Ca2+ and Calmodulin Modulate DNA-Binding Activity of Maize Heat Shock Transcription Factor in Vitro

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

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Constitutive nuclear NFκB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate

Biochemical Journal ◽

10.1042/bj3120833 ◽

1995 ◽

Vol 312 (3) ◽

pp. 833-838 ◽

Cited By ~ 19

Author(s):

A F G Slater ◽

M Kimland ◽

S A Jiang ◽

S Orrenius

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Gradient Centrifugation ◽

Binding Activity ◽

Pyrrolidine Dithiocarbamate ◽

Nf Kappa B ◽

Dna Binding Activity ◽

Apoptotic Activity

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

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Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.1983 ◽

1993 ◽

Vol 13 (4) ◽

pp. 1983-1997 ◽

Cited By ~ 164

Author(s):

A Nakai ◽

R I Morimoto

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Dna Binding ◽

Heat Shock Transcription Factor ◽

Heat Shock Gene ◽

Regulatory Sequences ◽

Regulatory Pathway ◽

Binding Properties ◽

Dna Binding Properties

We have cloned three avian heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and the avian homologs of mammalian HSF1 and HSF2. The predicted amino acid sequence of HSF3 is approximately 40% related to the sequence of HSF1 and HSF2. The sequences for all three factors exhibit extensive identify in the DNA binding motifs and the heptad repeats of hydrophobic amino acids which are common to all eukaryotic HSFs. Despite these overall similarities, each avian HSF exhibits distinct DNA binding properties. HSF2 when expressed in vitro binds constitutively to the heat shock element promoter sequence, whereas neither HSF1 nor HSF3 expressed in vitro binds to DNA. HSF1 DNA binding is induced upon heat shock or treatment with nonionic detergents, whereas the DNA binding properties of HSF3 are not induced by these conditions in vitro. These results suggest that HSF3 activation may involve an induction pathway distinct from the traditional forms of heat shock gene induction. HSF3 DNA binding activity, however, is obtained when the carboxyl-terminal region including the distal heptad repeat is deleted, indicating the presence of negative cis-regulatory sequences. The HSF3 message, like HSF1 and HSF2 messages, is coexpressed during development and in most tissues, which suggests a general role for the regulatory pathway involving HSF3.

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In vitro activation of heat shock transcription factor DNA-binding by calcium and biochemical conditions that affect protein conformation.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.10.3748 ◽

1990 ◽

Vol 87 (10) ◽

pp. 3748-3752 ◽

Cited By ~ 173

Author(s):

D. D. Mosser ◽

P. T. Kotzbauer ◽

K. D. Sarge ◽

R. I. Morimoto

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Dna Binding ◽

Protein Conformation ◽

Heat Shock Transcription Factor ◽

In Vitro Activation

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Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1

Biochemical Journal ◽

10.1042/bj3530591 ◽

2001 ◽

Vol 353 (3) ◽

pp. 591-601 ◽

Cited By ~ 7

Author(s):

Olivier LAROCHELLE ◽

Gale STEWART ◽

Pierre MOFFATT ◽

Véronique TREMBLAY ◽

Carl SÉGUIN

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Binding Activity ◽

Dependent Manner ◽

Rnase Protection ◽

Dna Binding Activity ◽

Cell Extracts ◽

Metal Element ◽

Transcription Factor 1

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn2+-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1–MRE and the MEP-1–MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn2+ ions but not by Cd2+, UV irradiation or PMA, and occurred on ice as well as at 21°C. In control and Zn2+-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn2+ or a preincubation at 37°C. However, recombinant MTF-1 produced in vitro required Zn2+ activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

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The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans

PeerJ ◽

10.7717/peerj.4755 ◽

2018 ◽

Vol 6 ◽

pp. e4755 ◽

Cited By ~ 1

Author(s):

Kyle K. Biggar ◽

Kenneth B. Storey

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Dna Binding ◽

Complex Dynamics ◽

Binding Activity ◽

Trachemys Scripta ◽

Model Organisms ◽

Trachemys Scripta Elegans ◽

Dna Binding Activity

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.

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