Zinc controls operator affinity of human transcription factor YY1 by mediating dimerization via its N-terminal region.

Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

The effect of D380Y pathogenic mutation in human Yin Yang 1 on the protein's structure and function.

Yin Yang 1 is a human transcription factor that controls a number of genes and takes part in the regulation of cell cycle, proliferation, differentiation, and neuronal development. Yin Yang 1 is composed of an N-terminal intrinsically disordered fragment and a C-terminal domain responsible for binding to DNA, composed of four zinc fingers. Recently, various alterations in the Yin Yang 1's DNA binding domain were linked with an unexplained intellectual disability named Gabriele-de Vries syndrome. In this paper, a repetitively occurring substitution of aspartate-380 for tyrosine was analyzed to assess its impact on Yin Yang 1's structure and DNA binding. The substitution was found to affect Yin Yang 1's secondary and tertiary structure to a limited extent and to impair the specificity of its interaction with DNA.

The transcription factor YY2 has less momentous properties of an intrinsically disordered protein than its paralog YY1.

The transcription factor YY2 is a recently discovered paralog of YY1. The two proteins exhibit substantial sequence similarity and partially similar transcriptional activity. They recognize the same DNA sequence in vitro yet bind different promoters in vivo. YY1 comprises two structurally distinct parts: an intrinsically disordered regulatory part and a compact DNA-binding domain. The structure of YY2 is yet unknown. We show that YY2 is structurally similar to YY1, although the conformational state of YY2 is more ordered, as shown by its composition, hydrodynamic properties, spectroscopic signal, and proteolytic susceptibility. As such, YY2's range of molecular partners might be distinct from that of YY1. This could explain different effects of YY1 and YY2 on gene expression patterns and the mechanism of YY proteins in transcriptional regulation.

Isolation stress impacts Met-enkephalin in the hypothalamo-pituitary-adrenocortical axis in growing Polish Mountain sheep: a possible role of the opioids in modulation of HPA axis.

It was hypothesized that there is cross-talk between the classical constituents of the hypothalamo-pituitary-adrenocortical axis (HPA) and Met-enkephalin in the HPA axis. The study examined effects of isolation stress, sex, and age on concentrations of native Met-enkephalin and pro-enkephalin (PENK) gene expression in tissues of the HPA (hypothalamus, pituitary gland and adrenal cortex) in 3-, 6- and 9-month old female and male lambs. In addition, the effects of isolation stress on in vitro release Met-enkephalin from fragments of the hypothalamus or adrenal cortex were examined. Isolation stress was followed by decreases in the concentration of Met-enkephalin in both the pituitary gland and adrenal cortex. There were also increases in the hypothalamic concentration of Met-enkephalin together with increases in PENK gene expression in the HPA in 6- and 9-months old females and males. There were reductions in release of Met-enkephalin from hypothalamic and adrenocortical tissue in vitro after isolation stress. In the presence of naltrexone, there were increases in basal release in vitro of Met-enkephalin from hypothalamic tissue from control and stressed female lambs but a decrease in tissue from stressed male lambs. In a somewhat similar manner, the presence of naltrexone was associated with increases in the basal release of Met-enkephalin from adrenocortical tissue from control female lambs but a decrease with tissue from stressed female and both stressed and control male lambs. Lay summary The present studies examine the impact of isolation stress on Met-enkephalin in growing female and male lambs. The results clearly showed the involvement of Met-enkephalin modulation of the psychological stress response in growing female and male lambs.

Site-directed fluorescence labeling of intrinsically disordered region of human transcription factor YY1: The inhibitory effect of zinc ions.

Site-specific labeling of proteins with fluorescent dyes allows the study of protein structure and function using a wide variety of fluorescent techniques. However, specific labeling is not trivial in the case of proteins containing multiple cysteine residues. An example of such a protein is transcription factor Yin Yang 1, which comprises eight cysteine residues in four C2H2 type zinc fingers in the C-terminal region. Kinetic measurements of the labeling process allowed us to develop preparative labeling of three cysteine residues differently introduced to the N-terminal, disordered fragment of the protein. The protocol developed in the present study allows to prepare the protein with high recovery yield and high selectivity of the labeling. This was confirmed using proteolytic digestion and spectroscopic approach. The labeling process was significantly affected by the presence of zinc ions and was dependent on the localization of the engineered cysteine residue. This is the first known example of the use of cysteine metal protection and labeling (CyMPL) technology for the labeling of protein regions with no stable secondary structures.

Sulpiride, Amisulpride, Thioridazine, and Olanzapine: Interaction with Model Membranes. Thermodynamic and Structural Aspects.

Neuroleptic drugs are widely applied in effective treatment of schizophrenia and related disorders. The lipophilic character of neuroleptics means that they tend to accumulate in the lipid membranes, impacting their functioning and processing. In this paper, the effect of four drugs, namely, thioridazine, olanzapine, sulpiride, and amisulpride, on neutral and negatively charged lipid bilayers was examined. The interaction of neuroleptics with lipids and the subsequent changes in the membrane physical properties was assessed using several complementary biophysical approaches (isothermal titration calorimetry, electron paramagnetic resonance spectroscopy, dynamic light scattering, and ζ potential measurements). We have determined the thermodynamic parameters, that is, the enthalpy of interaction and the binding constant, to describe the interactions of the investigated drugs with model membranes. Unlike thioridazine and olanzapine, which bind to both neutral and negatively charged membranes, amisulpride interacts with only the negatively charged one, while sulpiride does not bind to any of them. The mechanism of olanzapine and thioridazine insertion into the bilayer membrane cannot be described merely by a simple molecule partition between two different phases (the aqueous and the lipid phase). We have estimated the number of protons transferred in the course of drug binding to determine which of its forms, ionized or neutral, binds more strongly to the membrane. Finally, electron paramagnetic resonance results indicated that the drugs are localized near the water-membrane interface of the bilayer and presence of a negative charge promotes their burying deeper into the membrane.

Intrinsic disorder of human Yin Yang 1 protein.

YY1 (Yin Yang 1) is a zinc finger protein with an essential role in various biological functions via DNA- and protein-protein interactions with numerous partners. YY1 is involved in the regulation of a broad spectrum of cellular processes such as embryogenesis, proliferation, tumorigenesis, and snRNA transcription. The more than 100 reported targets of the YY1 protein suggest that it contains intrinsically disordered regions that are involved in such diverse interactions. Here, we present a study of the structural properties of human YY1 using several biochemical and biophysical techniques (fluorescence, circular dichroism, gel filtration chromatography, proteolytic susceptibility) together with various bioinformatics approaches. To facilitate our exploration of the YY1 structure, the full-length protein as well as an N-terminal fragment (residues 1-295) and the C-terminal DNA binding domain were used. We found the N-terminus to be a non-compact fragment of YY1 with little residual secondary structure and lacking a well-defined tertiary structure. The results of our study indicate that YY1 belongs to the family of intrinsically disordered proteins (IDPs), which exist natively in a partially unfolded conformation.

Effect of desipramine on gene expression in the mouse frontal cortex - microarray study.

BACKGROUND: These studies aimed to identify the genes differentially expressed in the frontal cortex of mice treated repeatedly with either saline or desipramine (DMI). METHODS: Differences in gene expression in the mouse frontal cortex were studied using a whole-genome microarray approach. RESULTS: The analyses revealed a group of 88 transcripts (18 genes) that were differentially expressed between the mice treated with saline and those treated with DMI. These genes include Spnb2, Mef2c, Ncam1, Hsp90ab1, Kif1b, Ddx6 and Gsk3b, which were connected in the gene relationship network. CONCLUSIONS: It appears that one week of DMI administration measurably altered the expression of a small number of genes, including genes connected with neuroplasticity and cytoskeletal changes, the regulation of calcium levels in the cell or translation processes.

Intrahepatic expression of genes related to metabotropic receptors in chronic hepatitis.

AIM: To screen for genes related to metabotropic receptors that might be involved in the development of chronic hepatitis. METHODS: Assessment of 20 genes associated with metabotropic receptors was performed in liver specimens obtained by punch biopsy from 12 patients with autoimmune and chronic hepatitis type B and C. For this purpose, a microarray with low integrity grade and with oligonucleotide DNA probes complementary to target transcripts was used. Evaluation of gene expression was performed in relation to transcript level, correlation between samples and grouping of clinical parameters used in chronic hepatitis assessment. Clinical markers of chronic hepatitis included alanine and aspartate aminotransferase, γ-glutamyltranspeptidase, alkaline phosphatase and cholinesterase activity, levels of iron ions, total cholesterol, triglycerides, albumin, glucose, hemoglobin, platelets, histological analysis of inflammatory and necrotic status, fibrosis according to METAVIR score, steatosis, as well as anthropometric body mass index, waist/hip index, percentage of adipose tissue and liver size in ultrasound examination. Gender, age, concomitant diseases and drugs were also taken into account. Validation of oligonucleotide microarray gene expression results was done with the use of quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The highest (0.002 < P < 0.046) expression among genes encoding main components of metabotropic receptor pathways, such as the α subunit of G-coupled protein, phosphoinositol-dependent protein kinase or arrestin was comparable to that of angiotensinogen synthesized in the liver. Carcinogenesis suppressor genes, such as chemokine ligand 4, transcription factor early growth response protein 1 and lysophosphatidic acid receptor, were characterized by the lowest expression (0.002 < P < 0.046), while the factor potentially triggering hepatic cancer, transcription factor JUN-B, had a 20-fold higher expression. The correlation between expression of genes of protein kinases PDPK1, phosphoinositide 3-kinase and protein kinase A (Spearman's coefficient range: 0.762-0.769) confirmed a functional link between these enzymes. Gender (P = 0.0046) and inflammation severity, measured by alanine aminotransferase activity (P = 0.035), were characterized by diverse metabotropic receptor gene expression patterns. The Pearson's coefficient ranging from -0.35 to 0.99 from the results of qRT-PCR and microarray indicated that qRT-PCR had certain limitations as a validation tool for oligonucleotide microarray studies. CONCLUSION: A microarray-based analysis of hepatocyte metabotropic G-protein-related gene expression can reveal the molecular basis of chronic hepatitis.

An investigation of the affinities, specificity and kinetics involved in the interaction between the Yin Yang 1 transcription factor and DNA.

Human transcription factor Yin Yang 1 (YY1) is a four zinc-finger protein that regulates a large number of genes with various biological functions in processes such as development, carcinogenesis and B-cell maturation. The natural binding sites of YY1 are relatively unconserved and have a short core sequence (CCAT). We were interested in determining how YY1 recognizes its binding sites and achieves the necessary sequence selectivity in the cell. Using fluorescence anisotropy, we determined the equilibrium dissociation constants for selected naturally occurring YY1 binding sites that have various levels of similarity to the consensus sequence. We found that recombinant YY1 interacts with its specific binding sites with relatively low affinities from the high nanomolar to the low micromolar range. Using a fluorescence anisotropy competition assay, we determined the affinity of YY1 for non-specific DNA to be between 30 and 40 µm, which results in low specificity ratios of between 3 and 220. Additionally, surface plasmon resonance measurements showed rapid association and dissociation rates, suggesting that the binding strength is regulated through changes in both k(a) and k(d). In conclusion, we propose that, in the cell, YY1 may achieve higher specificity by associating with co-regulators or as a part of multi-subunit complexes.

Efficient overexpression and purification of active full-length human transcription factor Yin Yang 1 in Escherichia coli.

The Yin Yang 1 protein is a zinc finger transcription factor involved in the regulation of diverse cellular processes through DNA and protein-protein interactions. Here we present an improved method for the expression and purification of the human full-length YY1 protein from Escherichia coli. The protein was first purified using denaturing conditions, refolded using optimized conditions and then purified using a DNA-affinity column to ≥ 95% purity; this process provided a high final yield and highly active protein. The protein was active in EMSA and the fluorescence anisotropy assays. The protein retained its full activity and its initial concentration for several months when stored at -80° C. Thus, we have obtained YY1 protein with levels of activity and concentration that are suitable for spectroscopic and other biochemical studies.

Flow cytometry application for studies on adenosine A2A receptors expression.

Adenosine A2A receptors belong to the heptaspanning membrane receptors family A, also known as G protein-coupled receptors. In human brain they are highly expressed in striatum, where they co-exist and co-function with adenosine A1, glutamate mGlu5 and dopamine D2 receptors. As glutaminergic neurotransmission modulators in GABAergic enkephalinergic neurons, adenosine A2A receptors are attractive targets for new, alternative therapies of neurodegenerative disorders, like Parkinson's disease and Huntington's disease. The aim of the research was to obtained fluorescently tagged adenosine A2A receptors. Gene encoding human adenosine A2A receptor was inserted into plasmid pEYFP-N1, bearing enhanced yellow fluorescent protein (EYFP). The construct was expressed in HEK 293 cells. Fluorescence was observed by flow cytometry and epifluorescence microscopy. Functional ligand binding properties were investigated by saturation binding analysis of adenosine A2A receptors specific agonist [3H] CGS 21680.