Fanconi anemia (FA) is a genome instability syndrome that is clinically manifested by bone marrow failure, congenital defects, and elevated cancer susceptibility. The FA pathway is known to regulate the repair of DNA interstrand crosslinks in part through DNA homologous recombination (HR) repair. Up to today 16 FA proteins have been discovered that may participate in the common pathway. Cells that have mutations in the FA genes are hypersensitive to DNA damaging agents and display chromosome instability. A key regulatory event in the FA pathway is monoubiquitination of FANCD2-FANCI heterodimer that is mediated by a multi-component E3 ubiquitin ligase complex called FA core complex. Current model suggests that once the FANCD2-FANCI heterodimer is monoubiquitinated it relocates to chromatin where it interacts with other key repair proteins to facilitate DNA repair. More than 90% of the FA cases are presumed to be associated with defects in the monoubiquitination reaction, suggesting the significance of the modification in the pathogenesis of the disease. Despite the significance, the molecular interplay between the FA core complex and the FANCD2-FANCI heterodimer remains enigmatic. We are interested in the assembly mechanism of the various FA subcomplexes into the core complex, and we are actively investigating how the FANCD2-FANCI heterodimer is recruited to these putative subcomplexes. As the FA pathway is a crucial determinant for cellular resistance to DNA damaging agents, there have been hypotheses that disruption of this pathway may be beneficial in enhancing chemosensitivity of certain cancer cells. In collaboration with Dr. Cai’s chemistry lab, we will develop a screen platform to identify a small molecules to interrupt the monoubiquitination reaction. Completion of these studies will enhance the much-needed knowledge of the key enzymatic reaction in the pathway, and perhaps the information can be used for development of novel chemotherapeutic strategies.

Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer predisposition. Cell lines derived from FA patient exhibit chromosomal instability and sensitivity to DNA interstand crosslinkers (ICLs) like mitomycin (MMC). The key event in Fanconi anemia pathway is the regulated ubiquitination and deubiquitination of FANCD2 and FANCI. Upon DNA damage, FANCD2 and FANCI are monoubiquitinated by FA core complex. They then move into the chromatin and serve as the landing site for downstream players, like FANCP/SLX4 and FAN1. USP1, the deubiquitinating enzyme (DUB), removes ubiquitin from FANCD-Ub/FANCI-Ub, and this step is required for the integrity of FA pathway. This dissertation addresses how USP1 is regulated in the cell. In Chapter 2, we discovered UAF1/WDR48 as a critical binding partner for USP1, by activating its enzymatic activity in vitro and in vivo. We then generated DT40 knockout cell lines of USP1 and UAF1. We showed that USP1/UAF1 complex is functionally required for homologous recombination (HR). Interestingly, PCNA-Ub is also a substrate for USP1. We discovered that hELG1, through its binding to USP1/UAF1 complex, regulates the deubiquitination of PCNA-Ub and translesion DNA synthesis (TLS). Then in Chapter 3, we discovered a tandem repeat of SUMO-like domains (SLD1 and SLD2) in the C terminus of UAF1. SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 of UAF1 or mutation of the SIM of FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination. The SLD2 sequence of UAF1 also binds to a SIM on hELG1, and targets the USP1/UAF1 complex to its PCNA-Ub substrate. We proposed the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates HR and TLS. Originating from USP1/UAF1 complex, we worked out a general mechanism of DUB regulation by WD40 proteins, which involved in two more DUBs, USP12 and USP46 (discussed in Chapter 4 and Appendix A). Lastly in Chapter 5, through bioinformatic analysis we identified a series of novel proteins containing ubiquitin-binding zinc fingers (UBZ). We then focused on SNM1A and FAAP20/C1orf86, and characterized their function in DNA crosslink repair.

The effect of a single genetic mutation can vary greatly between different types of cells. The mutated gene may not be expressed in one tissue but may cause a devastating loss of function in another. To learn about disease mechanisms and generate novel therapies, genetic disorders must be studied in the types of cells where the mutations are most deleterious. Recently, scientists have begun manipulating cellular identity to create the cell types most affected by various genetic diseases. This dissertation describes the experience of generating reprogramming models for three genetic disorders: Ring 14 syndrome, Pearson syndrome, and Fanconi anemia.

Resumo: A Anemia de Fanconi (AF) é uma doença genética rara caracterizada por instabilidades cromossômicas que geram anormalidades somáticas de graus variáveis, falência medular progressiva e susceptibilidade aumentada a neoplasias. O único tratamento curativo é o transplante de células tronco hematopoéticas (TCTH), entretanto a toxicidade e falha de pega ainda são limitantes para sua realização. A falha de pega do enxerto ou rejeição é uma complicação grave e potencialmente fatal que ocorre em até 30% dos pacientes com AF submetidos ao TCTH, dependendo do tipo de doador e regime de condicionamento utilizado. Entre Janeiro de 1985 e Outubro de 2011, 238 pacientes com AF realizaram TCTH nessa instituição. Duzentos e doze pacientes foram avaliáveis quanto à pega medular e divididos em 3 grupos. O grupo 1 foi constituído por 25 pacientes que tiveram falha primária de pega (FPP). O grupo 2 por 9 pacientes com falha secundária de pega (FSP) ou evolução para leucemia e o grupo 3, com 178 casos com pega medular adequada. Os pacientes com falha primária e secundária de pega apresentaram maior duração de doença e maior número de transfusões sanguíneas prévias ao TCTH do que o grupo com pega medular (p=0,001 e p<0,001). Doador não aparentado (NAP) foi utilizado em 84% dos pacientes do G1 e apenas 33% do G3 (p<0,001); houve ainda menor número de transplantes totalmente compatíveis no G1 do que nos outros grupos (p<0,001). A fonte de células foi sangue de cordão umbilical (SCU) em 56% dos pacientes do G1 e 13% no G3. Na análise multivariada os fatores associadas a uma maior FPP foram maior número de transfusões (p=0,003), a utilização de doadores NAP (p=0,001) ou com incompatibilidades HLA (p=0,025) ou a ausência de fludarabina no regime de condicionamento (p=0,005). O fator predisponente para pega medular não sustentada ou evolução para leucemia foi maior número de transfusões (p=0,032). Vinte e quatro pacientes foram submetidos a um novo TCTH, sendo 20 do G1 e 4 do G2 e apenas seis pacientes estão vivos. A incidência cumulativa para a falha de pega foi de 18,9%, sendo maior nos transplantes com doadores NAP (36,2%) e com SCU (40%). A falha de pega ou rejeição é uma complicação extremamente grave e mais frequente nos transplantes não aparentados, com incompatibilidades e em pacientes mais transfundidos. Novos regimes de condicionamento, imunomodulação ou redução do tempo para o encontro de doadores NAP podem ser alternativas na tentativa de reduzir a incidência da rejeição nos pacientes com AF.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22002号 / 医博第4516号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 松田 文彦, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18906号 / 医科博第62号 / 新制||医科||4(附属図書館) / 31857 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 野田 亮, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The dawn of the genetic era has allowed for investigation of gene transfer therapy as a treatment for certain diseases. Fanconi anemia (FA) is a rare genetic disorder in which the majority of patients develops progressive bone marrow failure (BMF) and require bone marrow transplantation. A possible alternative treatment is autologous gene therapy; however, original clinical trials involving gene transfer for FA were unsuccessful. This has led to re-evaluation of the gene transfer protocols, the vectors and also a deeper investigation of the FA pathway itself. My work has focused on illuminating these areas to further advance gene transfer therapy for FA. Many gene transfer protocols require the hematopoietic stem and progenitor cells (HSC/HPC) to be collected and then transduced ex vivo. The most common collection method is mobilization of the HSC/HPC to the peripheral blood (PB) using granulocyte colony-stimulating factor (G-CSF) and collection via apheresis. In FA patients G-CSF fails to mobilize a sufficient number of HSC/HPC. This has led to research into agents such as AMD3100, a CXCR4 antagonist, which may replace or augment G-CSF mobilization. These data show in two FA murine models that AMD3100 synergizes with G-CSF resulting in a significant increase in mobilization as compared to G-CSF alone. Previous work in our lab has shown that prototype foamy virus (FV) is an efficient gene transfer vector. Here a modified FV vector is used to transduce mobilized FA cells. The data indicate that long-term repopulating cells mobilized with both G-CSF and AMD3100 can be efficiently transduced by our FV vector. Clinically, FA is characterized mainly by BMF, but also by myelodysplasia (MDS) and acute myeloid leukemia (AML). However, current FA murine models do not display these disease phenotypes. These data show that double-mutant Fancc-/-;Fancg-/- mice spontaneously develop BMF, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous BMF and MDS in FA and novel gene transfer protocols for FA.

Since the dawn of the genomics era, the genetics of numerous human disorders has been understood which has led to improvements in targeted therapeutics. However, the focus of most research has been primarily on protein coding genes, which account for only 2% of the entire genome, leaving much of the remaining genome relatively unstudied. In particular, repetitive sequences, called microsatellites (MST), which are tandem repeats of 1 to 6 bases, are known to be mutational hotspots and have been linked to diseases, such as Huntington disease and Fragile X syndrome. This work represents a significant effort towards closing this knowledge gap. Specifically, we developed a next generation sequencing based enrichment method along with the supporting computational pipeline for detecting novel MST sequences in the human genome. Using this global MST enrichment protocol, we have identified 790 novel sequences. Analysis of these novel sequences has identified previously unknown functional elements, demonstrating its potential for aiding in the completion of the euchromatic DNA. We also developed a disease risk diagnostic using a novel target specific enrichment method that produces high resolution MST sequencing data that has the potential to validate, for the first time, the link between MST genotype variation and cancer. Combined with publicly available exome datasets of non-small cell lung cancer and 1000 genomes project, the target specific MST enrichment method uncovered a signature set of 21 MST loci that can differentiate between lung cancer and non-cancer control samples with a sensitivity ratio of 0.93. Finally, to understand the molecular causes of MST instability, we analyzed genomic variants and gene expression data for an autosomal recessive disorder, Fanconi anemia (FA). This first of its kind study quantified the heterogeneity of FA cells and demonstrated the possibility of utilizing the DNA crosslink repair dysfunctional FA cells as a suitable system to further study the causes of MST instability. / Ph. D.