scholarly journals Transcriptional activity of Npas4l controls endothelial lineage specification and restriction in zebrafish

2021 ◽  
Author(s):  
◽  
Kenny Mattonet
Keyword(s):  
Transcriptional Activity ◽  
Lineage Specification ◽  
Endothelial Lineage

Die Bildung von Blutgefäßen ist essentiell für die Entwicklung und Homöostase von Wirbeltieren und die Endothelzellspezifikation ist ein wichtiger erster Schritt in diesem Prozess. Das früheste bekannte Ereignis bei der Endothelzellspezifikation im Zebrafisch ist die Expression des bHLH-PAS-Transkriptionsfaktor-Gens npas4l. Ich habe eine transgene V5-Linie zum Nachweis des markierten Npas4l auf Proteinebene und eine Gal4-VP16-Reporterlinie zur Visualisierung und Verfolgung von npas4l exprimierenden Zellen in vivo generiert. Beide Linien können bereits in frühen Entwicklungsstadien nachgewiesen werden und komplementieren auch starke npas4l-Mutanten Allele. Um npas4l Reporter exprimierende Zellen in npas4l Mutanten zu verfolgen, habe ich anschließend eine mutierte Variante der Gal4-Reporterlinie erzeugt. Diese Mutante trägt eine Insertion in der Region, die die DNA-Bindedomäne kodiert. Dadurch stört sie die Npas4l-Funktion, aber nicht die Reporterexpression. Dieses mutierte Reporterallel komplementiert nicht die npas4l-Mutanten und zeigt einen starken Phänotyp, was darauf hindeutet, dass es sich um ein funktionelles Nullallel handelt. Phänotypische Analysen zeigten, dass npas4l-Reporter positive Zellen in npas4l-Mutanten nicht spezifizieren oder zur Mittelachse wandern. Stattdessen tragen sie zu den vom intermediären Mesoderm abgeleiteten pronephrischen Tubuli und dem vom paraxialen Mesoderm abgeleiteten Skelettmuskel bei. Ich habe diese Phänotypen durch Einzelzell-RNAseq an den npas4l-Reporter positiven Zellen in npas4l+/- und npas4l-/- Embryonen bestätigt. Zusammen erklären diese beiden alternativen Zellschicksale den Großteil der beobachteten Veränderungen zwischen den Genotypen. Npas4l ist dafür bekannt die Expression der drei Transkriptionsfaktorgene etsrp, tal1 und lmo2 zu fördern. Ich stellte die Hypothese auf, dass das Fehlen jedes dieser Transkriptionsfaktoren in npas4l-Mutanten verschiedene Aspekte des npas4l-Phänotyps verursacht. Daher habe ich Mutantenlinien für alle drei Gene generiert und sie sowohl in vaskulären Reporterlinien als auch im npas4l-Reporterhintergrund analysiert. Die Daten legen nahe, dass verschiedene Gene unterschiedliche Prozesse während der frühen Endothelentwicklung regulieren. In npas4l-/- und etsrp-/- Embryonen differenzieren npas4l-Reporter exprimierende Zellen nicht zu Endothelzellen und tragen stattdessen zur Skelettmuskelzellpopulation bei. In npas4l-/- und tal1-/- Embryonen können npas4l-Reporter exprimierende Zellen nicht migrieren und tragen stattdessen zu der Bildung der pronephrischen Tubuli bei. Um die Beziehung zwischen diesen Faktoren besser zu verstehen, habe ich getestet, ob die Injektion von etsrp-, tal1- oder lmo2-mRNA verschiedene Aspekte des npas4l-Phänotyps retten würde. npas4l-, etsrp- und tal1-Mutanten zeigen alle schwere vaskuläre Phänotypen. Einige Endothelzellen und vaskuläre Strukturen bleiben jedoch in jeder Mutante erhalten. Der Phänotyp ist am stärksten in npas4l-/- Embryonen, aber selbst in diesen Embryonen können einige fli1a-positive Endothelzellen in der Schwanzregion beobachtet werden. Es war unklar, ob sich diese Population von Endothelzellen unabhängig von der Npas4l-, Tal1- und Etsrp-Funktion entwickelt oder als Folge einer restlichen tal1- oder etsrp-Expression unabhängig von Npas4l. Um diese Frage zu untersuchen, habe ich Doppelmutanten generiert und nach dem Vorhandensein von fli1a-positiven Endothelzellen in diesen Mutanten gesucht. Während fli1a-positive Endothelzellen in npas4l-/- und npas4l-/-;tal1-/- Embryonen deutlich vorhanden sind, können keine solchen Zellen in npas4l-/-;etsrp-/- oder etsrp-/-;tal1-/- Embryonen beobachtet werden. Diese Daten deuten darauf hin, dass sich im Zebrafisch keine Endothelzellen entwickeln können, wenn zugleich npas4l und etsrp oder etsrp und tal1 gestört sind. Während der Verlust von etsrp zu stärkeren Defekten in npas4l-Mutanten führt, gibt es keinen zusätzlichen Phänotyp, der durch den Verlust von tal1verursacht wird, was darauf hindeutet, dass die Expression von etsrp, aber nicht die von tal1, unabhängig von Npas4l auftreten kann. Diese Idee wird durch die Beobachtung unterstützt, dass etsrp, aber nicht tal1-Expression in den meisten fli1a-exprimierenden Zellen in npas4l-/- Embryonen beobachtet wird. Dennoch wird der Großteil -Expression durch Npas4l reguliert. tal1-mRNA-Injektionen reichten aus, um eine Wildtyp-ähnliche vaskuläre Musterbildung im Bauchbereich der npas4l-/- Embryonen wiederherzustellen, einschließlich der Rettung sowohl der Zellmigration als auch der Differenzierung. Da Npas4l mehrere unterschiedliche transkriptionelle Effektoren hat, war eine so starke Rettung durch nur einen dieser Effektoren unerwartet. In den geretteten Mutanten wurde die bilaterale Population von npas4l-Reporter-positiven pronephrischen Tubuluszellen nicht entdeckt, aber die Anzahl der ektopischen npas4l-Reporter exprimierenden Muskelzellen war im Vergleich zu nicht injizierten npas4l-Mutanten gleichbleibend. ...

scholarly journals Hormonal regulation of cholesterol 7alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat

1997 ◽  
Vol 38 (12) ◽  
pp. 2483-2491 ◽  
Author(s):  
W M Pandak ◽  
D M Heuman ◽  
K Redford ◽  
R T Stravitz ◽  
J Y Chiang ◽  
...  
Keyword(s):  
Hormonal Regulation ◽  
Transcriptional Activity ◽  
Specific Activity ◽  
Mrna Levels

scholarly journals USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Oncogenesis ◽  
2021 ◽  
Vol 10 (7) ◽  
Author(s):  
Ruize Gao ◽  
David Buechel ◽  
Ravi K. R. Kalathur ◽  
Marco F. Morini ◽  
Mairene Coto-Llerena ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Transcriptional Activity ◽  
Kinase Inhibitor ◽  
Aerobic Glycolysis ◽  
Hypoxia Inducible Factor ◽  
Aberrant Expression ◽  
Key Enzyme ◽  
Hcc Cells

AbstractUnderstanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

scholarly journals Transcriptional Regulation of PEN-2, a Key Component of the γ-Secretase Complex, by CREB

2006 ◽  
Vol 26 (4) ◽  
pp. 1347-1354 ◽  
Author(s):  
Ruishan Wang ◽  
Yun-wu Zhang ◽  
Ping Sun ◽  
Runzhong Liu ◽  
Xian Zhang ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Activity ◽  
Start Codon ◽  
Gamma Secretase ◽  
Mobility Shift ◽  
Endoproteolytic Cleavage ◽  
Notch Cleavage ◽  
Translational Start

ABSTRACT Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's β-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the γ-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPα and Aβ without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

1997 ◽  
Vol 19 (2) ◽  
pp. 163-172 ◽  
Author(s):  
K Chu ◽  
HH Zingg
Keyword(s):  
Receptor Binding ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Fine Tuning ◽  
Orphan Receptor ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals Tat-SF1 Protein Associates with RAP30 and Human SPT5 Proteins

1999 ◽  
Vol 19 (9) ◽  
pp. 5960-5968 ◽  
Author(s):  
Jae B. Kim ◽  
Yuki Yamaguchi ◽  
Tadashi Wada ◽  
Hiroshi Handa ◽  
Phillip A. Sharp
Keyword(s):  
Recombinant Proteins ◽  
Transcriptional Activity ◽  
Transcription Activation ◽  
Cellular Proteins ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Cellular Factors ◽  
Immunodeficiency Virus

ABSTRACT The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

scholarly journals Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi
Keyword(s):  
Transcriptional Activity ◽  
Mapk Pathway ◽  
Skeletal Development ◽  
Critical Role ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

Depression of nuclear transcription and extension of mRNA half-life under anoxia in Artemia franciscana embryos

2000 ◽  
Vol 203 (7) ◽  
pp. 1123-1130 ◽  
Author(s):  
F. van Breukelen ◽  
R. Maier ◽  
S.C. Hand
Keyword(s):  
Half Life ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Artemia Franciscana ◽  
Mrna Levels ◽  
Compensatory Mechanisms ◽  
Depressed State ◽  
Initiation Of Transcription ◽  
Mrna Half Life

Transcriptional activity, as assessed by nuclear run-on assays, was constant during 10 h of normoxic development for embryos of the brine shrimp Artemia franciscana. Exposure of embryos to only 4 h of anoxia resulted in a 79.3+/−1 % decrease in levels of in-vivo-initiated transcripts, and transcription was depressed by 88. 2+/−0.7 % compared with normoxic controls after 24 h of anoxia (means +/− s.e.m., N=3). Initiation of transcription was fully restored after 1 h of normoxic recovery. Artificially lowering the intracellular pH of aerobic embryos to the value reflective of anoxia (pH 6.7) showed that acidification alone explained over half the transcriptional arrest. Initiation of transcription was not rescued by application of 80 % carbon monoxide under anoxia, which suggests that heme-based oxygen sensing is not involved in this global arrest. When these transcriptional data are combined with the finding that mRNA levels are unchanged for at least 6 h of anoxia, it is clear that the half-life of mRNA is extended at least 8.5-fold compared with that in aerobic embryos. In contrast to the activation of compensatory mechanisms to cope with anoxia that occurs in mammalian cells, A. franciscana embryos enter a metabolically depressed state in which gene expression and mRNA turnover are cellular costs apparently not compatible with survival and in which extended tolerance supercedes the requirement for continued metabolic function.

Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3405-3413 ◽  
Author(s):  
Adi Inbal ◽  
Naomi Halachmi ◽  
Charna Dibner ◽  
Dale Frank ◽  
Adi Salzberg
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Genetic Evidence ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Native Form ◽  
Downstream Target ◽  
Activating Function

Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.

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