Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

Role of Pseudomonas aeruginosa PA4157in Quorum Sensing and Iron Transport Regulation

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a pnmary contributing factor responsible for the morbidity and mortality in patients with cystic fibrosis. One of the trademarks of P. aeruginosa is its ability to resist antibiotics. P. aeruginosa does so in part through the LysR-type transcription factor, AmpR. To identify additional members of the AmpR regulon, a new algorithm called iterative enhancement of motifs was used to identify putative AmpR binding sites upstream of open reading frames in the P. aeruginosa genome. The surprising primary hit of this analysis was the promoter of an uncharacterized open reading frame, P A 415 7. P A 415 7 is located upstream ofthefep operon, which is known to be involved in iron acquisition. PA4157 shares high homology to the IclR family of transcriptional regulators which are known to regulate quorum sensing (QS), an elaborate cell-cell communication signaling system that uses quoromones. We postulated two hypotheses: 1) AmpR regulation of QS genes is mediated by PA4157, and 2) PA4157 may be involved in iron acquisition. To address the role of P A 415 7 we generated an in-frame chromosomal deletion of P A 415 7 in P. aeruginosa PA01 (PA0 PA4157). We compared PA0 PA4157 with its parent strain P A0 1 for its ability to produce quoromones using Chromobacterium violaceum as an indicator strain and LasA proteases using Staphylococcus aureus. We also tested its role in virulence using a Caenorhabditis elegans killing assay. Growth in iron-deficient media was also examined to determine if P A4157 has a potential role in iron uptake regulation. Our preliminary results suggest that P A 415 7 is not involved in quorum sensing regulation but does seem to exert a negative regulatory effect on iron uptake in P. aeruginosa P A0 1.

A Case-Only Genome-wide Association Study of Gender- and Age-specific Risk Markers for Childhood Leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.

Caractérisation d'un membre de la famille XKLF dans le développement cardiaque

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models

BACKGROUND: The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. METHODS: Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. RESULTS: Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. CONCLUSIONS: These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression.

Caractérisation d'un membre de la famille XKLF dans le développement cardiaque

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Insights into the multistep transformation of MGUS to myeloma using microarray expression analysis.

To define specific pathways important in the multistep transformation process of normal plasma cells (PCs) to monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM), we have applied microarray analysis to PCs from 5 healthy donors (N), 7 patients with MGUS, and 24 patients with newly diagnosed MM. Unsupervised hierarchical clustering using 125 genes with a large variation across all samples defined 2 groups: N and MGUS/MM. Supervised analysis identified 263 genes differentially expressed between N and MGUS and 380 genes differentially expressed between N and MM, 197 of which were also differentially regulated between N and MGUS. Only 74 genes were differentially expressed between MGUS and MM samples, indicating that the differences between MGUS and MM are smaller than those between N and MM or N and MGUS. Differentially expressed genes included oncogenes/tumor-suppressor genes (LAF4, RB1, and disabled homolog 2), cell-signaling genes (RAS family members, B-cell signaling and NF-kappaB genes), DNA-binding and transcription-factor genes (XBP1, zinc finger proteins, forkhead box, and ring finger proteins), and developmental genes (WNT and SHH pathways). Understanding the molecular pathogenesis of MM by gene expression profiling has demonstrated sequential genetic changes from N to malignant PCs and highlighted important pathways involved in the transformation of MGUS to MM.

Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

MyT1 Counteracts the Neural Progenitor Program to Promote Vertebrate Neurogenesis

The generation of neurons from neural stem cells requires large-scale changes in gene expression that are controlled to a large extent by proneural transcription factors, such as Ascl1. While recent studies have characterized the differentiation genes activated by proneural factors, less is known on the mechanisms that suppress progenitor cell identity. Here, we show that Ascl1 induces the transcription factor MyT1 while promoting neuronal differentiation. We combined functional studies of MyT1 during neurogenesis with the characterization of its transcriptional program. MyT1 binding is associated with repression of gene transcription in neural progenitor cells. It promotes neuronal differentiation by counteracting the inhibitory activity of Notch signaling at multiple levels, targeting the Notch1 receptor and many of its downstream targets. These include regulators of the neural progenitor program, such as Hes1, Sox2, Id3, and Olig1. Thus, Ascl1 suppresses Notch signaling cell-autonomously via MyT1, coupling neuronal differentiation with repression of the progenitor fate.

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.

Early growth response genes 2 and 3 regulate the expression of Bcl6 and differentiation of T follicular helper cells

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GC) and Tfh differentiation requires the function of B cell lymphoma 6 (Bcl6). We have now discovered that early growth response gene (Egr) 2 and 3 directly regulate the expression of Bcl6 in Tfh cells which is required for their function in regulation of GC formation. In the absence of Egr2 and 3, the expression of Bcl6 in Tfh cells is defective leading to impaired differentiation of Tfh cells resulting in a failure to form GCs following virus infection and defects in production of anti-viral antibodies. Enforced expression of Bcl6 in Egr2/3 deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of Egr2/3 which is important for Tfh cell development and Tfh cell mediated B cell immune responses.

A case-only genome-wide association study of gender- and age-specific risk markers for childhood leukemia

Males and age group 1 to 5 years show a much higher risk for childhood acute lymphoblastic leukemia (ALL). We performed a case-only genome-wide association study (GWAS), using the Illumina Infinium HumanCoreExome Chip, to unmask gender- and age-specific risk variants in 240 non-Hispanic white children with ALL recruited at Texas Children’s Cancer Center, Houston, Texas. Besides statistically most significant results, we also considered results that yielded the highest effect sizes. Existing experimental data and bioinformatic predictions were used to complement results, and to examine the biological significance of statistical results. ^ Our study identified novel risk variants for childhood ALL. The SNP, rs4813720 (RASSF2), showed the statistically most significant gender-specific associations (P < 2 x 10-6). Likewise, rs10505918 (SOX5) yielded the lowest P value (P < 1 x 10-5 ) for age-specific associations, and also showed the statistically most significant association with age-at-onset (P < 1 x 10-4). Two SNPs, rs12722042 and 12722039, from the HLA-DQA1 region yielded the highest effect sizes (odds ratio (OR) = 15.7; P = 0.002) for gender-specific results, and the SNP, rs17109582 (OR = 12.5; P = 0.006), showed the highest effect size for age-specific results. Sex chromosome variants did not appear to be involved in gender-specific associations. ^ The HLA-DQA1 SNPs belong to DQA1*01:07and confirmed previously reported male-specific association with DQA1*01:07. Twenty one of the SNPs identified as risk markers for gender- or age-specific associations were located in the transcription factor binding sites and 56 SNPs were non-synonymous variants, likely to alter protein function. Although bioinformatic analysis did not implicate a particular mechanism for gender- and age-specific associations, RASSF2 has an estrogen receptor-alpha binding site in its promoter. The unknown mechanisms may be due to lack of interest in gender- and age-specificity in associations. These results provide a foundation for further studies to examine the gender- and age-differential in childhood ALL risk. Following replication and mechanistic studies, risk factors for one gender or age group may have a potential to be used as biomarkers for targeted intervention for prevention and maybe also for treatment.^

A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land Plants

Post-print version of the article.