The Role of PTEN in Chemoresistance Mediated by the HIF-1α/YY1 Axis in Pediatric Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Current chemotherapy treatment regimens have improved survival rates to approximately 80%; however, resistance development remains the primary cause of treatment failure, affecting around 20% of cases. Some studies indicate that loss of the phosphatase and tensin homolog (PTEN) leads to deregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, increasing the expression of proteins involved in chemoresistance. PTEN loss results in deregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces hypoxia-inducible factor 1-alpha (HIF-1α) expression in various cancers. Additionally, it triggers upregulation of the Yin Yang 1 (YY1) transcription factor, leading to chemoresistance mediated by glycoprotein p-170 (Gp-170). The aim of this study was to investigate the role of the PTEN/NF-κB axis in YY1 regulation via HIF-1α and its involvement in ALL. A PTEN inhibitor was administered in RS4;11 cells, followed by the evaluation of PTEN, NF-κB, HIF-1α, YY1, and Gp-170 expression, along with chemoresistance assessment. PTEN, HIF-1α, and YY1 expression levels were assessed in the peripheral blood mononuclear cells (PBMC) from pediatric ALL patients. The results reveal that the inhibition of PTEN activity significantly increases the expression of pAkt and NF-κB, which is consistent with the increase in the expression of HIF-1α and YY1 in RS4;11 cells. In turn, this inhibition increases the expression of the glycoprotein Gp-170, affecting doxorubicin accumulation in the cells treated with the inhibitor. Samples from pediatric ALL patients exhibit PTEN expression and higher HIF-1α and YY1 expression compared to controls. PTEN/Akt/NF-κB axis plays a critical role in the regulation of YY1 through HIF-1α, and this mechanism contributes to Gp-170-mediated chemoresistance in pediatric ALL.

Binding of YY1/CREB to an Enhancer Region Triggers Claudin 6 Expression in H. pylori LPS-Stimulated AGS Cells.

Aberrant expression of the tight junction protein claudin 6 (CLDN6) is a hallmark of gastric cancer progression. Its expression is regulated by the cAMP response element-binding protein (CREB). In gastric cancer induced by Helicobacter pylori (H. pylori) there is no information regarding what transcription factors induce/upregulate the expression of CLDN6. We aimed to identify whether CREB and Yin Yang1 (YY1) regulate the expression of CLDN6 and the site where they bind to the promoter sequence. Bioinformatics analysis, H. pylori lipopolysaccharide (LPS), YY1 and CREB silencing, Western blot, luciferase assays, and chromatin immunoprecipitation experiments were performed using the stomach gastric adenocarcinoma cell line AGS. A gen reporter assay suggested that the initial 2000 bp contains the regulatory sequence associated with CLDN6 transcription; the luciferase assay demonstrated three different regions with transcriptional activity, but the -901 to -1421 bp region displayed the maximal transcriptional activity in response to LPS. Fragment 1279-1421 showed CREB and, surprisingly, YY1 occupancy. Sequential Chromatin Immunoprecipitation (ChIP) experiments confirmed that YY1 and CREB interact in the 1279-1421 region. Our results suggest that CLDN6 expression is regulated by the binding of YY1 and CREB in the 901-1421 enhancer, in which a non-described interaction of YY1 with CREB was established in the 1279-1421 region.

A de novo YY1 missense variant expanding the Gabriele-de Vries syndrome phenotype and affecting X-chromosome inactivation.

Yin and Yang 1 gene (YY1; MIM#600,013) is recognized as a dual transcriptional activating and repressing factor, RNA-binding protein, and 3D chromatin regulator, with multi roles in neurodevelopmental and maintenance pathways. YY1 haploinsufficiency caused either by heterozygous sequence variants or deletions involving the whole gene has been recently associated with Gabriele-de Vries syndrome (GADEVS), a rare congenital autosomal dominant condition, leading to intellectual disability (ID) and multiple physical/behavioural abnormalities. Herein, we describe clinical and molecular findings from a Brazilian female harbouring a de novo missense pathogenic variant in YY1 gene (NM_003403.5:c.1106A > G; p.Asn369Ser) found by whole exome sequencing with potential implications for protein structure and function. Undescribed or uncommon clinical features in this patient included non-febrile seizures, severe scoliosis, hearing impairment, and chorioretinitis. Further bioinformatics analyses using YY1-other protein interaction networks reinforced the involvement of YY1 interactors in such phenotypes, in exception of chorioretinitis. Moreover, X-chromosome inactivation (XCI) skewing was evidenced in the patient and attributed to the haploinsufficiency of YY1, which direct and indirectly interacts with numerous XCI key regulators. Besides expanding the mutational and phenotype spectrum of GADEVS, our results highlight the role of YY1 as an essential autosomal regulator of XCI epigenetic process.

Transcriptional Regulation of Yin-Yang 1 Expression through the Hypoxia Inducible Factor-1 in Pediatric Acute Lymphoblastic Leukemia.

Yin-Yang transcription factor 1 (YY1) is involved in tumor progression, metastasis and has been shown to be elevated in different cancers, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood. Bioinformatics analysis reveal three Hypoxia-inducible factor 1-alpha (HIF-1α) putative binding sites in the YY1 promoter region. The regulation of YY1 by HIF-1α in leukemia was analyzed. Mutation of the putative YY1 binding sites in a reporter system containing the HIF-1α promoter region and CHIP analysis confirmed that these sites are important for YY1 regulation. Leukemia cell lines showed that both proteins HIF-1α and YY1 are co-expressed under hypoxia. In addition, the expression of mRNA of YY1 was increased after 3 h of hypoxia conditions and affect several target genes expression. In contrast, chemical inhibition of HIF-1α induces downregulation of YY1 and sensitizes cells to chemotherapeutic drugs. The clinical implications of HIF-1α in the regulation of YY1 were investigated by evaluation of expression of HIF-1α and YY1 in 108 peripheral blood samples and by RT-PCR in 46 bone marrow samples of patients with pediatric acute lymphoblastic leukemia (ALL). We found that the expression of HIF-1α positively correlates with YY1 expression in those patients. This is consistent with bioinformatic analyses of several databases. Our findings demonstrate for the first time that YY1 can be transcriptionally regulated by HIF-1α, and a correlation between HIF-1α expression and YY1 was found in ALL clinical samples. Hence, HIF-1α and YY1 may be possible therapeutic target and/or biomarkers of ALL.

Expression of YY1 in pro-B and T phenotypes correlation with poor survival in pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, constituting 80% of all acute leukemias in minors. Despite the increase in the success of therapies, disease-free survival is over 80% in most cases. For the remaining 20% of patients, new strategies are needed to allow us to know and select those at greatest risk of relapse. We evaluated by immunohistochemistry the expression of the transcription factor YY1 and found that it is overexpressed in peripheral blood leukemia cells of pediatric patients with ALL with Pro-B and T phenotype compared to control samples. Over expression of YY1 was associated with a significantly lower chance of survival. We also evaluated by RT-PCR in bone marrow samples from ALL pediatric patients the association of YY1 expression with the percentage of blasts. High levels of YY1 were present in samples with higher percent of blasts in these patients. In addition, ALL pediatric patients with a poor response to therapy had higher levels at the nuclear level of YY1 than those who responded well to chemotherapy. In conclusion, our data suggest that YY1 could serve in pediatric ALL as markers of evolution and response for this disease, mainly in patients with pro-B and T immunophenotype. It is also suggested that YY1 is implicated in the expanse of blast in these patients.

Roles Played by YY1 in Embryonic, Adult and Cancer Stem Cells.

Accumulating evidence strongly indicates that the presence of cancer stem cells (CSCs) leads to the emergence of worse clinical scenarios, such as chemo- and radiotherapy resistance, metastasis, and cancer recurrence. CSCs are a highly tumorigenic population characterized by self-renewal capacity and differentiation potential. Thus, CSCs establish a hierarchical intratumor organization that enables tumor adaptation to evade the immune response and resist anticancer therapy. YY1 functions as a transcription factor, RNA-binding protein, and 3D chromatin regulator. Thus, YY1 has multiple effects and regulates several molecular processes. Emerging evidence indicates that the development of lethal YY1-mediated cancer phenotypes is associated with the presence of or enrichment in cancer stem-like cells. Therefore, it is necessary to investigate whether and to what extent YY1 regulates the CSC phenotype. Since CSCs mirror the phenotypic behavior of stem cells, we initially describe the roles played by YY1 in embryonic and adult stem cells. Next, we scrutinize evidence supporting the contributions of YY1 in CSCs from a number of various cancer types. Finally, we identify new areas for further investigation into the YY1-CSCs axis, including the participation of YY1 in the CSC niche.

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion / YY1 induce la disminución de la apoptosis a través de Fas en la leucemia linfoblástica aguda en condiciones hipóxicas: implicaciones en la evasión de la respuesta inmunitaria

Abstract Background: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL. Methods: Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson’s correlation. Results: Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas. Conclusions: These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis.

Resumen Introducción: La leucemia linfoblástica aguda (LLA) es una enfermedad con alta prevalencia en la población pediátrica. El mecanismo por el cual el receptor de Fas participa en la regulación inmunitaria en los tumores es desconocido, pero se sabe que está subexpresado en LLA. El factor de transcripción Ying-Yang-1 (YY1) está involucrado en la resistencia a la apoptosis y la progresión tumoral; se encuentra aumentado en diferentes tumores, incluida la LLA. Aunque los mecanismos que regulan la expresión de YY1 en LLA son desconocidos, se sabe que YY1 regula la expresión del receptor de Fas. El objetivo de este trabajo fue evaluar el efecto de YY1 en la expresión de Fas en condiciones de hipoxia en la LLA. Métodos: Se cultivaron células RS4;11 en condiciones de hipoxia y se analizó la expresión de YY1, receptor de Fas y HIF-1α. La apoptosis fue inducida usando un agonista de Fas (DX2) y se analizó con la detección de caspasa 3 activa. Los datos se analizaron mediante correlación de Pearson. Resultados: Las células RS4;11 coexpresaron HIF-1αy YY1 en hipoxia, lo cual correlaciona con una baja expresión de Fas. La apoptosis se encontró disminuida durante condiciones de hipoxia, después del tratamiento con DX2. El análisis bioinformático mostró que los pacientes con altos niveles de HIF-1αpresentan YY1 elevado y bajos niveles del receptor de Fas. Conclusiones: Estos resultados sugieren que YY1 regula negativamente la expresión del receptor de Fas, lo cual podría estar involucrado en el escape de las células leucémicas a la respuesta inmunitaria, contribuyendo a la patogénesis de la LLA.

Yin-Yang-1 decreases Fas-induced apoptosis in acute lymphoblastic leukemia under hypoxic conditions: its implications in immune evasion.

Background: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL. Methods: Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson's correlation. Results: Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas. Conclusions: These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis.

Introducción: La leucemia linfoblástica aguda (LLA) es una enfermedad con alta prevalencia en la población pediátrica. El mecanismo por el cual el receptor de Fas participa en la regulación inmunitaria en los tumores es desconocido, pero se sabe que está subexpresado en LLA. El factor de transcripción Ying-Yang-1 (YY1) está involucrado en la resistencia a la apoptosis y la progresión tumoral; se encuentra aumentado en diferentes tumores, incluida la LLA. Aunque los mecanismos que regulan la expresión de YY1 en LLA son desconocidos, se sabe que YY1 regula la expresión del receptor de Fas. El objetivo de este trabajo fue evaluar el efecto de YY1 en la expresión de Fas en condiciones de hipoxia en la LLA. Métodos: Se cultivaron células RS4;11 en condiciones de hipoxia y se analizó la expresión de YY1, receptor de Fas y HIF-1α. La apoptosis fue inducida usando un agonista de Fas (DX2) y se analizó con la detección de caspasa 3 activa. Los datos se analizaron mediante correlación de Pearson. Resultados: Las células RS4;11 coexpresaron HIF-1αy YY1 en hipoxia, lo cual correlaciona con una baja expresión de Fas. La apoptosis se encontró disminuida durante condiciones de hipoxia, después del tratamiento con DX2. El análisis bioinformático mostró que los pacientes con altos niveles de HIF-1αpresentan YY1 elevado y bajos niveles del receptor de Fas. Conclusiones: Estos resultados sugieren que YY1 regula negativamente la expresión del receptor de Fas, lo cual podría estar involucrado en el escape de las células leucémicas a la respuesta inmunitaria, contribuyendo a la patogénesis de la LLA.

Expression of YY1 in Wilms tumors with favorable histology is a risk factor for adverse outcomes.

AIM: To investigate the role of the transcription factor YY1 in Wilms tumor (WT). PATIENTS & METHODS: We measured YY1 expression using tissue microarray from patients with pediatric renal tumors, mainly WT and evaluated correlations with the predicted clinical evolution. YY1 expression was measured using immunohistochemical and protein expression was determined by digital pathology. RESULTS & CONCLUSION: YY1 significantly increased in WT patients. In addition, an increase in YY1 expression had a greater risk of adverse outcomes in WT patients with favorable histology. YY1 expression was higher in the blastemal component of tumors, and high nuclear expression positively correlated with metastasis. YY1 may be considered as a metastasis risk factor in WT.

Cytotoxic effect caspase activation dependent of a genetically engineered fusion protein with a CD154 peptide mimetic (OmpC-CD154p) on B-NHL cell lines is mediated by the inhibition of bcl-6 and YY1 through MAPK p38 activation.

The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.

YY1 negatively regulates the XAF1 gene expression in prostate cancer.

XAF1 is a tumor suppressor gene with low or absent expression in cancer. Since transcriptional reactivation or ectopic-mediated expression of XAF1 inhibits tumor growth, it is of great interest to elucidate the molecular mechanisms leading to XAF1 silencing. YY1 is a transcription factor that acts as a repressor or an activator to modulate several cancer-associated cellular processes. Both YY1 and XAF1 have key roles in prostate cancer (PCa) progression and are associated with worse clinical outcomes. To assess whether YY1 regulates the transcriptional activation of the XAF1 gene, we performed gene-reporter assays coupled with site-directed mutagenesis, which showed that YY1 is able to mediate XAF1 silencing. Concordantly, ChIP-qPCR assays showed that YY1 interacts with the XAF1 promoter in PC3 cells that lacks XAF1 expression. This association was lost after exposure to epigenetic modulators that induce XAF1 expression. Further supporting the YY1's repressive role, we found transcriptional reactivation of the XAF1 gene by YY1 downregulation. As expected by previous reports showing that HDAC1 is needed for YY1-mediated repressive actions, we observed XAF1 re-expression after either inhibition or downregulation of the HDAC1 gene. Finally, expression data retrieved from the TCGA consortium showed that PCa samples presented lower XAF1 and higher HDAC expression levels than normal tissues. Thus, our results support a model in which YY1 is able to silence tumor suppressor genes such as XAF1 through HDAC1 in PCa.

Role of Yin Yang-1 (YY1) in the transcription regulation of the multi-drug resistance (MDR1) gene.

Resistance to chemotherapy hinders the successful treatment of acute lymphoblastic leukemia (ALL). The multi-drug resistance-1 (MDR1/ABCB1) gene encodes P-glycoprotein (P-gp), which plays an important role in chemoresistance; however, its transcriptional regulation remains unclear. We investigated the role of YY1 in the regulation of MDR1 and its relation to ALL outcomes. Analysis of the MDR1 promoter revealed four putative YY1-binding sites, which we analyzed using a reporter system and ChIP analysis. YY1 silencing resulted in the inhibition of MDR1 expression and function. The clinical roles of YY1 and MDR1 expression were evaluated in children with ALL. Expression of both proteins was increased in ALL patients compared to controls. We identified a positive correlation between YY1 and MDR1 expression. High levels of YY1 were associated with decreased overall survival. Our results demonstrated that YY1 regulates the transcription of MDR1. Therefore, YY1 may serve as a useful prognostic and/or therapeutic target.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis.

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick­end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers.

Association between nuclear expression of retinoic acid receptor alpha and beta and clinicopathological features and prognosis of advanced non-small cell lung cancer.

BACKGROUND: Transcription factors such as retinoic acid receptor alpha (RARα) and beta (RARß) and Yin Yang 1 (YY1) are associated with the progression of non-small cell lung cancer (NSCLC). In particular, a lack of RARß expression is associated with NSCLC development. The aim of this study was to analyze the expression of RARα, RARß and YY1 and their relationship with prognosis in patients with advanced NSCLC. METHODS: The expression of RARα, RARß and YY1 was assessed by immunohistochemistry and quantitative computerized image software. RESULTS: Eighty-five patients treated with platinum-based chemotherapy were included in the analysis. The mean and standard deviation of the nuclear expression of RARα, RARß and YY1 were 184.5 ± 124.4, 18 ± 27 and 16.6 ± 20.5, respectively. The nuclear expression of RARß was associated with the nuclear expression of YY1 (R 2 = 0.28; p value < 0.0001). Patients with high nuclear expression of YY1 were likely to be non-smokers (61.9 vs 40.5 %). Median progression-free survival (PFS) was 5.9 months (3.48-8.28). Low expression of RARα was independently associated with worse PFS following chemotherapy (10.3 vs 5.46 months p = 0.040). Median overall survival (OS) was 15.6 months (4.5-26.7), and lower nuclear expression of RARß was independently associated with shorter OS (27.5 vs 8.7 months; p = 0.037). CONCLUSION: Our study suggests that the loss of RARs is associated with a worse prognosis and these receptors could be a potential molecular target for NSCLC.

A novel role of Yin-Yang-1 in pulmonary tuberculosis through the regulation of the chemokine CCL4.

Mycobacterium tuberculosis (M. tb) is the etiological agent of pulmonary tuberculosis (TB); this disease remains a worldwide health problem. Yin-Yang-1 (YY1) plays a major role in the maintenance and progression of some pulmonary diseases, including pulmonary fibrosis. However, the role of YY1 in TB remains unknown. The aim of this study was to elucidate the role of YY1 in the regulation of CCL4 and its implication in TB. We determined whether YY1 regulates CCL4 using reporter plasmids, ChIP and siRNA assays. Immunohistochemistry and digital pathology were used to measure the expression of YY1 and CCL4 in a mouse model of TB. A retrospective comparison of patients with TB and control subjects was used to measure the expression of YY1 and CCL4 using tissue microarrays. Our results showed that YY1 regulates the transcription of CCL4; moreover, YY1, CCL4 and TGF-ß were overexpressed in the lung tissues of mice with TB during the late stages of the disease and the tissues of TB patients. The expression of CCL4 and TGF-ß correlated with YY1 expression. In conclusion, YY1 regulates CCL4 transcription; moreover, YY1 is overexpressed in experimental and human TB and is positively correlated with CCL4 and TGF-ß expression. Therefore, treatments that decrease YY1 expression may be a new therapeutic strategy against TB.

Death receptor 5 expression is inversely correlated with prostate cancer progression.

Prostate carcinoma (PCa) is one of the most common cancers in men. Prostate-specific antigen (PSA) has been widely used to predict the outcome of PCa and screening with PSA has resulted in a decline in mortality. However, PSA is not an optimal prognostic tool as its sensitivity may be too low to reduce morbidity and mortality. Consequently, there is a demand for additional robust biomarkers for prostate cancer. Death receptor 5 (DR5) has been implicated in the prognosis of several cancers and it has been previously shown that it is negatively regulated by Yin Yang 1 (YY1) in prostate cancer cell lines. The present study investigated the clinical significance of DR5 expression in a prostate cancer patient cohort and its correlation with YY1 expression. Immunohistochemical analysis of protein expression distribution was performed using tissue microarray constructs from 54 primary PCa and 39 prostatic intraepithelial neoplasia (PIN) specimens. DR5 expression was dramatically reduced as a function of higher tumor grade. By contrast, YY1 expression was elevated in PCa tumors as compared with that in PIN, and was increased with higher tumor grade. DR5 had an inverse correlation with YY1 expression. Bioinformatic analyses corroborated these data. The present findings suggested that DR5 and YY1 expression levels may serve as progression biomarkers for prostate cancer.

Sequential chromatin immunoprecipitation protocol: ChIP-reChIP.

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.

Valproate-dependent transcriptional regulation of GLAST/EAAT1 expression: involvement of Ying-Yang 1.

Valproate, a widely used anti-epileptic drug also employed in the treatment of neurological diseases such as bipolar disorder and migraine, regulates the glutamatergic and GABAergic systems, although its effects in cell physiology have not been thoroughly characterized. High concentrations of glutamate reached during abnormal neurotransmission if not removed properly, become neurotoxic. Glutamate clearance is carried out by high affinity Na(+)-dependent glutamate transporter systems. The glutamate/aspartate transporter GLAST/EAAT1 plays the major role in glutamate removal and is regulated at different levels: transcription, post-translational modifications and cytoplasmic trafficking. The aim of this work was to gain insight into a plausible effect of valproate in GLAST function. Using cultured Bergmann glia cells from chick cerebellum we demonstrate here that valproate exposure elicits a dual regulatory effect on GLAST. In the short-term, valproate increases its Na(+)-dependent [(3)H]-d-aspartate uptake activity in a cytochalasin B-sensitive manner. Interestingly, a synergism between valproate and a histone deacetylase inhibitor was observed. Long-term valproate treatment up-regulates chglast promoter activity, GLAST mRNA levels, GLAST molecules at the plasma membrane and its uptake activity. Furthermore, valproate induces histone 3 lysine 14 acetylation and regulates Ying-Yang 1 (YY1) transcriptional repression on the chglast promoter. These results suggest that valproate elicits its effect through its histone deacetylase inhibitor properties.

Characterization of human collagen XVIII promoter 2: interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes.

Different levels of Collagen XVIII expression have been associated with several pathological processes such as cancer, liver fibrosis, diabetic retinopathy and Alzheimer's disease. Understanding the transcriptional regulation of Collagen XVIII might elucidate some pathways related to the progression of these diseases. The promoter 2 of COL18A1 gene is poorly understood and is responsible for the transcription of this gene in several adult tissues such as liver, eyes and brain. This study focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of SNPs in this region in different ethnic groups. Our results show that there are five conserved regions (I to V) between human and mouse promoter 2 and that the human COL18A1 core promoter is located between nucleotides -186 and -21. Sp1 and Sp3 bind to conserved regions I and V, while Sp3 and YY1 interact with region II. We have verified that the SNP at position -700 (T>G) is embedded in two common haplotypes, which have different frequencies between European and African descendents. The allele -700G increases transcription and binding for a still unknown transcription factor. SNP -700 affects Sp3 and YY1 interaction with this region, even though it is not part of these transcription factors' predicted binding sites. Therefore, our results show for the first time that Sp3 and YY1 interact with human COL18A1 promoter 2, and that nucleotide -700 is part of a binding motif for a still unknown TF that is involved in the expression of this gene in hepatocytes. In addition, we also confirm the involvement of Sp1 in the regulation of this gene.

A functional SNP in the promoter region of TCOF1 is associated with reduced gene expression and YY1 DNA-protein interaction.

Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial malformation caused by null mutations in the TCOF1 gene. High inter and intra familial clinical variability, ranging from mild malar hypoplasia to perinatal death due to airway collapse is observed, but, to date, no genotype-phenotype correlation has been reported. Considering haploinsufficiency as the molecular mechanism underlying the disease, we have hypothesized that mutations in the promoter region of the gene, which has never been previously characterized, in trans with a pathogenic mutation, could modulate the phenotype. Therefore, the aims of the present study were to determine the TCOF1 gene's core promoter and to identify mutations in this region that could contribute to the phenotypic variation observed in this syndrome. We have delimitated the minimal promoter to a region of less than 150 bp, with 63% of identity among 5 different species. We screened 1.2 kbp of the TCOF1 5' flanking sequence in the DNA obtained from 21 patients and 51 controls and identified four new single nucleotide polymorphisms (SNPs), one of which (-346C>T), was proved to be functional, as it decreased the promoter activity by 38%. Electrophoretic mobility shift assay (EMSA) analysis demonstrated that the -346T allele impairs DNA-binding to the YY1 transcription factor. This promoter variant represents a candidate allele to explain the clinical variability in patients bearing TCS.