Consensus Sequences For Transcription Factors Research Articles

Molecular basis of facilitated target search and sequence discrimination of TALE homeodomain transcription factor Meis1

Transcription factors specifically bind to their consensus sequence motifs and regulate transcription efficiency. Transcription factors are also able to non-specifically contact the phosphate backbone of DNA through electrostatic interaction. The homeodomain of Meis1 TALE human transcription factor (Meis1-HD) recognizes its target DNA sequences via two DNA contact regions, the L1-α1 region and the α3 helix (specific binding mode). This study demonstrates that the non-specific binding mode of Meis1-HD is the energetically favored process during DNA binding, achieved by the interaction of the L1-α1 region with the phosphate backbone. An NMR dynamics study suggests that non-specific binding might set up an intermediate structure which can then rapidly and easily find the consensus region on a long section of genomic DNA in a facilitated binding process. Structural analysis using NMR and molecular dynamics shows that key structural distortions in the Meis1-HD–DNA complex are induced by various single nucleotide mutations in the consensus sequence, resulting in decreased DNA binding affinity. Collectively, our results elucidate the detailed molecular mechanism of how Meis1-HD recognizes single nucleotide mutations within its consensus sequence: (i) through the conformational features of the α3 helix; and (ii) by the dynamic features (rigid or flexible) of the L1 loop and the α3 helix. These findings enhance our understanding of how single nucleotide mutations in transcription factor consensus sequences lead to dysfunctional transcription and, ultimately, human disease.

Genome-wide chromatin occupancy of BRDT and gene expression analysis suggest transcriptional partners and specific epigenetic landscapes that regulate gene expression during spermatogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple-Negative Breast Cancer.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein-coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. IMPLICATIONS: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

Sequencing of lipoprotein lipase gene in the Mediterranean river buffalo identified novel variants affecting gene expression

Lipoprotein lipase (LPL) is a key enzyme for lipid metabolism, playing a fundamental role in the composition of fat in adipose tissue and milk. The LPL gene has been seldom investigated in dairy ruminants and barely studied in river buffalo (Bubalus bubalis). The aim of this work was to explore the genetic diversity of LPL and its promoter and to identify functional mutations, using a combined approach based on sequencing, dual-color electrophoretic mobility shift assay, and quantitative PCR. Thirteen consensus sequences for transcription factors were found in the promoter. Eleven SNP were detected, and the attention was focused on the SNP with potential functional effects: g.-446A>G, because the presence of G created a consensus motif for the transcription factor Sp1, and g.107A>G, which was the only exonic SNP. We developed PCR-RFLP methods for genotyping the 2 SNP and calculated the allele frequencies. A strong linkage disequilibrium (D' = 1; r2 = 0.903) was found between the 2 SNP. The dual-color electrophoretic mobility shift assay demonstrated that only genotype g.-446GG allowed the binding of the Sp1 transcription factor, resulting in overexpression of the gene (~2.5 fold), as confirmed by the quantitative PCR results. Haploinsufficiency is proposed as a regulation mechanism. This study adds further knowledge on the structure of the LPL gene and its expression in river buffalo, with potential effects on milk qualitative and quantitative production.

Establishment of regulatory elements during erythro-megakaryopoiesis identifies hematopoietic lineage-commitment points

BackgroundEnhancers and promoters are cis-acting regulatory elements associated with lineage-specific gene expression. Previous studies showed that different categories of active regulatory elements are in regions of open chromatin, and each category is associated with a specific subset of post-translationally marked histones. These regulatory elements are systematically activated and repressed to promote commitment of hematopoietic stem cells along separate differentiation paths, including the closely related erythrocyte (ERY) and megakaryocyte (MK) lineages. However, the order in which these decisions are made remains unclear.ResultsTo characterize the order of cell fate decisions during hematopoiesis, we collected primary cells from mouse bone marrow and isolated 10 hematopoietic populations to generate transcriptomes and genome-wide maps of chromatin accessibility and histone H3 acetylated at lysine 27 binding (H3K27ac). Principle component analysis of transcriptional and open chromatin profiles demonstrated that cells of the megakaryocyte lineage group closely with multipotent progenitor populations, whereas erythroid cells form a separate group distinct from other populations. Using H3K27ac and open chromatin profiles, we showed that 89% of immature MK (iMK)-specific active regulatory regions are present in the most primitive hematopoietic cells, 46% of which contain active enhancer marks. These candidate active enhancers are enriched for transcription factor binding site motifs for megakaryopoiesis-essential proteins, including ERG and ETS1. In comparison, only 64% of ERY-specific active regulatory regions are present in the most primitive hematopoietic cells, 20% of which containing active enhancer marks. These regions were not enriched for any transcription factor consensus sequences. Incorporation of genome-wide DNA methylation identified significant levels of de novo methylation in iMK, but not ERY.ConclusionsOur results demonstrate that megakaryopoietic profiles are established early in hematopoiesis and are present in the majority of the hematopoietic progenitor population. However, megakaryopoiesis does not constitute a “default” differentiation pathway, as extensive de novo DNA methylation accompanies megakaryopoietic commitment. In contrast, erythropoietic profiles are not established until a later stage of hematopoiesis, and require more dramatic changes to the transcriptional and epigenetic programs. These data provide important insights into lineage commitment and can contribute to ongoing studies related to diseases associated with differentiation defects.

Hepcidin and DNA promoter methylation in hepatocellular carcinoma.

The liver hormone hepcidin regulates iron homoeostasis that is often altered in hepatocellular carcinoma (HCC). Epigenetic phenomena control gene expression through a dynamic fashion; therefore, considering the plasticity of both iron homoeostasis and epigenetic mechanisms and their role in liver carcinogenesis, we investigated whether hepcidin gene (HAMP) expression is modulated by DNA methylation, thus affecting iron status in human HCC. Thirty-two patients affected by nonviral HCC were enrolled, and their main clinical and biochemical characteristics were obtained. Neoplastic and homologous non-neoplastic liver tissues were analysed for HAMP promoter DNA methylation, for HAMP gene expression and for iron content. An invitro demethylation assay with a human hepatocarcinoma cell line was performed to evaluate the role of DNA methylation on HAMP transcriptional repression. Gene expression and DNA methylation analyses on tissues showed that HAMP was transcriptionally repressed in HCC tissues consensually with a promoter hypermethylation. Furthermore, patients with HCC had low serum hepcidin concentrations, and HCC tissues had relative iron depletion as compared to non-neoplastic liver tissues. The cell culture model showed the functional role of DNA hypermethylation by downregulating HAMP gene expression. Through a quantitative methylation analysis on HCC tissues, we then proved that methylation at definite CpG sites within consensus sequences for specific transcription factors is possibly the mechanism underlying HAMP repression. This study highlights a novel role for HAMP downregulation through DNA promoter hypermethylation and emphasises the significance of epigenetics in the regulation of iron metabolism in HCC.

MED12 regulates a transcriptional network of calcium-handling genes in the heart.

The Mediator complex regulates gene transcription by linking basal transcriptional machinery with DNA-bound transcription factors. The activity of the Mediator complex is mainly controlled by a kinase submodule that is composed of 4 proteins, including MED12. Although ubiquitously expressed, Mediator subunits can differentially regulate gene expression in a tissue-specific manner. Here, we report that MED12 is required for normal cardiac function, such that mice with conditional cardiac-specific deletion of MED12 display progressive dilated cardiomyopathy. Loss of MED12 perturbs expression of calcium-handling genes in the heart, consequently altering calcium cycling in cardiomyocytes and disrupting cardiac electrical activity. We identified transcription factors that regulate expression of calcium-handling genes that are downregulated in the heart in the absence of MED12, and we found that MED12 localizes to transcription factor consensus sequences within calcium-handling genes. We showed that MED12 interacts with one such transcription factor, MEF2, in cardiomyocytes and that MED12 and MEF2 co-occupy promoters of calcium-handling genes. Furthermore, we demonstrated that MED12 enhances MEF2 transcriptional activity and that overexpression of both increases expression of calcium-handling genes in cardiomyocytes. Our data support a role for MED12 as a coordinator of transcription through MEF2 and other transcription factors. We conclude that MED12 is a regulator of a network of calcium-handling genes, consequently mediating contractility in the mammalian heart.

Bioinformatics review of alcohol exposed neuronal stem cells and transcription factor consensus sequences in cell cycle progression genes.

Previous studies from microarray analysis of neuronal stem cells exposed to moderate levels of alcohol suggested that alterations in proliferation and induced apoptosis may well be associated with a distinct set of transcription factors and associated consensus sequences in key gene sets responsible for G1/S phase progression and DNA synthesis. The objective of the study was to gather existing information on the Fox1, E2F1, DP-1, DP-2, DP-3, AP-1, and SP-1 transcription factors and examine their interaction with the promoter regions of genes involved in cell cycle progression through a bioinformatics approach. To complete this review, data was compiled using the Unipro UGENE program. Using this program, the promoter regions of genes of interest were aligned with the known DNA consensus sequences targeted by the transcription factors. Based on the alignments – or lack thereof – conclusions were drawn as to which transcription factors exert influence on controlling proper cell cycle progression. Data showed interactions among several of the transcription factors with the multiple regions in the promoter regions of almost all the examined genes. Based on the discovered consensus sequences and their influence on the expression rates of genes necessary for cell cycle progression, this study provides plausible evidence of transcription factors worth targeting for controlling specific genes in cell cycle progression.

TERT Promoter Mutations Enhance Telomerase Activation by Long-Range Chromatin Interactions.

Although single-nucleotide somatic mutations in the proximal promoter of the human telomerase reverse transcriptase (TERT) gene create novel consensus sequences for transcription factors that enhance TERT expression, the precise mechanism of how telomerase is reactivated in cancer cells remains poorly understood. In this issue, Akıncılar and colleagues identify a potential mechanism of TERT reactivation that is mediated by a novel long-range chromatin interaction between the TERT promoter on chromosome 5p and a 300-kb upstream region. This permits recruitment of the transcription factor GABPA in mutant TERT promoters but not in wild-type promoters. Cancer Discov; 6(11); 1212-4. ©2016 AACRSee related article by Akıncılar and colleagues, p. 1276.

Abstract 4779: In silico design, synthesis and evaluation of a new family of C1-substituted pyrrolobenzodiazepines (PBDs)

Abstract The pyrrolobenzodiazepines (PBDs) are a class of naturally occurring and synthetic DNA minor-groove binding agents. They covalently bind to the C2-amino group of guanines, and can form mono-alkylated adducts and cross-links depending on the structure of the molecule. PBDs can be extremely cytotoxic, and there are reports of IC50 values in cell lines in the high femtomolar ranges. Synthetic A-ring-linked PBD dimers have found use as payloads in antibody drug conjugates (ADCs) due to their potent cytoxicity, and a number of ADCs are now in clinical trials. The C-ring of the PBD structure has been under-exploited as a means to improve cytotoxicity, and for joining PBD units together to create novel dimers. This lack of interest in the C-ring may have been due to literature reports that related C2-linked PBD dimers are poorly DNA interactive and cytotoxic. Using our proprietary molecular dynamics (MD) protocols, we undertook a series of MD simulations to establish a rationale for the poor DNA-binding affinity of C2-linked PBD dimers. We were able to determine a number of characteristics preventing efficient DNA-interaction including (a) their shape does not follow the curvature of the DNA minor groove, (b) if a molecule is attached to DNA via one imine moiety, the other imine moiety is not oriented in an appropriate position to effect the second covalent cross-linking event, (c) if a cross-linked model is produced, significant DNA distortion is observed, suggesting that rapid repair would occur in cells, perhaps contributing to the low cytotoxicity. During this study, our models and simulations suggested that, after covalent adduct formation, C1-substituents should extend along the minor groove in an appropriate orientation to form extensive van der Waals interactions with functional groups on the minor groove floor. This was reflected in free energy of binding calculations. For example, in the case of one C2-linked dimer, calculations suggested a weak binding affinity (i.e., -42.74 kcal/mol) with the DNA sequence 5’-GCGATACTCGC-3’, whereas under identical experimental conditions, an equivalent C1-linked dimer had a much higher binding affinity (i.e., -50.42 kcal/mol). Based on these promising in silico data, a small library of C1-PBD molecules was synthesised, and biophysical results indicated that they possessed significant DNA-binding affinity. HPLC-MS experiments using DNA sequences based on the STAT3 and NF-ÊB transcription factor consensus sequences showed that some library members converted up to 60% of the DNA to covalent adducts within 3 hours. Furthermore, in preliminary experiments in cell lines such as MDA-MB231, the same molecules were highly cytotoxic with IC50 values in the nanomolar region (e.g., 36 nM after 72 hour incubation for one compound). Studies are now underway to further functionalise the C1-position of PBDs to enhance cytotoxicity, and to assess their viability as novel payloads for use in ADCs. Citation Format: Paul J. M. Jackson, George Procopiou, Nicolas Veillard, Julia Mantaj, K Miraz Rahman, David E. Thurston. In silico design, synthesis and evaluation of a new family of C1-substituted pyrrolobenzodiazepines (PBDs). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4779.

Reactivation of telomerase in cancer.

Activation of telomerase is a critical step in the development of about 85 % of human cancers. Levels of Tert, which encodes the reverse transcriptase subunit of telomerase, are limiting in normal somatic cells. Tert is subjected to transcriptional, post-transcriptional and epigenetic regulation, but the precise mechanism of how telomerase is re-activated in cancer cells is poorly understood. Reactivation of the Tert promoter involves multiple changes which evolve during cancer progression including mutations and chromosomal re-arrangements. Newly described non-coding mutations in the Tert promoter region of many cancer cells (19 %) in two key positions, C250T and C228T, have added another layer of complexity to telomerase reactivation. These mutations create novel consensus sequences for transcription factors which can enhance Tert expression. In this review, we will discuss gene structure and function of Tert and provide insights into the mechanisms of Tert reactivation in cancers, highlighting the contribution of recently identified Tert promoter mutations.

Activation of Six1 Expression in Vertebrate Sensory Neurons.

SIX1 homeodomain protein is one of the essential key regulators of sensory organ development. Six1-deficient mice lack the olfactory epithelium, vomeronasal organs, cochlea, vestibule and vestibuloacoustic ganglion, and also show poor neural differentiation in the distal part of the cranial ganglia. Simultaneous loss of both Six1 and Six4 leads to additional abnormalities such as small trigeminal ganglion and abnormal dorsal root ganglia (DRG). The aim of this study was to understand the molecular mechanism that controls Six1 expression in sensory organs, particularly in the trigeminal ganglion and DRG. To this end, we focused on the sensory ganglia-specific Six1 enhancer (Six1-8) conserved between chick and mouse. In vivo reporter assays using both animals identified an important core region comprising binding consensus sequences for several transcription factors including nuclear hormone receptors, TCF/LEF, SMAD, POU homeodomain and basic-helix-loop-helix proteins. The results provided information on upstream factors and signals potentially relevant to Six1 regulation in sensory neurons. We also report the establishment of a new transgenic mouse line (mSix1-8-NLSCre) that expresses Cre recombinase under the control of mouse Six1-8. Cre-mediated recombination was detected specifically in ISL1/2-positive sensory neurons of Six1-positive cranial sensory ganglia and DRG. The unique features of the mSix1-8-NLSCre line are the absence of Cre-mediated recombination in SOX10-positive glial cells and central nervous system and ability to induce recombination in a subset of neurons derived from the olfactory placode/epithelium. This mouse model can be potentially used to advance research on sensory development.

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself. The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.

Abstract 11697: Hyperhomocysteinemia Induces CD40 + Monocyte and Inflammatory Monocyte in Chronic Kidney Disease Subjects via DNA Hypomethylation-Related Mechanism

Hyperhomocysteinenia (HHcy) is associated with chronic kidney disease (CKD) which has increased cardiovascular disease (CVD) mortality and mobility. Elevated inflammatory monocyte (inf. MC) is a cellular hallmark of chronic inflammation which contributes to the burden CVD. We previously reported that HHcy induces inf. MC differentiation in mice and DNA hypomethylation in vascular cells. We assesses whether HHcy causes MC differentiation in CKD and the underlying mechanism. Degree of CKD was determined by plasma creatinine from patients with peripheral vascular disease (VD). Estimated glomerular filtration rate was calculated with modification in age, race and gender. (VD, n=13; VD with CKD, n=13; Healthy donor without evidence of VD and CKD, n=13). Blood cells were assessed for phenotypic characterization by flow cytometry. We found that plasma Hcy levels are elevated in VD and CKD. CD14++CD16+ inf. MC are increased in VD subjects and mild HHcy(>15μM). Plasma Hcy levels are positively correlated with inf. MC, CD40, TNF receptor family 5. We identify that CKD patient serum and Hcy (50μM) treatment increased CD40 in purified human blood MC by RT-PCR. Hcy metabolites, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), are increased in CKD subjects. SAM/SAH ratio, an indicator of methylation status, is reduced and is negatively correlated with Hcy, inf. MC, and CD40+ inf. MC. Hcy induced MC-origin inflammatory cytokines IL-6 mRNA in MC isolated from healthy donor by RT-PCR, and potentiated inflammatory cytokine IL-6, TNFα, and IFN[[Unable to Display Character: ɤ]]-induced CD40 expression in cultured PBMCs. Hcy suppressed CD40 transcription and reduced DNA methyltransferase 1 protein levels in cultured human MC. We identified four DNA hypomethylation CpG dinucleotides at p65 and PU.1 transcription factor consensus sequences on CD40 promoter in white blood cells isolated from CKD patients by Bisulfite pyrosequencing. Finally, CD40L levels are positively correlated with plasma Hcy and inf. MC. CD40 ligation by CD40L treatment (0.4μg/l) in peripheral blood mononuclear cells (PBMC) induced inf. MC differentiation. We conclude that HHcy potentiates IFN[[Unable to Display Character: ɤ]]-mediated CD40 expression via CD40 DNA hypomethylation in CKD and promotes CD40-CD40L mediated inf. MC differentiation.

A new haplotype variability in complement C6 is marginally associated with resistance to Aeromonas hydrophila in grass carp

Aeromonas hydrophila, a widespread bacterium in the aquatic environment, causes haemorrhagic septicemia in fish. In the last decade, the disease has caused mass mortality and tremendous economic loss in cultured grass carp in the mainland China. The complement component C6 is a constituent of a biochemical cascade that serves as a major effector of the human innate and adaptor immunity, and eliminates infected cells. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the C6 gene and to assess their association with A. hydrophila resistance in grass carp. A resource population consisting of 186 susceptible and 191 resistant grass carp was constructed. The gcC6 genomic sequence is composed of 9292 bp, containing 18 exons and 17 introns. The promoter sequence of gcC6 gene contained several consensus sequences for hepatic-specific transcription factors. We sequenced a total of 9744 bp of the C6 gene from a diverse population of grass carp and identified 8 SNPs that were genotyped in the resource population. Statistical analysis revealed a lack of association between any individual SNPs and resistance to A. hydrophila in grass carp. The SNPs 1214G>A, 1380G>C, 2095A>C and 2167T>C were linked together (r2 > 0.8). The haplotype GCCC generated with these four SNPs was associated marginally with resistance to A. hydrophila in grass carp. These findings suggest a lack of strong association of the C6 polymorphisms with the A. hydrophila resistance in grass carp.

Insulin-Like 3 Exposure of the Fetal Rat Gubernaculum Modulates Expression of Genes Involved in Neural Pathways1

Insulin-like 3 (INSL3) signaling directs fetal gubernacular development and testis descent, but the actions of INSL3 in the gubernaculum are poorly understood. Using microarray gene expression profiling of fetal male rat gubernaculum explants exposed to 10 or 100 nM INSL3, significant changes in expression were identified for approximately 900 genes. Several of the genes showing the largest inductions regulate neuronal development or activity, including Pnoc (34-fold), Nptx2 (9-fold), Nfasc (4-fold), Gfra3 (3-fold), Unc5d (3-fold), and Crlf1 (3-fold). Bioinformatics analysis revealed BMP and WNT signaling pathways and several gene ontologies related to neurogenesis were altered by INSL3. Promoter response elements significantly enriched in the INSL3-regulated gene list included consensus sequences for MYB, REL, ATF2, and TEF transcription factors. Comparing in vivo gene expression profiles of male and female rat fetal gubernaculum showed expression of the Bmp, Wnt, and neurodevelopmental genes induced by INSL3 was higher in males. Using quantitative RT-PCR, the microarray data were confirmed, and the induction of Bmp3, Chrdl2, Crlf1, Nptx2, Pnoc, Wnt4, and Wnt5a mRNA levels were examined over a range of INSL3 concentrations (0.1-100 nM) in male and female gubernaculum. In both sexes, an increasing gene expression response was observed between 0.1 and 10 nM INSL3. These data suggest that INSL3 signaling in the fetal gubernaculum induces morphogenetic programs, including BMP and WNT signaling, and support other rodent data suggesting a role for these pathways in development of the gubernaculum.

Differentiated Evolutionary Conservatism and Lack of Polymorphism of Crucial Sex Determination Genes (SRY and SOX9) in Four Species of the Family Canidae

The sex determination process is under the control of several genes of which two (SRY and SOX9), encoding transcription factors, play a crucial role. It is well-known that mutations at these genes may cause the development of an intersexual phenotype. The aim of this study was to conduct a comparative analysis of the coding sequence and 5'-flanking regions of both genes in four species of the family Canidae (the dog, red fox, arctic fox and Chinese raccoon dog). Similarity of the coding sequence of the SOX9 gene among the studied species was higher (99.7-99.9%) than in the case of the SRY gene (96.7-97.3%). Only single nucleotide changes were found in the compared coding sequences, whereas in the 5'-flanking region of both genes nucleotide substitutions, as well as insertions and deletions were observed. None of the changes detected in the 5'-flanking region occurred within the potential consensus sequences for transcription factors. No polymorphism was found for either of these genes in any of the analyzed species.

Induction of endogenous γ-globin gene expression with decoy oligonucleotide targeting Oct-1 transcription factor consensus sequence

Human β-globin disorders are relatively common genetic diseases cause by mutations in the β-globin gene. Increasing the expression of the γ-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the γ-globin gene. The human γ-globin genes (both Aγ and Gγ-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing γ-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the γ-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, α2γ2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous γ-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the γ-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the γ-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and β-thalassemia.

Acute endotoxemia is associated with upregulation of lipocalin 24p3/Lcn2 in lung and liver

Acute endotoxemia is associated with production of acute phase proteins which regulate inflammatory responses to tissue injury. Consistent with DNA microarray experiments, we found that acute endotoxemia, induced by administration of lipopolysaccharide (LPS) to mice (1 mg/kg) or rats (5 mg/kg), resulted in increased expression of the hepatic acute phase protein, lipocalin 24p3, which was evident within 4 h and persisted for 24–48 h. Increases in 24p3 expression were also observed in the lung after LPS administration, as well as in isolated liver and lung macrophages, and Type II alveolar epithelial cells. The actions of LPS are dependent, in part, on Toll-like receptor (TLR) proteins. Macrophages from C3H/HeJ mice, which possess a nonfunctional TLR-4, expressed low levels of 24p3 mRNA when compared to cells from control C3H/OuJ mice. Whereas LPS administration increased 24p3 expression in lung and liver macrophages from control C3H/OuJ mice, minimal effects were observed in TLR-4 mutant mice demonstrating that TLR-4 is important in regulating 24p3 expression during acute endotoxemia. Promoters for genes encoding lipocalin proteins including 24p3 contain consensus sequences for transcription factors including NF-κB, and C/EBP. Acute endotoxemia resulted in NF-κB nuclear binding activity in both alveolar macrophages and Type II cells. In contrast, C/EBP activation was evident only in Type II cells, suggesting differential effects of LPS on these cell types. These data suggest that the acute phase response to acute endotoxemia involves induction of 24p3 in both the lung and liver. This protein may be important in restoring tissue homeostasis following LPS-induced injury.

Retrospective Analysis of Coagulation Factor II Receptor ( F2R ) Sequence Variation and Coronary Heart Disease in Hypertensive Patients

The purpose of this study was to evaluate the role of genetic variants within the coagulation factor II receptor (F2R) in the occurrence of coronary heart disease (CHD). Four SNPs (-1738 G/A, 2860 G/A, 2930 T/C, and 9113 C/A) and an ins/del polymorphism -506-/GGCCGCGGGAAGC (D/I), replicating a consensus sequence for Ets-1 transcription factor, and their related haplotypes were tested for association to CHD in 1600 hypertensive patients divided in 2 groups according to presence (cases, n=559) and absence (controls, n=1041) of CHD. Allele I at -506 locus was associated with increased risk of CHD under additive, dominant, and recessive models of inheritance (all P<0.01). Three haplotypes carrying I allele were consistently associated with an increased risk of CHD (all P<0.05). Patients homozygous for the C allele at the 2930 locus also showed an increased risk of CHD (P<0.05). To test the functionality of -506 locus, nuclear extracts were incubated with -506D and -506I sequences by EMSA and F2R promoter activity (F2R-A) were assessed in HUVECs transfected with vectors carrying -506D and -506I sequences and exposed to hypoxia. Presence of the -506I sequence was associated with a 26% reduction of affinity binding to nuclear proteins and to blunted F2R-A in response to hypoxia as compared with the -506D sequence (all P<0.05). F2R genetic variants may influence the natural history of CHD in patients at high risk of cardiovascular events.