Truncated variants of thyroid hormone receptor beta display disease-inflicting malfunctioning at cellular level.

Thyroid hormone receptor ß (THRß) is a member of the nuclear receptor superfamily of ligand-modulated transcription factors. Upon ligand binding, THRß sequentially recruits the components of transcriptional machinery to modulate target gene expression. In addition to regulating diverse physiological processes, THRß plays a crucial role in hypothalamus-pituitary-thyroid axis feedback regulation. Anomalies in THRß gene/protein structure are associated with onset of diverse disease states. In this study, we investigated disease-inflicting truncated variants of THRß using in-silico analysis and cell-based assays. We examined the THRß truncated variants on multiple test parameters, including subcellular localization, ligand-receptor interactions, transcriptional functions, interaction with heterodimeric partner RXR, and receptor-chromatin interactions. Moreover, molecular dynamic simulation approaches predicted that shortened THRß-LBD due to point mutations contributes proportionally to the loss of structural integrity and receptor stability. Deviant subcellular localization and compromised transcriptional function were apparent with these truncated variants. Present study shows that 'mitotic bookmarking' property of some THRß variants is also affected. The study highlights that structural and conformational attributes of THRß are necessary for normal receptor functioning, and any deviations may contribute to the underlying cause of the inflicted diseases. We anticipate that insights derived herein may contribute to improved mechanistic understanding to assess disease predisposition.

Mitotic genome bookmarking by nuclear receptor VDR advocates transmission of cellular transcriptional memory to progeny cells.

Mitosis is an essential process for the self-renewal of cells that is accompanied by dynamic changes in nuclear architecture and chromatin organization. Despite all the changes, the cell manages to re-establish all the parental epigenetic marks, post-mitotically. Recent reports suggest that some sequence-specific transcription factors remain attached to mitotic chromatin during cell division to ensure timely reactivation of a subset of transcription factors necessary to maintain cell identity. These mitotically associated factors are suggested to act as 'genome bookmarking factors' and the phenomenon is termed 'genome bookmarking'. Here, we studied this phenomenon with Vitamin D Receptor (VDR), a key regulator of calcium and phosphate homeostasis and a member of the nuclear receptor superfamily. This study, for the first time, has confirmed VDR as a mitotic bookmarking factor that may be playing a crucial role in the maintenance of cell identity and genome bookmarking. Full 'DNA binding domain (DBD)' present in VDR was identified as essential for enrichment of VDR on mitotic chromatin. Furthermore, the study also demonstrates that VDR evokes mitotic chromatin binding behaviour in its heterodimeric partner Retinoid X receptor (RXR). Interestingly, for promoting bookmarking behaviour in RXR, both DBD and/or ligand-binding domain (LBD) in conjunction with hinge region of VDR were required. Additionally, ChIP analysis showed that VDR remains associated with DR3 (direct repeat 3) region of its specific target gene promoter CYP24A1(Cytochrome P450 family 24 subfamily A member1), during mitosis. Altogether, our study illustrates a novel function of VDR in the epigenetic transmission and control of expression of target proteome for maintenance of cell identity and traits in progeny cells.

Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging.

Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a valuable tool for studying the NR-ligand interactions and their downstream effects. The translocation response of NRs can be studied irrespective of the nature of the interacting ligand (agonist, antagonist, or a small molecule modulator). These nuclear translocation studies offer an advantage over promoter-reporter-based transcription assays where transcription response is observed only with the activating hormones or agonistic ligands. Globally, milk serves as a major dietary source. However, suspected presence of endocrine/metabolism-disrupting chemicals like bisphenols, parabens, organochlorine pesticides, carbamates, non-steroidal anti-inflammatory drugs, chloramphenicol, brominated flame retardants, etc. has been reported. Considering that these chemicals may impart serious developmental and metabolism-related health concerns, it is essential to develop assays suitable for the detection of xenobiotics present at differing levels in milk. Since milk samples cannot be used directly on cultured cells or for microscopy, a combination of screening strategies has been developed herein based on the revelation that i) lipophilic NR ligands can be successfully retrieved in milk-fat; ii) milk-fat treatment of cells is compatible with live-cell imaging studies; and finally, iii) treatment of cells with xenobiotics-spiked and normal milk derived fat provides a visual and quantifiable response of NR translocation in living cells. Utilizing a milk-fat extraction method and Green Fluorescent Protein (GFP) tagged NRs expressed in cultured mammalian cells, followed by an assessment of NR response proved to be an effective approach for screening xenobiotics present in milk samples.HighlightsDiverse endocrine and metabolism-disrupting chemicals are suspected to contaminate milk.Nuclear receptors serve as 'xenosensors' for assessing the presence of xenobiotics in milk.Nuclear import of steroid receptors with (ant)agonist can be examined in live cells.Lipophilic xenobiotics are extracted and observed enriched in milk-fat fraction.A comprehensive cell-based protocol aids in the detection of xenobiotics in milk.

Global footprints of organochlorine pesticides: a pan-global survey.

Organochlorine pesticides (OCPs) are ubiquitous environmental contaminants widely used all over the world. These chlorinated hydrocarbons are toxic and often cause detrimental health effects because of their long shelf life and bioaccumulation in the adipose tissues of primates. OCP exposure to humans occurs through skin, inhalation and contaminated foods including milk and dairy products, whereas developing fetus and neonates are exposed through placental transfer and lactation, respectively. In 1960s, OCPs were banned in most developed countries, but because they are cheap and easily available, they are still widely used in most third world countries. The overuse or misuse of OCPs has been rising continuously which pose threats to environmental and human health. This review reports the comparative occurrence of OCPs in human and bovine milk samples around the globe and portrays the negative impacts encountered through the long history of OCP use.

Phosphorylation of HSP90 by protein kinase A is essential for the nuclear translocation of androgen receptor.

The androgen receptor (AR) is often activated in prostate cancer patients undergoing androgen-ablative therapy because of the activation of cellular pathways that stimulate the AR despite low androgen levels. In many of these tumors, the cAMP-dependent protein kinase A (PKA) pathway is activated. Previous studies have shown that PKA can synergize with low levels of androgen to enhance androgen signaling and consequent cell proliferation, leading to castration-resistant prostate cancer. However, the mechanism by which PKA causes AR stimulation in the presence of low/no androgen is not established yet. Here, using immunofluorescence immunoblotting assays, co-immunoprecipitation, siRNA-mediated gene silencing, and reporter gene assays, we demonstrate that PKA activation is necessary for the phosphorylation of heat shock protein (HSP90) that binds to unliganded AR in the cytoplasm, restricting its entry into the nucleus. We also found that PKA-mediated phosphorylation of the Thr89 residue in HSP90 releases AR from HSP90, enabling AR binding to HSP27 and its migration into the nucleus. Substitution of the Thr89 in HSP90 prevented its phosphorylation by PKA and significantly reduced AR transactivation and cellular proliferation. We further observed that the transcription of AR target genes, such as prostate-specific antigen (PSA), is also lowered in the HSP90 Thr89 variant. These results suggest that using a small-molecule inhibitor against the HSP90 Thr89 residue in conjunction with existing androgen-ablative therapy may be more effective than androgen-ablative therapy alone in the treatment of prostate cancer patients.

Influence of signaling kinases on functional dynamics of nuclear receptor CAR.

Constitutive androstane receptor (CAR) is a xenobiotic nuclear receptor known to regulate genes involved in key physiological processes like drug metabolism, maintenance of energy homeostasis, and cell proliferation. Owing to the diverse regulatory roles played by the receptor, it is critical to understand the precise cellular signals that dictate functional dynamics of CAR. With the objective of exploring the hitherto unknown regulatory pathways modulating CAR, we subjected the CAR protein sequence to a kinase prediction tool and identified several kinases recognizing CAR as a substrate. Using fluorescence live cell imaging and specific inhibitors it was observed that CAR functions under the regulation of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) signaling cascade. Additionally, insulin-like growth factor 1 (IGF1)-mediated inhibition of GSK3 also induced nuclear translocation of CAR linking CAR to the Akt signaling pathway. Identification of T38 residue of CAR as the GSK3 target site further substantiated our observations. Taking cues from these findings, we propose a hypothetical model elucidating the GSK3-mediated regulation of CAR dynamics through the involvement of Akt pathway. Further research into this area is expected to provide novel therapeutic targets in disease conditions like type 2 diabetes and hepatocellular carcinoma.

Nuclear localization signal region in nuclear receptor PXR governs the receptor association with mitotic chromatin.

In recent years, some transcription factors have been observed to remain associated with mitotic chromatin. Based on these observations, it is suggested that these chromatin-bound transcription factors may serve as 'epigenetic marks' for transmission of pattern of gene expression from progenitor to progeny cells. In this context, our laboratory has reported that nuclear receptor PXR, a master regulator of xenobiotic metabolism, remains constitutively associated with mitotic chromatin. However, the region responsible for this interaction with chromatin remained unknown. In this study, we have shown, for the first time, that mitotic chromatin association of this factor is mediated by the combined action of two zinc fingers present in the DNA-binding domain of PXR. Overall, the nuclear localization signal (NLS) region appears to play a major role in this interaction with mitotic chromatin. Also, we have identified a sub-region of 11 amino acid residues within NLS region of PXR (R66-76R) essential for receptor interaction with the mitotic chromatin. Interestingly, this minimal region is sequence-specific and independent of its basic charge. We have termed this minimal sub-region as 'mitotic chromatin binding-determining region' (MCBR). It is suggested that this receptor region is essential for activation of its target genes. Additionally, we have shown that PXR remains associated with the everted repeat (ER6) region of its major target gene, CYP3A4 promoter during mitosis implying its suggested role in 'gene bookmarking'.

Dynamic Hyaluronan drives liver endothelial cells towards angiogenesis.

BACKGROUND: Angiogenesis, the formation of new blood vessels from pre-existing vasculature is essential in a number of physiological processes such as embryonic development, wound healing as well as pathological conditions like, tumor growth and metastasis. Hyaluronic acid (HA), a high molecular weight polysaccharide, major component of extracellular matrix is known to associate with malignant phenotypes in melanomas and various other carcinomas. Hyaluronic acid binding protein 1 (HABP1) has been previously reported to trigger enhanced cellular proliferation in human liver cancer cells upon its over-expression. In the present study, we have identified the HA mediated cellular behaviour of liver endothelial cells during angiogenesis. METHODS: Endothelial cells have been isolated from perfused liver of mice. Cell proliferation was studied using microwell plates with tetrazole dye. Cell migration was evaluated by measuring endothelial monolayer wound repair as well as through transwell migration assay. Alterations in proteins and mRNA expression were estimated by immunobloting and quantitative real time PCR using Applied Biosystems. The paraformaldehyde fixed endothelial cells were used for immuno- florescence staining and F-actin detection with conjugated antibodies. The images were captured by using Olympus florescence microscope (IX71). RESULTS: We observed that administration of HA enhanced cell proliferation, adhesion, tubular sprout formation as well as migration of liver endothelial cells (ECs). The effect of HA in the rearrangement of the actins confirmed HA -mediated cytoskeleton re-organization and cell migration. Further, we confirmed enhanced expression of angiogenic factors like VEGF-A and VEGFR1 in endothelial cells upon HA treatment. HA supplementation led to elevated expression of HABP1 in murine endothelial cells. It was interesting to note that, although protein levels of ß- catenin remained unaltered, but translocation of this protein from membrane to nucleus was observed upon HA treatment, suggesting its role not only in vessel formation but also its involvement in angiogenesis signalling. CONCLUSIONS: The elucidation of molecular mechanism (s) responsible for HA mediated regulation of endothelial cells and angiogenesis contributes not only to our understanding the mechanism of disease progression but also offer new avenues for therapeutic intervention.

Heterodimerization of Retinoid X Receptor with Xenobiotic Receptor partners occurs in the cytoplasmic compartment: Mechanistic insights of events in living cells.

Retinoid X Receptor (RXR) serves as the heterodimeric partner of two major xenobiotic nuclear receptors, Pregnane and Xenobiotic Receptor (PXR) and Constitutive Androstane Receptor (CAR). These receptors are primarily involved in the metabolism and clearance of endobiotics and xenobiotics (including clinical drugs) from the body. Here, we report for the first time that intermolecular interactions between RXR-PXR and RXR-CAR occurs in the cytoplasmic compartment of the cell in a ligand-independent manner. These interactions lead to nuclear import of the heterodimeric complex thereby making them competent for chromatin binding and transactivation of target genes. To explore the cellular site involved in the process of heterodimerization we created various RFP- and GFP-tagged receptor chimeras and also the mutants of their nuclear localization signal (NLS). From the study it is apparent that NLS of PXR/CAR/RXR play a major role in the import of the heterodimeric complex from the cytoplasm to the nucleus in a ligand-independent manner. We observed that along with the heterodimeric partner and/or respective ligand a functional NLS is necessary for activation of target gene. The data suggests that RXR is the major driving force to import the heterodimeric complex into the nucleus since the mutation in the NLS region of RXR weakens this import process dramatically, whereas mutations in the NLS regions of PXR and CAR have little or no significant effect. This RXR-dependent nuclear translocation of the heterodimeric complex also modulates the individual transcriptional activity of PXR and CAR by increasing the basal transcriptional activity. Finally, it is documented that the heterodimerization of RXR with both the partners (PXR, CAR) occurs in the cytoplasm and implies that these dynamic interactions have functional and regulatory attributes in gene expression. In addition, this RXR-dependent enhancement of the transcriptional activity of PXR and CAR may be utilized for evaluating the receptor-drug interactions.

A comprehensive analysis and functional characterization of naturally occurring non-synonymous variants of nuclear receptor PXR.

Pregnane & Xenobiotic Receptor (PXR) acts as a xenosensing transcriptional regulator of many drug metabolizing enzymes and transporters of the 'detoxification machinery' that coordinate in elimination of xenobiotics and endobiotics from the cellular milieu. It is an accepted view that some individuals or specific populations display considerable differences in their ability to metabolize different drugs, dietary constituents, herbals etc. In this context we speculated that polymorphisms in PXR gene might contribute to variability in cytochrome P450 (CYP450) metabolizing enzymes of phase I, drug metabolizing components of phase II and efflux components of the detoxification machinery. Therefore, in this study, we have undertaken a comprehensive functional analysis of seventeen naturally occurring non-synonymous variants of human PXR. When compared, we observed that some of the PXR SNP variants exhibit distinct functional and dynamic responses on parameters which included transcriptional function, sub-cellular localization, mitotic chromatin binding, DNA-binding properties and other molecular interactions. One of the unique SNP located within the DNA-binding domain of PXR was found to be functionally null and distinct on other parameters. Similarly, some of the non-synonymous SNPs in PXR imparted reduced transactivation function as compared to wild type PXR. Interestingly, PXR is reported to be a mitotic chromatin binding protein and such an association has been correlated to an emerging concept of 'transcription memory' and altered transcription output. In view of the observations made herein our data suggest that some of the natural PXR variants may have adverse physiological consequences owing to its influence on the expression levels and functional output of drug-metabolizing enzymes and transporters. The present study is expected to explain not only the observed inter-individual responses to different drugs but may also highlight the mechanistic details and importance of PXR in drug clearance, drug-drug interactions and diverse metabolic disorders. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Influence of a critical single nucleotide polymorphism on nuclear receptor PXR-promoter function.

The Pregnane and Xenobiotic Receptor (PXR; NR1I2) is a ligand-modulated transcription factor that belongs to the nuclear receptor superfamily. It is expressed at higher levels primarily in liver and intestine as compared to the levels in several other organs. It is activated by a broad spectrum of xenobiotics and endobiotics. The primary function of PXR is to regulate the expression of drug metabolizing enzymes and transporters and prevent the accumulation of toxic chemicals in the body, thereby maintaining body's homeostasis. In this study, we identified a C/T single nucleotide polymorphism at position -831 from the transcriptional start site of the PXR gene promoter and examined the functional significance of this variant using both the luciferase reporter gene assays and electrophoretic mobility shift assays (EMSA). Transient transfection experiments showed that the T-allele was associated with significantly greater transcriptional activity than the C-allele of SNP rs3814055. These results indicate that the -831C/T polymorphism has a direct effect on transcriptional regulation of PXR gene. This allelic variation may be a potential genetic marker that can help identify individuals at higher risk for Inflammatory Bowel Disease (IBD).

Novel Application of Red Fluorescent Protein (DsRed-Express) for the Study of Functional Dynamics of Nuclear Receptors.

Arrivals of fluorescent proteins have revolutionized the way we do research in the areas of molecular cell biology. In the present study, we have successfully exploited the multimer-forming property of Red Fluorescent Protein (RFP) in living cells to preferentially shift the unliganded nuclear receptors from the nuclear to the cytoplasmic compartment. Subsequently, these cytoplasmic-shifted unliganded receptors could be induced to translocate into the nucleus by their ligands. Though the multimerization of RFP as a protein-tag is viewed as a disadvantage, we have exploited and projected this property towards novel applications in validating the clinical drugs, herbal compounds, metabolic disruptors etc. Such cytoplasmic shifted transcription factors can offer a unique opportunity to study receptor-ligand interactions and functional dynamics by analyzing ligand-mediated receptor translocation from cytoplasmic compartment to the nucleus of living cells.

Identification and interplay of sequence specific DNA binding proteins involved in regulation of human Pregnane and Xenobiotic Receptor gene.

Pregnane and Xenobiotic Receptor (PXR), a member of nuclear receptor superfamily, acts as a 'xenosensor' in our body and modulates a network of genes involved in xenobiotic metabolism and elimination. Expression levels of PXR in certain metabolic disorders including cancer are reported to be altered and its induced expression is associated with the development of resistance towards chemotherapy and adverse drug-drug interactions. Though the transcriptional regulation of PXR target genes have been elucidated in significant details, the structure and functional control of PXR promoter itself remains inadequately explored. In this work, we identify a Composite Element (CE) located within the proximal PXR promoter region that consists of multiple overlapping cis-elements and demonstrated that CE interacts specifically with some critical nuclear proteins. Subsequent DNA-protein interaction studies revealed mutually exclusive interactions on CE occurring between Sp1 and two unidentified DNA binding proteins with molecular masses of 50 and 54kDa. Here, we report the identification of 54kDa CE binding protein as a heterogeneous nuclear ribonucleoprotein K (hnRNPK) and demonstrate the effect of hnRNP K and Sp1 on PXR promoter transcriptional activity. Overall, the study indicates that PXR gene is tightly regulated to maintain a low receptor level.

Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-Jun.

Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling.

Nuclear localization signal in nuclear receptor VDR facilitates the mitotic genome bookmarking by involving distinct amino acid residues.

Mitotic genome-bookmarking preserves epigenetic information, re-establishing progenitor's gene expression profile through transcription factors, chromatin remodelers, and histone modifiers, thereby regulating cell fate and lineage commitment post-mitotically in progeny cells. Our recent study revealed that the constitutive association of VDR with mitotic chromatin involves its DNA-binding domain. However, amino acid residues in this domain, crucial for genome bookmarking, remain elusive. This study demonstrates that nuclear localization signal (NLS) residues between 49 and 55 amino acids in VDR are essential for receptor-chromatin interaction during mitosis. Furthermore, it is revealed that both bipartite nature of VDR-NLS region and N-terminally located positively charged arginine residues are critical for its 'genome-bookmarking' property. Since mitotic chromatin association of heterodimeric partner RXR depends on VDR-chromatin association, interventions in VDR binding also abort RXR-chromatin interaction. Overall, this study documents the mechanistic details underlying VDR-chromatin interactions in genome-bookmarking behavior, potentially aiding in comprehending VDR-mediated diseases attributed to certain SNPs.

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway.

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.

Mitotic genome-bookmarking by nuclear hormone receptors: A novel dimension in epigenetic reprogramming and disease assessment.

Arrival of multi-colored fluorescent proteins and advances in live cell imaging has immensely contributed to our understanding of intracellular trafficking of nuclear receptors and their roles in gene regulatory functions. These regulatory events need to be faithfully propagated from progenitor to progeny cells. This is corroborated by multiple converging mechanisms that include histone modifications and lately, the phenomenon of 'mitotic genome-bookmarking' by specific transcription factors. This phenomenon refers to the retention and feed-forward transmission of progenitor's architectural blueprint of active transcription status which is silenced and preserved during mitosis. Upon mitotic exit, this phenomenon ensures accurate reactivation of transcriptome, proteome, cellular traits and phenotypes in the progeny cells. In addition to diverse modes of genome-bookmarking by nuclear receptors, a correlation between disease-associated receptor polymorphism and disruption of this phenomenon is apparent. However, breakthrough technologies shall reveal finer details of this phenomenon to help achieve normalcy in receptor-specific diseases.

Hereditary Vitamin D-Resistant Rickets (HVDRR) associated SNP variants of vitamin D receptor exhibit malfunctioning at multiple levels.

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. It is a primary regulator of calcium and phosphate homeostasis required for skeleton and bone mineralization. Vitamin D in active form 1α,25 dihydroxyvitamin-D3 mediates its cellular functions by binding to VDR. Active VDR forms heterodimers with partner RXR (retinoid X receptor) to execute its physiological actions. HVDRR (Hereditary Vitamin D-Resistant Rickets) is a rare genetic disorder that occurs because of generalized resistance to the 1α,25(OH)2D3. HVDRR is caused by the polymorphic variations in VDR gene leading to defective intestinal calcium absorption and mineralization of newly forming bones. Using point and deletion SNPs of VDR we have studied several HVDRR-associated SNP variants for their subcellular dynamics, transcriptional functions, 'genome bookmarking', heterodimeric interactions with RXR, and receptor stability. We previously reported that VDR is a 'mitotic bookmarking factor' that remains constitutively associated with the mitotic chromatin to inherit 'transcriptional memory', however the mechanistic details remained unclear. We document that 'genome bookmarking' property by VDR is critically impaired by naturally occurring HVDRR-associated point and deletion variants found in patients. Furthermore, these HVDRR-associated SNP variants of VDR were found to be compromised in transcriptional function, nuclear translocation, protein stability and intermolecular interactions with its heterodimeric partner RXR. Intriguingly, majority of these disease-allied functional defects failed to be rescued by RXR. Our findings suggest that the HVDRR-associated SNP variations influence the normal functioning of the receptor, and this derived understanding may help in the management of disease with precisely designed small molecule modulators.

Retention and transmission of active transcription memory from progenitor to progeny cells via ligand-modulated transcription factors: elucidation of a concept by BIOPIT model.

Observations made in live cells have clearly demonstrated that agonist-activated steroid/nuclear receptors reorganize in the nucleoplasm into hundreds of discrete speckled structures commonly referred to as nuclear foci. Subsequent studies have shown that nuclear foci are formed only with agonist- and not with pure antagonist-bound receptors. Also, the other accessory components of transcriptional machinery co-localize in nuclear foci with the activated receptors, suggesting these to be active gene transcription sites. Recently, it has been observed that during mitosis nuclear foci present in interphase of progenitor cells co-migrate with condensing chromatin and are inherited into the progeny cells. Ensuing events imply that as memory, the cells inherit only a biomolecular blueprint of transcription status over to next generations to express and sustain their characteristic proteome. Thus, cells achieve self-renewal via mitosis but not without ensuring that the characteristic proteome and traits are distinctively preserved during this transcription phase. This mechanism, although somewhat analogous to epigenetic marking, differs in Nature since transcription factors themselves execute this transmission. To uphold the mechanistic distinctions the phenomenon has been termed BIOPIT (biomolecular imprints offered to progeny for inheritance of traits). The BIOPIT model proposed herein attempts to explain how the disruption of BIOPIT markings by therapeutic anti-hormones or endocrine disruptors over prolonged periods may lead to eradication of cellular transcription memory with deleterious cellular consequences. It is anticipated that our model has the potential to explain the concerted actions and consequences of ligand-receptor interactions with the chromatin in the perspective of normal and aberrant physiological situations.

Evaluation of (Anti)androgenic Activities of Environmental Xenobiotics in Milk Using a Human Liver Cell Line and Androgen Receptor-Based Promoter-Reporter Assay.

The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we investigated the possible presence of (anti)androgenic contaminants in raw and commercialized milk samples. For this purpose, a novel HepARE-Luc cell line that stably expresses human androgen receptor (AR) and the androgen responsive luciferase reporter gene was generated and used in the present study. Treatment of this cell line with androgens and corresponding antiandrogen (flutamide) stimulated or inhibited expression of reporter luciferase, respectively. Real time polymerase chain reaction and immunostaining results exhibited transcription response and translocation of AR from the cytoplasm to the nucleus in response to androgen. Observations implied that a cell-based xenobiotic screening assay via AR response can be conducted for assessing the (anti)androgenic ligands present in food chain including milk. Therefore, the cell line was further used to screen the (anti)androgenic activity of a total of 40 milk fat samples procured as raw or commercial milk. Some of the raw and commercial milk fat samples distinctly showed antiandrogenic activities. Subsequently, some commonly used environmental chemicals were also evaluated for their (anti)androgenic activities. Initial observations with molecular docking studies of experimental compounds were performed to assess their interaction with AR ligand binding domain. Furthermore, (anti)androgenic activities of these compounds were confirmed by performing luciferase assay using the HepARE-Luc cell line. None of the test compounds showed androgenic activities rather some of them like Bisphenol A (BPA) and rifamycin showed antiandrogenic activities. In conclusion, our results provide a valuable information about the assessment of (anti)androgenic activities present in milk samples. Overall, it is proposed that a robust cell-based CALUX assay can be used to assess the (anti)androgenic activities present in milk which can be attributed to different environmental chemicals present therein.