Doctor of Philosophy

dissertationEwing sarcoma is a highly aggressive pediatric malignancy that is characterized by a chromosomal translocation-derived fusion protein, Ewing sarcoma (EWS)/ Friend leukemia insertion (FLI), EWS/FLI. EWS/FLI is an aberrant transcription factor and its downstream targets contribute to oncogenic transformation in Ewing sarcoma. However, the mechanisms of transcriptional regulation by EWS/FLI and the full complement of direct targets of EWS/FLI were previously unknown. The work documented in this dissertation describes a novel basis for EWS/FLI function in gene activation, and also uncovers a mechanism by which EWS/FLI directly represses a subset of critical targets genes in Ewing sarcoma. Through the identification of direct in vivo targets of EWS/FLI, we made an unexpected discovery that EWS/FLI activates some of its critical target genes, including NR0B1 and GSTM4, by binding to microsatellite repeats. These findings suggest a new paradigm for cancer-relevant gene regulation by EWS/FLI, and perhaps other ETS family members. In addition to the microsatellite repeats, EWS/FLI regulates some of its target genes through the canonical high-affinity consensus E-26 oncogene (ETS) site. We focused on one such target gene, GLI1, and uncovered a novel role for GLI1 and its downstream target, KRT17, in coordinating two cancer-relevant functions: oncogenic transformation and cellular adhesion in Ewing sarcoma. iv The functional relationship between wild-type EWS and the EWS/FLI fusion protein in Ewing sarcoma was largely unknown. Through global transcription profiling and mechanistic studies we demonstrated that EWS and EWS/FLI coregulate a subset of genes in Ewing sarcoma and that EWS functions as a cofactor of the REST transcription factor to repress neuronal differentiation genes. These data suggest that EWS, and consequently EWS/FLI, have transcriptional repressive roles in Ewing sarcoma. We next focused on identifying and characterizing the mechanism underlying EWS/FLI-mediated direct transcriptional repression. We demonstrated that EWS/FLI interacts with the nucleosome remodeling and histone deacetylase (NuRD) corepressor complex to repress critical tumor suppressor genes in Ewing sarcoma. These data identify inhibitors of the NuRD complex components as potentially effective therapeutic agents for the treatment of Ewing sarcoma. Taken together, the work presented in this dissertation advances our molecular understanding of EWS/FLI-mediated gene regulation in Ewing sarcoma

Gene regulatory networks in neural cell fate acquisition from genome-wide chromatin association of Geminin and Zic1

Neural cell fate acquisition is mediated by transcription factors expressed in nascent neuroectoderm, including Geminin and members of the Zic transcription factor family. However, regulatory networks through which this occurs are not well defined. Here, we identified Geminin-associated chromatin locations in embryonic stem cells and Geminin- and Zic1-associated locations during neural fate acquisition at a genome-wide level. We determined how Geminin deficiency affected histone acetylation at gene promoters during this process. We integrated these data to demonstrate that Geminin associates with and promotes histone acetylation at neurodevelopmental genes, while Geminin and Zic1 bind a shared gene subset. Geminin- and Zic1-associated genes exhibit embryonic nervous system-enriched expression and encode other regulators of neural development. Both Geminin and Zic1-associated peaks are enriched for Zic1 consensus binding motifs, while Zic1-bound peaks are also enriched for Sox3 motifs, suggesting co-regulatory potential. Accordingly, we found that Geminin and Zic1 could cooperatively activate the expression of several shared targets encoding transcription factors that control neurogenesis, neural plate patterning, and neuronal differentiation. We used these data to construct gene regulatory networks underlying neural fate acquisition. Establishment of this molecular program in nascent neuroectoderm directly links early neural cell fate acquisition with regulatory control of later neurodevelopment

Follow-up of high risk neonates

The improvement in perinatal care has led to increase in survival as well as reduction of morbidity in sick newborns. These babies need to be followed up regularly to assess growth and neurodevelopmental outcome and for early stimulation and rehabilitation. We present a protocol describing the various components of a follow up program, and services

Uncovering the transcriptomic and epigenomic landscape of nicotinic receptor genes in non-neuronal tissues

Abstract Background Nicotinic acetylcholine receptors (nAChRs) play an important role in cellular physiology and human nicotine dependence, and are closely associated with many human diseases including cancer. For example, previous studies suggest that nAChRs can re-wire gene regulatory networks in lung cancer cell lines. However, the tissue specificity of nAChRs genes and their regulation remain unexplored. Result In this study, we integrated data from multiple large genomic consortiums, including ENCODE, Roadmap Epigenomics, GTEx, and FANTOM, to define the transcriptomic and epigenomic landscape of all nicotinic receptor genes across many different human tissues and cell types. We found that many important nAChRs, including CHRNA3, CHRNA4, CHRNA5, and CHRNB4, exhibited strong non-neuronal tissue-specific expression patterns. CHRNA3, CHRNA5, and CHRNB4 were highly expressed in human colon and small intestine, and CHRNA4 was highly expressed in human liver. By comparing the epigenetic marks of CHRNA4 in human liver and hippocampus, we identified a novel liver-specific transcription start site (TSS) of CHRNA4. We further demonstrated that CHRNA4 was specifically transcribed in hepatocytes but not transcribed in hepatic sinusoids and stellate cells, and that transcription factors HNF4A and RXRA were likely upstream regulators of CHRNA4. Our findings suggest that CHRNA4 has distinct transcriptional regulatory mechanisms in human liver and brain, and that this tissue-specific expression pattern is evolutionarily conserved in mouse. Finally, we found that liver-specific CHRNA4 transcription was highly correlated with genes involved in the nicotine metabolism, including CYP2A6, UGT2B7, and FMO3. These genes were significantly down-regulated in liver cancer patients, whereas CHRNA4 is also significantly down-regulated in cancer-matched normal livers. Conclusions Our results suggest important non-neuronally expressed nicotinic acetylcholine receptors in the human body. These non-neuronal expression patterns are highly tissue-specific, and are epigenetically conserved during evolution in the context of non-conserved DNA sequence

Growth and neurosensory outcomes of preterm very low birth weight infants at 18 months of corrected age

Objective: To determine the growth and neurosensory outcomes of infants with birth weight ≤1,500 g or gestation ≤32 wks at 18 months corrected age. This prospective cohort study was conducted at a Level III neonatal unit in India. The neonates with birth weight ≤1,500 g or gestation ≤32 wks were included in the study. Methods: The infants were followed up at 3,6,9,12 and 18 months corrected age. Weight, length and head circumference were plotted on WHO multisite growth reference study (MGRS) charts. Neurological examination was conducted by Amiel-Tison method, hearing was evaluated with brainstem auditory evoked responses, vision assessed with Teller acuity cards, and development assessed with Developmental Assessment Scales for Indian Infants II. Results: During the period from July 2006 through June 2007, there were 141 neonates born at gestation ≤32 wks or birth weight ≤1,500 g. Seven infants had major malformations, 30 died before discharge, 36 had residence >20 km and parents of four had refused consent. The remaining 64 neonates were enrolled for follow up. The mean gestation and birth weight were 31(2.4) wks and 1208 (365) g respectively. There were 38 (59%) small for gestation infants. Fifty-five infants completed 18 months follow up for growth outcomes. Seventeen (30.9%; 95% CI 18.3% to 43.5%) infants were undernourished, 28(50.9%; 95% CI 37.3% to 64.6%) were stunted, 8(14.5%; 95% CI 0 to 24) were wasted and 14(25.4%; 95% CI 13.6% to 37.3%) had microcephaly. Infants with birth weight < 1,000 g (n = 17) were significantly more affected. Ten (58.8%; p < 0.01) were undernourished, 13(76.5%; p <  0.01) were stunted and 10(58.8%; p < 0.01) had microcephaly. Complete formal neurological evaluation for development, hearing and vision was done in 31 infants. Six of these 31 (19.3%; 95% CI 4.6% to 34.1%) infants had one or more major disabilities. These included cerebral palsy (n = 3), developmental delay (development quotient < 70, n = 3), and deafness (n = 3). Conclusions: Very low birth weight infants are at a high risk of neurosensory disability and growth failure. There is a need to create a nation-wide database of these infants for neurodevelopment and growth outcomes

El Diario de Pontevedra : periódico liberal: Ano XXVIII Número 8206 - 1911 setembro 20

The epigenetic landscape around CHRNB4, CHRNA5, CHRNA3, CHRNB2, and CHRNA4 in human liver, CD34-HSC, brain, colon, and lung tissues. (PDF 288 kb

A Novel Role for Keratin 17 in Coordinating Oncogenic Transformation and Cellular Adhesion in Ewing Sarcoma

Oncogenic transformation in Ewing sarcoma is caused by EWS/FLI, an aberrant transcription factor fusion oncogene. Glioma-associated oncogene homolog 1 (GLI1) is a critical target gene activated by EWS/FLI, but the mechanism by which GLI1 contributes to the transformed phenotype of Ewing sarcoma was unknown. In this work, we identify keratin 17 (KRT17) as a direct downstream target gene upregulated by GLI1. We demonstrate that KRT17 regulates cellular adhesion by activating AKT/PKB (protein kinase B) signaling. In addition, KRT17 is necessary for oncogenic transformation in Ewing sarcoma and accounts for much of the GLI1-mediated transformation function but via a mechanism independent of AKT signaling. Taken together, our data reveal previously unknown molecular functions for a cytoplasmic intermediate filament protein, KRT17, in coordinating EWS/FLI- and GLI1-mediated oncogenic transformation and cellular adhesion in Ewing sarcoma