The effects of the DNA Demethylating reagent, 5-azacytidine on SMCHD1 genomic localization.

BACKGROUND: DNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC). RESULTS: Our data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression. CONCLUSION: These findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies.

Retinoblastoma protein (Rb) links hypoxia to altered mechanical properties in cancer cells as measured by an optical tweezer.

Hypoxia modulates actin organization via multiple pathways. Analyzing the effect of hypoxia on the biophysical properties of cancer cells is beneficial for studying modulatory signalling pathways by quantifying cytoskeleton rearrangements. We have characterized the biophysical properties of human LNCaP prostate cancer cells that occur in response to loss of the retinoblastoma protein (Rb) under hypoxic stress using an oscillating optical tweezer. Hypoxia and Rb-loss increased cell stiffness in a fashion that was dependent on activation of the extracellular signal-regulated kinase (ERK) and the protein kinase B (AKT)- mammalian target of rapamycin (MTOR) pathways. Pharmacological inhibition of MEK1/2, AKT or MTOR impeded hypoxia-inducible changes in the actin cytoskeleton and inhibited cell migration in Rb-deficient cells conditioned with hypoxia. These results suggest that loss of Rb in transformed hypoxic cancer cells affects MEK1/2-ERK/AKT-MTOR signalling and promotes motility. Thus, the mechanical characterization of cancer cells using an optical tweezer provides an additional technique for cancer diagnosis/prognosis and evaluating therapeutic performance.

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip.

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.

The aryl hydrocarbon receptor nuclear translocator (ARNT) family of proteins: transcriptional modifiers with multi-functional protein interfaces.

The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of transcription factors mediates cellular responses to a variety of internal and external stimuli. As functional transcription factors, these proteins act as bHLH-PAS heterodimers and can be further sub-classified into sensory/activated subunits and regulatory or ARNT-like proteins. This class of proteins act as master regulators of the bHLH-PAS superfamily of transcription factors that mediate circadian rhythm gene programs, innate and adaptive immune responses, oxygen-sensing mechanisms and compensate for deleterious environmental exposures. Some contribute to the etiology of human pathologies including cancer because of their effects on cell growth and metabolism. We will review the canonical roles of ARNT and ARNT-like proteins with an emphasis on coactivator selectivity and recruitment. We will also discuss recent advances in our understanding of noncanonical DNA-binding independent or off-target roles of ARNT that are uncoupled from its classic heterodimeric bHLH-PAS binding partners. Understanding the DNA binding-independent functions of ARNT may identify novel therapeutic options for the treatment of a large spectrum of disease states.

A polymorphic dinucleotide repeat in the human dopamine D5 receptor gene promoter.

Previously, we have reported the cloning and characterization of the 5'-flanking region of the human dopamine D5 receptor encoding gene (D5) and that the major transactivation domain was 119-182 bp upstream of the transcriptional start site [Beischlag, T.V. et al., Biochemistry, 34 (1995) 5960-5970]. Within this region existed a small dinucleotide repeat termed (TC)13. In this report, we describe the screening of genomic DNAs from 18 unrelated individuals by single-strand conformation polymorphism (SSCP) analysis. SSCP analysis revealed the existence of two additional alleles, termed (TC)12 and (TC)14. Neither form significantly altered D5 promoter-mediated luciferase activity when compared to that of the wild-type control, suggesting that small differences in the number of dinucleotide repeats are not likely of any functional consequence for D5 transactivation.

The human dopamine D5 receptor gene: cloning and characterization of the 5'-flanking and promoter region.

Genomic and overlapping cDNA clones encompassing the entire 5'-untranslated region of the human D5 receptor gene were cloned and sequenced. Comparison of these human D5 receptor genomic and cDNA clones revealed the presence of two exons separated by a small and variably sized intron (of either 179 or 155 bp). We have determined that the major site of transcription initiation of the D5 gene is 2125 bp upstream from the translational initiation start site. The region 5' to the transcription initiation site lacked conventional TATA and CAAT sequences, but contained several putative binding sites for transcription factors, such as Sp1 and Ap1. Luciferase reporter gene constructs containing D5 gene sequence information up to 500 bp 5' of the transcription initiation site were able to stimulate transcription only in SK-N-SH cells but not in COS-7, CHO, P19EC, NB41A3, and SK-N-MC cell lines. Promoter deletion analysis indicated that the D5 gene promoter contained a positive modulator at 119-182 and a negative modulator 251-500 bases upstream from the site of transcription initiation. In addition, in order to detect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, in situ hybridization analysis of monkey and human brain using a 5' D5-specific riboprobe revealed that D5 receptor mRNA was most abundant in discrete cortical areas (layers II, IV, and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense riboprobes labeled discrete cell bodies in the pars compacta of the substantia nigra. The characterization of the genomic organization of the D5 receptor gene and of those factors involved in its transcriptional regulation may aid in our understanding of the role this gene product plays in the generation and maintenance of dopamine D1-like receptor-mediated events.

Two gene duplication events in the human and primate dopamine D5 receptor gene family.

The human dopamine D5 receptor (DRD5) gene family consists of the DRD5-encoding gene (DRD5) and the pseudogenes psi DRD5-1 and psi DRD5-2. Analysis of the 5' UTR of DRD5 and homologous regions in the pseudogenes revealed that the nucleotide identity (approx. 95%) extended for 1.9 kb and terminated at a monomeric Alu sequence in each of the pseudogenes. The presence of Alu sequences in the pseudogenes, at this point of divergence with DRD5, suggests that Alu sequences were involved in the evolution of the DRD5 family. This report is the first to describe a possible mechanism involved in the duplication of genes in the G-protein-coupled receptor (GPCR) family. The pseudogenes continue to share identity (approx. 98%) beyond this 5' UTR point of divergence with DRD5 for at least another 6 kb. Analysis of the 3' UTR of DRD5 and homologous regions in the pseudogenes revealed that the identity (approx. 95%) extends at least 14 kb, and the identity between the pseudogenes (approx. 98%) extends for at least 18 kb. Thus, the duplication unit that produced the first pseudogene was at least 16 kb, whereas the second pseudogene was at least 28 kb. We have also located two DRD5 pseudogenes in gorilla demonstrating that these closely related pseudogenes were present in a common ancestor of human and gorilla.

A composite hormone response element mediates the transactivation of the rat oxytocin gene by different classes of nuclear hormone receptors.

Transcription factors of the steroid/thyroid hormone receptor superfamily are mediators of development and regulation of the brain. Previous studies have shown that the hypothalamic oxytocin (OT) gene is a potential target of these receptors, since its promoter is stimulated by estrogens and thyroid hormone. Here it is shown that the rat OT promoter is stimulated (at least 20-fold) by retinoic acid through two distinct regions in the 5'-flanking region. The major retinoic acid response element was located between nucleotides -172 and -148 and a minor one between nucleotides -112 and -77, as concluded from the transactivation of 5'-deletion mutants and binding to promoter elements by the retinoic acid receptor. Since the -172/-148 element also conferred estrogen and thyroid hormone responsiveness, it can be considered a composite hormone response element. This element contains a natural variant of the direct repeat of the half-site AGGTCA with spacing zero (DR-0) as well as a palindrome. Analysis of the core sequences of this element by site-directed mutagenesis showed that each of the three TGACC motifs integral to this element contributes to the multihormone sensitivity, but the contribution of each motif is different for the individual receptors. In neonatal rats, vitamin A deficiency and retinoic acid supplementation did not cause changes in hypothalamic OT mRNA levels and OT peptide levels in the pituitary gland and plasma. Gel-retarded protein-DNA complexes were formed between the composite hormone response element and extracts of the hypothalamic supraoptic and paraventricular nuclei. The composite hormone response element has a unique configuration and integrates responses of multiple members of the steroid/thyroid hormone receptor superfamily.

Determination of nonderivatized para-hydroxylated metabolites of diazepam in biological fluids with a GC Megabore column system.

A method was developed using gas chromatography (GC) and a Megabore column system capable of simultaneous detection of diazepam, N-desmethyldiazepam, temazepam, oxazepam, and their para-hydroxylated metabolites. This method does not require derivatization of para-hydroxylated metabolites. Standard curves for pure reference compounds were linear, with the minimum detectable concentration of diazepam and its metabolites as low as 0.13 ng/injection.

Diazepam metabolism by rat and human liver in vitro: inhibition by mephenytoin.

1. Diazepam metabolism and its association with mephenytoin hydroxylase were studied in vitro using human and rat livers. 2. Enzyme kinetic parameters were obtained for the formation of p-hydroxydiazepam (p-hydroxy-DZP), N-desmethyldiazepam (NDZ), and temazepam (TMZ) from diazepam (DZP) in rat liver fractions. The Km values for formation in rat of p-hydroxy-DZP, NDZ and TMZ were 14 +/- 3 (SEM) microM, 44 +/- 4 and 63 +/- 8, respectively; clearance values calculated from Vmax/Km were 5.7, 3.2 and 4.9 ml/g per min, respectively. 3. Mephenytoin (MP) competitively inhibited, in rat liver, the formation of NDZ, but not the formation of p-hydroxy-DZP or TMZ; in human liver neither NDZ nor TMZ formation was inhibited by MP. 4. In seven different human livers the formation of p-hydroxy-DZP represented a minor pathway compared to the formation of NDZ and TMZ.