The Cre–loxP System: A Versatile Tool for Targeting Genes in a Cell- and Stage-Specific Manner

The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a–CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the −183 to −137 OL2 promoter region forms DNA–protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the −183 to −137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the −183 to −137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the −183 to −137 promoter region together with gene silencing factors, including HDAC.

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Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

Canadian Journal of Microbiology ◽

10.1139/m68-005 ◽

1968 ◽

Vol 14 (1) ◽

pp. 25-31 ◽

Cited By ~ 169

Author(s):

G. W. Strandberg ◽

P. W. Wilson

Keyword(s):

Color Change ◽

Azotobacter Vinelandii ◽

Nitrogenase Activity ◽

Color Difference ◽

Potassium Nitrate ◽

Whole Cells ◽

System A ◽

Growth System ◽

A Cell ◽

Enzyme Formation

The formation and activity of nitrogenase2 in Azotobacter vinelandii OP was examined using a cell-free assay system. A lag period of about 30 min occurred between the exhaustion of the combined nitrogen source and growth on N2. Cells grown on ammonium acetate or potassium nitrate had no detectable nitrogenase activity. Nitrogenase activity appeared in cells, grown under a flowing gas phase of 20% O2 – 60% He, about 45 min after the exhaustion of ammonia. Nitrogenase formation was inhibited in a closed system with an atmosphere containing 40% O2 but not by one containing 20% O2. Hydrogen did not inhibit enzyme formation. The question of whether N2 is required for the formation of the enzyme could not be answered as this gas could not be completely eliminated from the growth system. Chloramphenicol prevented the formation of the enzyme and inhibited nitrogen fixation in whole cells, but had no effect on cell-free enzyme activity. A brief rise in turbidity which occurred during nitrogenase formation appeared to be due to a color change in the cells from reddish brown to dark brown. Spectrophotometric examination of extracts from ammonia- and N2-grown cells did not reveal any components responsible for this color difference, but this result may reflect only the presence of interfering substances in the crude extract.

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Rel Induces Interferon Regulatory Factor 4 (IRF-4) Expression in Lymphocytes

Journal of Experimental Medicine ◽

10.1084/jem.191.8.1281 ◽

2000 ◽

Vol 191 (8) ◽

pp. 1281-1292 ◽

Cited By ~ 146

Author(s):

Raelene J. Grumont ◽

Steve Gerondakis

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Nuclear Factor Κb ◽

Wild Type ◽

Cell Type ◽

Regulatory Factor ◽

Specific Manner ◽

A Cell ◽

Cell Type Specific ◽

Interferon Regulatory Factor 4

In lymphocytes, the Rel transcription factor is essential in establishing a pattern of gene expression that promotes cell proliferation, survival, and differentiation. Here we show that mitogen-induced expression of interferon (IFN) regulatory factor 4 (IRF-4), a lymphoid-specific member of the IFN family of transcription factors, is Rel dependent. Consistent with IRF-4 functioning as a repressor of IFN-induced gene expression, the absence of IRF-4 expression in c-rel−/− B cells coincided with a greater sensitivity of these cells to the antiproliferative activity of IFNs. In turn, enforced expression of an IRF-4 transgene restored IFN modulated c-rel−/− B cell proliferation to that of wild-type cells. This cross-regulation between two different signaling pathways represents a novel mechanism that Rel/nuclear factor κB can repress the transcription of IFN-regulated genes in a cell type–specific manner.

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Development of RNAi-based genetic tools to silence MKP3 expression in a cell-specific manner

10.17918/etd-7168 ◽

2021 ◽

Author(s):

Sayani Patra

Keyword(s):

Genetic Tools ◽

Specific Manner ◽

A Cell ◽

Mkp3 Expression

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trans-Activation of a globin promoter in nonerythroid cells

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.843-853.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 843-853

Author(s):

T Evans ◽

G Felsenfeld

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Gene Activation ◽

Specific Factor ◽

Erythroid Cells ◽

Specific Manner ◽

Alpha Globin ◽

A Cell ◽

Globin Promoter ◽

In Erythroid Cells

We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor.

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