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Two polypyrimidine tracts in the nitric oxide synthase 2 gene: similar regulatory sequences with different properties
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
2010 (English)In: Molecular Biology Reports, ISSN 0301-4851, E-ISSN 1573-4978, Vol. 37, no 4, 2021-2030 p.Article in journal (Refereed) Published
Abstract [en]

We reported previously that the polymorphic polypyrimidine CCTTT-microsatellite in the regulatory region of nitric oxide synthase 2 (NOS2) bound nuclear proteins in vitro. In the present work, we aimed to characterize and investigate a potential regulatory role of the CCTTT-microsatellite in NOS2 expression. Therefore, we performed gel-shift, S1-nuclease, and chromatin immunoprecipitation (ChIP) assays. In vitro experiments showed that the microsatellite formed triplex-DNA both with and without superhelical constraint. We also found that the CCTTT-microsatellite and an apparently similar CT-repeat in the first intron of NOS2 were specifically cleaved by S1-nuclease, when cloned into a supercoiled plasmid. In vitro data suggested that the CCTTT-microsatellite bound both polypyrimidine tract-binding protein (PTBP1) and heterogeneous nuclear ribonucleoprotein K (hnRNPK). On the contrary, ChIP revealed binding of PTBP1 and hnRNPK rather to the CT-repeat in the first intron than to the CCTTT-microsatellite. Enrichment for RNA polymerase II and acetylated histones H3 and H4 was also detected at the intronic site. We suggest that both PTBP1 and hnRNPK binds the single strand of the triplex-DNA formed at the CT-repeat in the first intron and that this interaction could be involved in the regulation of NOS2 expression.

Place, publisher, year, edition, pages
2010. Vol. 37, no 4, 2021-2030 p.
Keyword [en]
HnRNPs, NOS2, Polypyrimidines, Transcriptional regulation, Triplex DNA
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97862DOI: 10.1007/s11033-009-9653-9ISI: 000275161700040PubMedID: 19669598OAI: oai:DiVA.org:uu-97862DiVA: diva2:172951
Available from: 2008-11-18 Created: 2008-11-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Genetic and Genomic Analysis of Transcriptional Regulation in Human Cells
Open this publication in new window or tab >>Genetic and Genomic Analysis of Transcriptional Regulation in Human Cells
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There are around 20.000 genes in the human genome all of which could potentially be expressed. However, it is obvious that not all of them can be active at the same time. Thus, there is a need for coordination achieved through the regulation of transcription. Transcriptional regulation is a crucial multi-component process involving genetic and epigenetic factors, which determine when and how genes are expressed. The aim of this thesis was to study two of these components, the transcription factors and the DNA sequence elements with which they interact.

In papers I and II, we tried to characterize the regulatory role of repeated elements in the regulatory sequences of nitric oxide synthase 2 gene. We found that this type of repeat is able to adopt non B-DNA conformations in vitro and that it binds nuclear factors, in addition to RNA polymerase II. Therefore it is probable that these types of repeats can participate in the regulation of genes.

In papers III-V, we intended to analyze the genome-wide binding sites for six transcription factors involved in fatty acid and cholesterol metabolism and the sites of an epigenetic mark in a human liver cell line. For this, we applied the chromatin immunoprecipitation (ChIP) method together with detection on microarrays (ChIP-chip) or by detection with the new generation massively parallel sequencers (ChIP-seq). We found that all of these transcription factors are involved in other liver-specific processes than metabolism, for example cell proliferation. We were also able to define two sets of transcription factors depending on the position of their binding relative to gene promoters. Finally, we demonstrated that the patterns of the epigenetic mark reflect the structure and transcriptional activity of the promoters.

In conclusion, this thesis presents experiments, which moves our view from genetics to genomics, from in vitro to in vivo, and from low resolution to high resolution analysis of transcriptional regulation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 403
Keyword
Transcription, ChIP-chip, ChIP-seq, genome-wide, transcription factors, microsatellite, epigenetic
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-9407 (URN)978-91-554-7355-6 (ISBN)
Public defence
2008-12-12, Rudbecksalen, Rudbecklaboratoriet, Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-11-18 Created: 2008-11-18 Last updated: 2013-09-20Bibliographically approved

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