory Cascade ◽  
And Function ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1−/− mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.

Sign in / Sign up

Export Citation Format

Share Document