Small-molecule inducible transcriptional control in mammalian cells

In response to UV irradiation, mammalian cells elicit a gene expression programme designed to repair damage and control cell proliferation and apoptosis. Important members of this stress response include the NF-κB (nuclear factor-κB) family. However, the mechanisms by which UV irradiation activates NF-κB are not well understood. In eukaryotes, a variety of environmental stresses are recognized and remediated by a family of protein kinases that phosphorylate the α subunit of eIF2 (eukaryotic initiation factor-2). In the present study we show that NF-κB in MEF (murine embryo fibroblast) cells is activated by UV-C and UV-B irradiation through a mechanism requiring eIF2α phosphorylation. The primary eIF2α kinase in response to UV is GCN2 (general control non-derepressible-2), with PEK/PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic-reticulum kinase) carrying out a secondary function. Our studies indicate that lowered protein synthesis accompanying eIF2α phosphorylation, combined with eIF2α kinase-independent turnover of IκBα (inhibitor of κBα), reduces the levels of IκBα in response to UV irradiation. Release of NF-κB from the inhibitory IκBα would facilitate NF-κB entry into the nucleus and targeted transcriptional control. We also find that loss of GCN2 in MEF cells significantly enhances apoptosis in response to UV exposure similar to that measured in cells deleted for the RelA/p65 subunit of NF-κB. These results demonstrate that GCN2 is central to recognition of UV stress, and that eIF2α phosphorylation provides resistance to apoptosis in response to this environmental insult.

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Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response

Physiological Genomics ◽

10.1152/physiolgenomics.00160.2012 ◽

2013 ◽

Vol 45 (4) ◽

pp. 127-137 ◽

Cited By ~ 14

Author(s):

Lingchen Fu ◽

Michael S. Kilberg

Keyword(s):

Amino Acid ◽

Transcriptional Activation ◽

Translational Control ◽

Mammalian Cells ◽

Transcriptional Control ◽

Present Report ◽

Binding Activity ◽

Maximal Response ◽

Protein Levels ◽

Amino Acid Response

Mammalian cells respond to amino acid deprivation through multiple signaling pathways referred to as the amino acid response (AAR). Transcription factors mediate the AAR after their activation by several mechanisms; examples include translational control (activating transcription factor 4, ATF4), phosphorylation (p-cJUN), and transcriptional control (ATF3). ATF4 induces ATF3 transcription through a promoter-localized C/EBP-ATF response element (CARE). The present report characterizes an ATF/CRE site upstream of the CARE that also contributes to AAR-induced ATF3 transcription. ATF4 binds to the ATF/CRE and CARE sequences and both are required for a maximal response to ATF4 induction. ATF3, which antagonizes ATF4 and represses its own gene, also exhibited binding activity to the ATF/CRE and CARE sequences. The AAR resulted in elevated total cJUN and p-cJUN protein levels and both forms exhibited binding activity to the ATF/CRE and CARE ATF3 sequences. Knockdown of AAR-enhanced cJUN expression blocked induction of the ATF3 gene and mutation of either the ATF/CRE or the CARE site prevented the cJUN-dependent increase in ATF3-driven luciferase activity. The results indicate that both increased cJUN and the cis-acting ATF/CRE sequence within the ATF3 promoter contribute to the transcriptional activation of the gene during the AAR.

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Control of Transcription Factor Activity and Osteoblast Differentiation in Mammalian Cells Using an Evolved Small-Molecule-Dependent Intein

Journal of the American Chemical Society ◽

10.1021/ja062980e ◽

2006 ◽

Vol 128 (27) ◽

pp. 8939-8946 ◽

Cited By ~ 36

Author(s):

Courtney M. Yuen ◽

Stephen J. Rodda ◽

Steven A. Vokes ◽

Andrew P. McMahon ◽

David R. Liu

Keyword(s):

Transcription Factor ◽

Small Molecule ◽

Mammalian Cells ◽

Osteoblast Differentiation ◽

Transcription Factor Activity ◽

Factor Activity

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Transcription of brain chromatin by ribonucleic acid polymerases from brain nucleic and from Escherichia coli

Biochemical Journal ◽

10.1042/bj1301095 ◽

1972 ◽

Vol 130 (4) ◽

pp. 1095-1099 ◽

Cited By ~ 6

Author(s):

Vijendra K. Singh ◽

S. C. Sung

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Transcriptional Control ◽

Polymerase Activity ◽

Control Mechanisms ◽

E Coli ◽

Bacterial Enzyme ◽

Rna Polymerase Activity

1. Transcription of ox brain chromatin by brain nuclear RNA polymerase II and Escherichia coli RNA polymerase was studied. 2. The soluble chromatin prepared from brain nuclei contained DNA, RNA, histone and non-histone proteins. Such chromatin preparations did not display any endogenous RNA polymerase activity, when assayed in the presence of concentrations of KCl as high as 0.4m. 3. The chromatin-templated activity of brain nuclear polymerase II was stimulated by KCl, with an optimum around 0.25m. 4. The template activity of brain chromatin for brain nuclear polymerase II and E. coli enzyme was about 20–25% of that of pure DNA. This greatly repressed templatecapacity of chromatin was probably due to the acid-soluble chromosomal proteins. 5. Brain nuclear polymerase II was 3–4 times more active with dehistonized chromatin than with pure DNA as template, whereas bacterial enzyme was almost equally active with either of these two templates, reflecting the specificity of the transcriptional control mechanisms in mammalian cells.

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Identification of novel small molecule enhancers of protein production by cultured mammalian cells

Biotechnology and Bioengineering ◽

10.1002/bit.21839 ◽

2008 ◽

Vol 100 (6) ◽

pp. 1193-1204 ◽

Cited By ~ 39

Author(s):

Martin J. Allen ◽

James P. Boyce ◽

Michael T. Trentalange ◽

David L. Treiber ◽

Brian Rasmussen ◽

...

Keyword(s):

Small Molecule ◽

Mammalian Cells ◽

Protein Production ◽

Small Molecule Enhancers

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A rational blueprint for the design of chemically-controlled protein switches

Nature Communications ◽

10.1038/s41467-021-25735-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sailan Shui ◽

Pablo Gainza ◽

Leo Scheller ◽

Che Yang ◽

Yoichi Kurumida ◽

...

Keyword(s):

Small Molecule ◽

Protein Design ◽

Protein Interactions ◽

Mammalian Cells ◽

Chemical Space ◽

Domain Architecture ◽

Receptor Protein ◽

Drug Side Effects ◽

Protein Protein Interactions ◽

Drug Receptors

AbstractSmall-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

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A general strategy to construct small molecule biosensors in eukaryotes

eLife ◽

10.7554/elife.10606 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 83

Author(s):

Justin Feng ◽

Benjamin W Jester ◽

Christine E Tinberg ◽

Daniel J Mandell ◽

Mauricio S Antunes ◽

...

Keyword(s):

Metabolic Engineering ◽

Ligand Binding ◽

Small Molecules ◽

Small Molecule ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Dynamic Range ◽

Plant Cells ◽

General Strategy ◽

Target Molecule

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

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Control of lipid metabolism by regulated intramembrane proteolysis of sterol regulatory element binding proteins (SREBPs)

Biochemical Society Symposium ◽

10.1042/bss0700221 ◽

2003 ◽

Vol 70 ◽

pp. 221-231 ◽

Cited By ~ 37

Author(s):

Robert B Rawson

Keyword(s):

Mammalian Cells ◽

Transcriptional Control ◽

Binding Proteins ◽

Regulatory Element ◽

Lipid Level ◽

Transmembrane Helices ◽

Intramembrane Proteolysis ◽

Terminal Domain ◽

Regulated Intramembrane Proteolysis ◽

Sterol Regulatory Element

In mammalian cells, the supply of lipids is co-ordinated with demand through the transcriptional control of genes encoding proteins required for synthesis or uptake. The sterol regulatory element binding proteins (SREBPs) are responsible for increased transcription of these genes when lipid level fall. Mammals have three SREBPs (-1a, -1c and -2), which are the products of two distinct genes. Synthesized as approximately 120 kDa precursors, they are inserted into membranes of the endoplasmic reticulum (ER) in a hairpin fashion. Both the N-terminal transcription factor domain and the C-terminal regulatory domain face the cytoplasm. These are connected by two transmembrane helices separated by a short loop projecting into the ER lumen. The C-terminal domain of SREBP interacts with the C-terminal domain of SREBP-cleavage-activating protein (SCAP). The N-terminal half of SCAP contains eight transmembrane helices, five of which (helices 2-6) form the sterol-sensing domain. In response to cellular demand for lipid, this complex exits the ER and transits to the Golgi apparatus, where two distinct proteases cleave the SREBP precursor to release the transcriptionally active N-terminus. This process was the first example of regulated intramembrane proteolysis for which the proteases were identified. Recent work has additionally uncovered integral membrane proteins, insig-1 and insig-2, that are required to retain the SREBP-SCAP complex in the ER in the presence of sterols, thus providing a more complete understanding of the control of proteolysis in this complex regulatory pathway.

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