Early Growth Response Protein Egr-1 Research Articles

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Transcriptomic changes in Ca2+-depleted cells: Role of elevated intracellular [Na+]/[K+] ratio

Previously, we reported that Ca2+ depletion increased permeability of the plasma membrane for Na+. This study examined the relative impact of [Na+]i/[K+]i-mediated signaling on transcriptomic changes in cultured vascular smooth muscle cells from rat aorta (VSMC) subjected to Ca2+-depletion by extra-(EGTA) and intracellular (BAPTA-AM) Ca2+ chelators. Na+,K+-ATPase inhibition in K+-free medium during 3h led to elevation of [Na+]i and attenuation of [K+]i by ∼7- and 10-fold, whereas Ca2+-depletion resulted in alteration of these parameters by ∼3- and 2-fold, respectively. Augmented VSMC permeability for Na+ and elevation of the [Na+]i/[K+]i ratio was triggered by addition to Ca2+-free medium 50μM EGTA and was not affected by 10μM BAPTA-AM. Na+,K+-ATPase inhibition and Ca2+-depletion changed expression of 3677 and 4610 mRNA transcripts, respectively. We found highly significant (p<10−12) positive (R2>0.51) correlation between levels of expression of 2071 transcripts whose expression was affected by both stimuli. Among genes whose expression in Ca2+-depleted cells was augmented by more than 7-fold we noted cyclic AMP-dependent transcription factor Atf3, early growth response protein Egr1 and nuclear receptor subfamily 4, group A member Nr4a1. Dissipation of transmembrane gradients of monovalent cations in high-K+, low-Na+-medium abolished the increments of the [Na+]i/[K+]i ratio as well as the augmented expression of these genes triggered by incubation of VSMC in EGTA containing medium. Thus, our results demonstrate, for the first time, that robust transcriptomic changes triggered by Ca2+-depletion in the presence of extracellular Ca2+-chelators are at least partially mediated by elevation of the [Na+]i/[K+]i ratio and activation of Ca2+i-independent, [Na+]i/[K+]i-mediated mechanism of excitation–transcription coupling. These results shad a new light on analysis of data obtained in cells subjected to long-term exposure to Ca2+ chelators.

An autoregulatory loop reverts the mechanosensitive Sirt1 induction by EGR1 in skeletal muscle cells

Muscle contraction is associated with the production of reactive oxygen species (ROS). Mechanisms of ROS scavenging are fundamental to avoid muscle damage. We had previously discovered a stretch-induced genetic program in myotubes that triggers an antioxidant defense. At the core of this mechanism, transcriptional activation of SIRT1 by the early growth response protein EGR1 results in increased MnSOD activity through the activation of Sod2 by SIRT1/FOXO pathway. In this report, we show experimental evidence that; a) EGR1 and SIRT1 proteins physically interact at the time of maximal Sirt1 induction, b) SIRT1 has a negative effect on the activation of the Sirt1 promoter by EGR1. Thus, the interaction between EGR1 and SIRT1 describes an autoregulatory loop that shuts down the stretch-induced Sirt1 expression.

Single-Molecule Detection of Transcription Factor Binding to DNA in Real Time: Specificity, Equilibrium, and Kinetic Parameters

Specificity and temporal control of transcriptional machinery are encoded within sequence-specific transcription factors, of which there are thousands in mammalian genomes. Efforts to completely decipher this code will require an understanding of the DNA binding thermodynamic and kinetic properties displayed by each transcription factor, a daunting task given the current methodologies for measuring these interactions. Here, we present a novel methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device. Using this technology, we measured DNA binding by fluorescently tagged domains of four distinct transcription factors, namely, human early growth response protein Egr-1, vertebrate GATA-1, Drosophila GAGA factor, and lambda bacteriophage Cro repressor. These proteins represent different structural classes (zinc-finger and helix-turn-helix), quaternary states (monomeric and dimeric), and relative affinities (high, intermediate, and low). Specific binding of each protein to its cognate DNA target was demonstrated at low picomolar concentrations. The equilibrium (Kd) and kinetic (kon and koff) constants governing DNA binding by one of these transcription factors, that of Egr-1, were measured using this approach. Kd values obtained from three different types of saturation titrations were reproducible and consistent, yielding values between 10 and 14 pM that, along with the kinetic constants, agree closely with literature values. Because this methodology offers several significant advantages over other existing approaches, namely, real-time determination, requirement for small amounts of reagents, high reproducibility, exquisite sensitivity, and amenability to high-throughput analysis, it is suitable for characterizing DNA-binding proteins as well as other interacting pairs of molecules that can be fluorescently tagged.

Bombesin Regulates Cyclin D1 Expression through the Early Growth Response Protein Egr-1 in Prostate Cancer Cells

Our previous studies indicate that the activation of mitogen-activated protein kinase (MAPK) pathway is involved in bombesin-induced cell proliferation in prostate cancer cells. Cyclin D1 is a critical regulator involved in cell cycle progression through the G1 phase into the S phase, thereby contributing to cell proliferation. Mostly, mitogen-stimulated expression of cyclin D1 is attributed to the extracellular signal-regulated kinase (ERK) activation. Here, we found that bombesin induced human cyclin D1 expression on both mRNA and protein levels in DU-145 prostate cancer cells. Mutational analyses showed that bombesin-enhanced cyclin D1 transcription required the binding of nuclear proteins to the -143 to -105 region of the human cyclin D1 promoter, which contains binding sites for transcription factors Sp-1 and early growth response protein (Egr-1). Do novo protein synthesis was requisite for bombesin-induced cyclin D1 expression. Further studies showed Egr-1 was induced upon bombesin stimulation. The induction of Egr-1 expression and its binding to the cyclin D1 promoter were essential for bombesin-enhanced cyclin D1 transcription. Inhibition of MAPK pathway with either the MEK1 inhibitor PD98059 or a dominant-negative Ras mutant, RasN17, abolished bombesin-induced cyclin D1 activation. Taken together, bombesin-induced cyclin D1 expression in prostate cancer cells is mediated by Egr-1 activation and the interaction of Egr-1 with the Egr-1/Sp1 motif of the cyclin D1 promoter through the activation of MAPK pathway. These findings represent a novel mechanism of bombesin-dependent stimulation of mitogenesis by regulating directly the cell cycle in prostate cancer.

O-069. The synergy between the orphan nuclear receptor steroidogenic factor 1 (SF-1) and the early growth response protein EGR-1 (NGFI-A) plays an important role in gonadotrophin releasing hormone modulation of the luteinizing hormone (LH)β gene

O-069. The synergy between the orphan nuclear receptor steroidogenic factor 1 (SF-1) and the early growth response protein EGR-1 (NGFI-A) plays an important role in gonadotrophin releasing hormone modulation of the luteinizing hormone (LH)β gene

A Regulatory Element in the Human Interleukin 2 Gene Promoter Is a Binding Site for the Zinc Finger Proteins Sp1 and EGR-1

Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity.