The Role of RNA Polymerase II Contiguity and Long-Range Interactions in the Regulation of Gene Expression in Human Pluripotent Stem Cells

The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between the POU5F1 gene and genes previously identified as being POU5F1 enhancer-interacting, namely, CDYL, TLE2, RARG, and MSX1 (all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene, RUNX1 was used, which is expressed during hematopoietic differentiation and not associated with pluripotency. To reveal how these long-range interactions between POU5F1 and the selected genes change with the onset of differentiation and upon RNAP II inhibition, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) followed by computational simulation analysis. Our analysis showed that the numbers of long-range interactions between specific genes decrease during differentiation, suggesting that the transcription of monitored genes is associated with pluripotency. In addition, we showed that upon inhibition of RNAP II, long-range associations do not disintegrate and remain constant. We also analyzed the distance distributions of these genes in the context of their positions in the nucleus and revealed that they tend to have similar patterns resembling normal distribution. Furthermore, we compared data created in vitro and in silico to assess the biological relevance of our results.

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Identification of stable reference genes in differentiating human pluripotent stem cells

Physiological Genomics ◽

10.1152/physiolgenomics.00130.2014 ◽

2015 ◽

Vol 47 (6) ◽

pp. 232-239 ◽

Cited By ~ 11

Author(s):

Gustav Holmgren ◽

Nidal Ghosheh ◽

Xianmin Zeng ◽

Yalda Bogestål ◽

Peter Sartipy ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Gene Expression Data ◽

Pluripotent Stem Cells ◽

Reference Genes ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Expression Data ◽

Large Variability ◽

The Stability

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

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DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression

Journal of General Virology ◽

10.1099/vir.0.029587-0 ◽

2011 ◽

Vol 92 (7) ◽

pp. 1710-1720 ◽

Cited By ~ 15

Author(s):

Shilpi Tyagi ◽

Alex Ochem ◽

Mudit Tyagi

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

Protein Kinase ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dependent Protein Kinase ◽

Immunodeficiency Virus ◽

Target Sites ◽

Dependent Protein ◽

Rnap Ii

DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat–DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription.

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341. Highly Efficient Transient Gene Expression and Gene Targeting in Human Pluripotent Stem Cells with Helper-Dependent Adenoviral Vectors

Molecular Therapy ◽

10.1016/s1525-0016(16)38699-3 ◽

2009 ◽

Vol 17 ◽

pp. S132

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Gene Targeting ◽

Pluripotent Stem Cells ◽

Transient Gene Expression ◽

Adenoviral Vectors ◽

Human Pluripotent Stem Cells ◽

Highly Efficient

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Abstract 319: Structural Reorganization of Cardiac Transcription Factories Mediates Transcriptional Changes in Response to Stress

Circulation Research ◽

10.1161/res.119.suppl_1.319 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Elaheh Karbassi ◽

Manuel Rosa Garrido ◽

Douglas J Chapski ◽

Yong Wu ◽

Emma Monte ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Three Dimensional ◽

Pressure Overload ◽

Structural Features ◽

Cardiac Cells ◽

Structural Reorganization ◽

Transcription Factories ◽

Response To Stress

The heart’s response to stress entails precise gene expression changes to affect the metabolic and structural features of the cardiomyocyte. The changes in gene expression are mediated by structural alterations in the packaging of the genome. However, the manner in which the three-dimensional architecture of the genome is established is unknown. In non-cardiac cells, genes that are actively transcribed are thought to reside in transcriptionally permissive compartments called transcription factories. The structural principles for achieving cardiac-specific transcription are not understood. We sought to understand the functional nature of cardiac transcription factories: whether they are stable structures (to which genes move in and out of) or are transiently formed around genes in response to cardiac stimuli. Using 5-fluorouridine incorporation into nascent RNA, we quantified changes in RNA polymerase II-mediated transcription in cardiomyocytes upon hypertrophic stress. Furthermore, we characterized the spatial distribution of transcription factories, marked by RNA polymerase II, from adult mice subjected to pressure overload. Using super-resolution microscopy, our analyses revealed reorganization of RNA polymerase II, evidenced by a significant increase in the distance between clusters (130nm in sham to 132.5nm in failing hearts, p=0.02) and a 38% increase in cluster intensity in failing hearts. To understand regulation of cardiac gene expression, we used DNA fluorescence in situ hybridization to map the nuclear position of the gene for SERCA2a (atp2a2), which is down regulated in disease. In failing hearts, we measured increased association of atp2a2 with the nuclear envelope (0/159 loci in sham to 11/278 loci in failure) and increased colocalization with heterochromatin (53/160 loci in sham versus 139/290 loci in failure), providing a structural mechanism for the decrease in SERCA2a expression. In contrast, atp2a2 positioning in the liver remained unaffected, with the majority of loci colocalizing with heterochromatin. These findings show that RNA polymerase II is redistributed to affect transcriptional programming and characterize for the first time the structural rearrangements in chromatin that underpin cardiac pathology.

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Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells

Stem Cells and Development ◽

10.1089/scd.2016.0099 ◽

2016 ◽

Vol 25 (24) ◽

pp. 1884-1897 ◽

Cited By ~ 10

Author(s):

Kana Yanagihara ◽

Yujung Liu ◽

Kei Kanie ◽

Kazuo Takayama ◽

Minako Kokunugi ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Differentiation Tendency

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Single-Cell Gene Expression Profiles Define Self-Renewing, Pluripotent, and Lineage Primed States of Human Pluripotent Stem Cells

Stem Cell Reports ◽

10.1016/j.stemcr.2014.04.014 ◽

2014 ◽

Vol 2 (6) ◽

pp. 881-895 ◽

Cited By ~ 60

Author(s):

Shelley R. Hough ◽

Matthew Thornton ◽

Elizabeth Mason ◽

Jessica C. Mar ◽

Christine A. Wells ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Single Cell ◽

Pluripotent Stem Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Human Pluripotent Stem Cells ◽

Cell Gene Expression ◽

Cell Gene

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O-GlcNAcylation and O-GlcNAc Cycling Regulate Gene Transcription: Emerging Roles in Cancer

Cancers ◽

10.3390/cancers13071666 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1666

Author(s):

Matthew Parker ◽

Kenneth Peterson ◽

Chad Slawson

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Transcription ◽

Regulatory Elements ◽

Post Translational Modification ◽

Hexosamine Biosynthetic Pathway ◽

Rnap Ii ◽

Tata Box Binding Protein

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar post-translational modification (PTM) of intracellular proteins linking nutrient flux through the Hexosamine Biosynthetic Pathway (HBP) to the control of cis-regulatory elements in the genome. Aberrant O-GlcNAcylation is associated with the development, progression, and alterations in gene expression in cancer. O-GlcNAc cycling is defined as the addition and subsequent removal of the modification by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) provides a novel method for cells to regulate various aspects of gene expression, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. We will focus on the complex relationship between phosphorylation and O-GlcNAcylation in the regulation of the RNA Polymerase II (RNAP II) pre-initiation complex and the regulation of the carboxyl-terminal domain of RNAP II via the synchronous actions of OGT, OGA, and kinases. Additionally, we discuss how O-GlcNAcylation of TATA-box binding protein (TBP) alters cellular metabolism. Next, in a non-exhaustive manner, we will discuss the current literature on how O-GlcNAcylation drives gene transcription in cancer through changes in transcription factor or chromatin remodeling complex functions. We conclude with a discussion of the challenges associated with studying O-GlcNAcylation and present several new approaches for studying O-GlcNAc regulated transcription that will advance our understanding of the role of O-GlcNAc in cancer.

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DNA-PK facilitates HIV transcription by regulating the activity of RNA polymerase II and the recruitment of transcription machinery at HIV LTR

10.1101/174573 ◽

2017 ◽

Author(s):

Sonia Zicari ◽

Geetaram Sahu ◽

Larisa Dubrovsky ◽

Lin Sun ◽

Han Yue ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mononuclear Cells ◽

Hiv Replication ◽

Peripheral Blood Mononuclear ◽

Hiv Transcription ◽

Severe Impairment ◽

Rnap Ii ◽

Latent Hiv

ABSTRACTDespite the use of highly effective antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents cure and eradication of HIV infection. These transcriptionally silent proviruses are well protected from both the immune system and HAART regimens. Thus, in order to tackle the problem of latent HIV reservoirs, it is a prerequisite to define all the pathways that regulate HIV transcription. We have previously reported that DNA-PK facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. To extend those studies further, here we demonstrate that DNA-PK promotes HIV transcription by supporting it at several stages, including initiation, pause-release and elongation. We discovered that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by both directly catalyzing and by augmenting the recruitment of P-TEFb at HIV LTR. We found that DNA-PK facilitates the establishment of euchromatin structure at HIV LTR, which further supports HIV gene expression. DNA-PK inhibition or knockdown leads to the severe impairment of HIV gene expression and conversion of euchromatin to heterochromatin at HIV LTR. It also profoundly restricts HIV replication and reactivation of latent provirus. DNA-PK promotes the recruitment of TRIM28 at LTR and facilitates the release of paused RNAP II through TRIM28 phosphorylation. The results were reproduced in cell lines belonging to both lymphoid and myeloid lineages and were confirmed in primary CD4+T cells and peripheral blood mononuclear cells (PBMCs) from HIV-infected patients.IMPORTANCE:Our results reveal the important role of DNA-PK in supporting HIV transcription, replication and latent proviral reactivation. Intriguingly, this study sheds light on an important pathway that affects HIV gene expression. These findings provide strong rationale for developing and using transcriptional inhibitors, such as DNA-PK inhibitors, as supplement to HAART regimens in order to further enhance their effectiveness and to suppress toxicity due to HIV proteins.

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An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

10.1101/106112 ◽

2017 ◽

Cited By ~ 2

Author(s):

Jianying Guo ◽

Dacheng Ma ◽

Rujin Huang ◽

Jia Ming ◽

Min Ye ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Genetic Manipulation ◽

Inner Cell Mass ◽

Cell Mass ◽

Human Pluripotent Stem Cells ◽

Hesc Line ◽

Transcription Activator

AbstractHuman pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level did not only promote naïve pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

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