Discovery and functional characterization of self-renewal regulatory factors constitute one of the central themes in the field of human hematopoietic stem and progenitor cell (HSPC) biology both for advancing the fundamental science as well as harnessing the knowledge to expand hematopoietic stem cells (HSCs) for regenerative therapeutics purposes. Musashi-2 (MSI2) is a marker and essential positive regulator of HSCs but knowledge of the transcription factors (TFs) that ensure its appropriate HSC-specific expression is lacking. I believed that uncovering the transcriptional circuitry of MSI2 could also offer opportunities for the discovery of candidate TFs with similar or superior functions in human HSPCs. To that end, initial investigations were set out to identify a conserved minimal promoter region bound by TFs upstream of the translation start site of human MSI2. In silico analysis of the minimal promoter region and series of TF binding-site mutagenesis biochemical assays identified USF2 and PLAG1 as key co-regulators of MSI2 expression and function in the model K562 cell line and cord blood HSPCs. The approach identified PLAG1 as a candidate TF whose function in HSPCs was not previously reported. I have characterized PLAG1 as an important regulator of HSPCs self-renewal using in vitro and xenotransplantation functional genomics coupled with RNA sequencing and downstream pathway analysis. In vitro, overexpression of PLAG1 (PLAG1OE) imparts sustained pro-survival advantage to progenitors and produces significantly more CD34+ cells compared to control culture. PLAG1OE enhances BFU-E and total colony forming unit (CFU) output. Complementary in vitro assays using shRNA-mediated knockdown (PLAG1KD) elucidated impaired CFU output, increased apoptosis and significantly reduced CD34+ cell counts. In vivo transplantation of PLAG1OE CD34+ cells into NSG mice at limiting doses robustly enhances the frequency and absolute number of HSCs by a magnitude of 24.1-fold compared to control. Serial transplantation of bone marrow cells into secondary recipients demonstrated enhanced self-renewal of HSCs compared to control. Mechanistically, transcriptome-wide gene-set enrichment and network analysis revealed that ribosome biogenesis and proteostasis pathways are significantly attenuated. PLAG1OE up-regulates expression of H19 and MEG3 imprinted long non-coding RNA (lncRNA)-derived miR-675 and miR-127 species respectively. In silico overlap analysis with experimentally validated targets of both miR-127 and miR-675 revealed ribosome biogenesis and proteins synthesis pathways components as prime targets. PLAG1 also represses the pan-ribosome biogenesis transcriptional regulator MYC. Down regulation of MYC demonstrates another layer of PLAG1-mediated transcriptional attenuation of protein synthesis. Additionally, we observed dampening of IGF1R/PI3K/AKT/mTOR signaling pathway. Conversely, PLAG1KD down regulated H19 and MEG3 expression and reverses global gene-set enrichment observed by PLAG1OE in reciprocal fashion. Taken together, our study presents PLAG1 as master regulator of ribosome biogenesis and protein synthesis from multiple checkpoints to enhance clonogenicity, survival and long-term self-renewal capacity of HSCs. / Thesis / Doctor of Philosophy (PhD)

No description available.

Submitted by YURI TANI UTSUNOMIYA null (yuri.tani@yahoo.com.br) on 2018-01-08T12:45:05Z No. of bitstreams: 1 UTSUNOMIYA.pdf: 11784469 bytes, checksum: 9fc7d258b6e5e683b47b78ad25853acc (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-01-09T09:34:15Z (GMT) No. of bitstreams: 1 utsunomiya_yt_dr_jabo.pdf: 11784469 bytes, checksum: 9fc7d258b6e5e683b47b78ad25853acc (MD5) / Made available in DSpace on 2018-01-09T09:34:15Z (GMT). No. of bitstreams: 1 utsunomiya_yt_dr_jabo.pdf: 11784469 bytes, checksum: 9fc7d258b6e5e683b47b78ad25853acc (MD5) Previous issue date: 2017-12-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O gene 1 do adenoma pleomórfico (PLAG1) apresenta evidência de seleção positiva recente e associação com tamanho corporal e fertilidade em um grande número de raças bovinas ao redor do mundo. Tendo em vista sua recentemente descoberta função como fator de transcrição para o gene do fator de crescimento semelhante à insulina 2 (IGF2), o PLAG1 possui papel emergente como um dos principais reguladores do crescimento e da reprodução em bovinos. Apesar de sua importância, a variante de sequência de DNA responsável pelos efeitos pleiotrópicos atribuídos ao PLAG1 em bovinos permanece desconhecida. Também não está claro se a mesma mutação explica as associações fenótipo-genótipo encontradas em diferentes populações bovinas. Além disso, ainda é incerto onde e quando ocorreu a pressão de seleção responsável pelo aumento da frequência da mutação do PLAG1. No presente trabalho, reportamos o desenvolvimento de um pacote para o software estatístico R, o qual é direcionado à análise de haplótipos como preditores para variantes genéticas não observadas. Através da aplicação desta ferramenta a dados genômicos de bovinos oriundos de diversas regiões do mundo, encontramos evidência indicando que um único alelo derivado do PLAG1 aumentou em frequência rapidamente em bovinos Bos taurus do noroeste europeu entre os séculos XVI e XVIII. Este período é reconhecido como a última onda de aumento de estatura em bovinos por meio de registros arqueológicos. Os dados também sugerem que o alelo foi introgredido em B. taurus não europeu e raças Bos indicus entre os séculos XIX e XX, adquirindo uma distribuição quase global no último século. Análises de DNA antigo revelaram que esta mutação segrega em gado do noroeste europeu há pelo menos 1.000 anos. Em conjunto, estes resultados implicam um papel central da mutação do PLAG1 em recentes mudanças de tamanho corporal em bovinos. / The pleomorphic adenoma gene 1 (PLAG1) presents both evidence of recent positive selection and association with body size and fertility in a wide range of worldwide cattle breeds. Considering its recently uncovered function as a transcription factor for the insulin-like growth factor 2 gene (IGF2), PLAG1 is emerging as a major regulator of bovine growth and reproduction. In spite of its importance, the causal DNA sequence variant underlying the pleiotropic effects of PLAG1 in cattle remains unknown. It is also unclear whether the same mutation accounts for the phenotype-genotype associations detected across different cattle populations. Furthermore, when and where the selective pressure responsible for increasing the frequency of the PLAG1 mutation occurred is still uncertain. Here, we report the development of a package for the R statistical software to analyze haplotypes as surrogates for unobserved genetic variants. By applying this tool to genomic data of worldwide cattle breeds, we found evidence that a single bovine PLAG1 derived allele increased rapidly in frequency in Northwestern European Bos taurus populations between the 16th and 18th centuries. This period is recognized as the last wave of increase in bovine stature from archaeological data. The data also suggested that the allele was introgressed into non-European B. taurus and Bos indicus breeds towards the 19th and 20th centuries, achieving an almost global distribution in the last century. Ancient DNA analyses further revealed that this mutation has been segregating in Northwestern European cattle for at least 1,000 years. Altogether, these results implicate a major role of the PLAG1 mutation in recent changes in body size in cattle. / 2014/01095-8 / 2016/07531-0