Mechanisms Regulating Epigenetic Information Passage and Development-Associated Chromatin Remodeling

Gao, Yuan ProQuest Dissertations & Theses College of Medicin 2018 해외박사(DDOD)

The proper development of multicellular organisms depends on the specification of different cell types. Despite having identical genomes, cells display distinct gene expression patterns and phenotypes, which can persist after numerous cell divisions. The term ‘Epigenetics’ is the study of inheritable changes in gene function without changes in DNA sequence. How epigenetic states of chromatin are faithful propagated to daughter cells during mitosis and precisely modified during cell fate change, are challenging questions in the epigenetics field. In this dissertation, I describe studies investigating mechanisms regulating these two important processes.Inheritance of epigenetic information in the context of chromatin is a challenge, in terms of both accessibility to the compact structure and maintenance of chromatin states. The basic building blocks of chromatin is nucleosome, which consists of 145–147 base pairs of DNA wrapped around a histone octamer containing one histone (H3–H4)2 tetramer and two histone H2A–H2B dimers. Two distinct processes are involved in maintaining chromatin structure during DNA replication. The first is the disruption of the pre-existing nucleosomes ahead of replication forks and their transfer to nascent DNA, which is a process known as parental histone segregation. The second is the deposition of newly synthesized histones through a pathway called replication-coupled de novo nucleosome assembly. How these two events are coordinated during S phase has a major impact on the ability of cells to maintain genome stability and to reproduce epigenetic information. Here, we show that CAF-1, which is the primary histone chaperone for new (H3–H4)2 deposition on nascent DNA, could bind to DNA through a winged helix domain. This DNA binding ability functions synergistically with CAF-1-PCNA interaction to stabilize CAF-1 at replication fork, and is important for CAF-1 function in the replication-coupled de novo nucleosome assembly pathway. During cell differentiation, the specification of a given cell type is governed by the establishment of specific transcription program. Chromatin structure and PTMs play important roles in this process. It has been shown that defects in nucleosome assembly affect cell identity and cell fate change. To understand the detailed mechanism, we tested the role of nucleosome assembly factors in embryonic stem cell identity maintenance, as well as during differentiation. Interestingly, we uncovered a novel role of histone chaperone Asf1a in resolving bivalent chromatin domains, which act as barriers for specific transcription factor recruitment, to induce lineage specific gene expression during cell differentiation, through its function in nucleosome disassembly. Together, the studies described here furthered our understandings of the molecular mechanisms regulating proliferating cells to propagate or modify epigenetic landscape under different cell context. Insights into these mechanisms will contribute to the study of epigenetic principles in human diseases and the development of regenerative medicine.

The Role of TNFα in Chronic Lymphocytic Leukemia Hematopoietic Dysfunction

Manso, Bryce Alexander ProQuest Dissertations & Theses College of Medicin 2020 해외박사(DDOD)

Immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the well-described peripheral immune abnormalities of these patients are not able to consistently explain these complications. This thesis examines the nature of bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. Numbers and function of BM hematopoietic stem and progenitor cells (HSPCs) are diminished in untreated CLL patients. Consistent with this reduction, BM resident monocytes and natural killer cells are reduced, a deficiency recapitulated in blood. Flow cytometry revealed increased expression of the cross-antagonizing transcription factors PU.1, GATA-2, and GATA-1 in CLL patient-derived HSPC subsets. CLL B cell-derived tumor necrosis factor alpha (TNFα) has been implicated in CLL disease progression and immune suppression. TNFα, which is constitutively produced by CLL B-cells, is a negative regulator of BM hematopoiesis and directly modulates PU.1, GATA-2, and GATA-1 expression. Short-term co-culture of patient-derived CLL cells with control human BM HSPCs directly altered expression levels of PU.1 and GATA-2 at the single cell level. The in vitro increases in PU.1 and GATA-2 precisely mimicked ex vivo expression patterns in all hematopoietic progenitor subsets analyzed and neutralizing TNFα abrogated the molecular alterations induced by CLL cells. Importantly, chronic exposure of control HSPCs to TNFα recapitulated the functional deficiency of CLL-patient derived BM progenitors observed ex vivo. Together, the findings presented in this thesis reveal BM hematopoietic dysfunction in untreated CLL patients and provides new insight into the etiology of the complex immunodeficiency state in CLL. Further, the experimental findings presented here clearly establish the role of CLL B cell-derived TNFα as a major driver of BM hematopoietic dysfunction in CLL disease.

Zebrafish Hearing Science: Paving the Way to Better Patient Care

Koleilat, Alaa ProQuest Dissertations & Theses College of Medicin 2020 해외박사(DDOD)

Background: Hearing loss is the second most prevalent health issue globally and the most common impairment of the senses. The World Health Organization reports that over 6% of the world’s population experience hearing loss (https://www.who.int/health-topics/hearing-loss accessed Feb. 25th, 2020). Genetics is the most common cause of hearing loss among pediatric patients. About ten percent of genetic hearing loss is due to Usher Syndrome, a condition where children are born deaf or hard of hearing, but then develop retinitis pigmentosa. Usher Syndrome is the most common cause of deafness and blindness in children. Currently, there are no cures for Usher Syndrome; however, management options include hearing aids and cochlear implants. Other causes of hearing loss include excessive noise exposure, aging, ototoxic medications and infection. All of these various causes of hearing loss manifest differently and require different evaluation and management needs.Objectives: The thesis takes a bench to bedside approach to hearing loss and aims to address two objectives.1. To characterize hair cell synaptic elements and behavioral phenotypes in a zebrafish model of Usher Syndrome Type 12. To determine if L-type voltage-gated calcium channel agonists can improve abnormal hair cell and behavioral phenotypes observed in the zebrafish model of Usher Syndrome Type 1 due to variants in MYO7A (the myo7aa-/- mutant)3. To develop a clinically validated and accurate hearing assessment tool that can empower the patient to test outside of a sound treated booth and can be used to assess hearing thresholds as part of a hearing health pathway.Methods: This thesis addressed two studies: one preclinical study and one clinical study to address our objectives. The preclinical study characterized the phenotype of a zebrafish model of human Usher Syndrome and assessed the efficacy of three compounds that act either directly or indirectly on the L-type voltage-gated calcium channel located in inner ear hair cells of the myo7aa-/- mutant. We quantified the compounds’ effect on hair cell ultrastructure, distribution of proteins important in hair cell synaptic activity and postsynaptic cell activity, swimming behavior and acoustic startle responses.The clinical study was conducted in the Division of Audiology at Mayo Clinic and included 24 adults, ages 18 to 65 years, tested with GoAudio (the newly developed technology) versus the “gold-standard” clinical audiometry for eight frequencies to evaluate “real-world” applications. Participants utilized active noise cancelling headphones combined with a tablet-based application for the GoAudio assessment.Results: In the preclinical study we discovered that myo7aa-/- mutant zebrafish have fewer glutamatergic vesicles tethered to hair cell ribbon synapses, yet maintain a comparable ribbon area. We identified that myo7aa-/- mutants have fewer total ribbon containing hair cells, fewer total CTBP2 puncta, and an altered distribution of CTBP2 puncta compared to wildtype. We also identified that myo7aa-/- mutants have fewer MAGUK containing cells and fewer total MAGUK puncta, compared to wildtype. Behaviorally, myo7aa-/- mutant fish have abnormal swimming as measured by larger absolute smooth orientations and have little to no acoustic startle. Treatment with L-type voltage-gated calcium channel agonists altered the abnormal synaptic machinery and behavioral phenotypes more towards wildtype.In the clinical study testing GoAudio as a clinical hearing assessment tool results suggest that GoAudio is comparable to clinical audiometry for the identification of hearing loss at most frequencies (except 1 kHz for both ears and 2 kHz in the right ear). Upon stratifying data based on age we identified that GoAudio is capable of identifying suspected age-related hearing loss or hearing thresholds greater than 30 dB HL at higher frequencies in both ears.Conclusions: Our preclinical data supports that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse—in both the number of tethered vesicles and the distribution of CTBP2 puncta, shifts swimming behavior towards wildtype swimming and improves acoustic startle response.The clinical study results support that GoAudio can be used as an accurate hearing assessment tool for the identification of hearing loss at the majority of frequencies outside of a sound-treated booth and can detect characteristics of age-related hearing loss.

Role of DNA Replication-coupled Nucleosome Assembly in Cell Fate Determination During Development

Cheng, Liang ProQuest Dissertations & Theses College of Medicin 2018 해외박사(DDOD)

One long-standing yet still mysterious question in developmental biology is how a multicellular organism with different cell types is derived from a single fertilized egg. Almost all the cells from adult organism share same DNA content yet exhibiting diversified gene expression programs to execute different functions, suggesting that in additional to genetic information, epigenetic mechanisms, initially coined to describe this developmental process, have been increasingly corroborated as critical players for regulating and maintaining cell fate. In eukaryotes, DNA is packaged into chromatin. Chromatin landscapes determine the transcription status of genetic information encoded in DNA sequence. Duplication of chromatin during S phase is critical for maintaining cell-type-specific gene expression pattern and cell fate through cell divisions. Chromatin duplication requires additional supply of newly synthesized histones in addition to the parental histones. Restoration of chromatin status after duplication necessitates the cooperation between newly synthesized histones and parental histones. Moreover, chromatin duplication process provides a window of opportunity for alterations in chromatin state and cell fate. Here in the dissertation, we studied the function of CAF-1, a chaperone for newly synthesize histone H3-H4, in embryonic stem cell differentiation. We show that p150, a subunit of CAF-1, is required for pluripotency dissolution. Depletion of p150 impairs silencing of pluripotent genes on the induction of differentiation, and p150’s function in DNA replication coupled nucleosome assembly is critical in this process. Deficiency of p150 disrupts establishment of repressive chromatin with H3K27me3 at promoter regions of pluripotent genes. Furthermore, we identified that p150 interacts with PRC2 and histone H3 co-purified with p150 is mono- and di-methylated at lysine27 (H3K27me1 and H3K27me2). Our data support a model whereby CAF-1 mediated histone deposition facilitates formation of silencing chromatin at pluripotent genes and cell fate change during differentiation. The study presented here provides evidence supporting the idea that alterations in the CAF-1 mediated nucleosome assembly process can impact cell fate determination. These insights could contribute to our understanding of epigenetic inheritance and cell fate determination as well as development of optimized differentiation strategy for regenerative medicine application.

IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

Hernandez, Danielle Marie ProQuest Dissertations & Theses College of Medicin 2019 해외박사(DDOD)

Pathogenic fibrotic diseases, including Idiopathic Pulmonary Fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two-thirds of IPF patients die within 2-5 years from this progressive interstitial lung disease. Transforming Growth Factor-β (TGFβ) and Insulin-like Growth Factor-1 (IGF-1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF-1 in the initiation and progression of TGFβ-induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF-1 via TGFβ in myofibroblasts, lung epithelial cells, and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) evaluates TGFβ’s effects on the transcription of profibrotic-IGFBPs and pappasylins; (3) identifies TGFβ’s utilization of the Class-1 alternative promoter to increase transcription of Igf-1; (4) determines that SMAD2 and mTOR signaling are required for TGFβ-dependent Igf-1 expression in myofibroblasts; (6) profiles IPF tissue and TGFβ-stimulated myofibroblast transcription of extracellular matrix (ECM) gene expression patterns; (7) demonstrates IGF-1R activation is essential to support TGFβ-driven profibrotic myofibroblast functions and excessive wound-healing; and (8) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF-1R inhibitor OSI-906. These findings expand our knowledge of IGF-1’s role as a novel fibrotic-switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

End of Life Care and Decision Making for Patients with Limited English Proficiency

Barwise, Amelia ProQuest Dissertations & Theses College of Medicin 2019 해외박사(DDOD)

Background: The population with Limited English Proficiency (LEP) in the United States (US) continues to grow and age. Previous work done by our group has demonstrated that differences exist between patients with LEP and those who speak English, in the context of decision-making and end-of-life care in the Intensive Care Unit (ICU) setting. Patients with LEP in the ICU are less likely to change their code status from full code to do-not-resuscitate (DNR), have Advance Care Directives, or receive comfort care measures only orders and tend to get more aggressive interventions at the end of life. Decisions to switch to DNR and comfort measures only orders are not only are less frequent but also take longer. Patients with LEP face multiple barriers with decision making at end of life in the ICU. There are a variety of factors contributing to this challenging problem but this issue has not been studied sufficiently to date.Objectives: To determine and understand the reasons for differences in end of life care and decision making in the ICU for patients with LEP.Methods: For our initial study we conducted secondary data analysis of the Health Information National Trends Survey (HINTS 5 cycle 2 iteration). HINTS is fielded by the National Cancer Institute and is an annual, cross-sectional, national representative survey that provides a unique and reliable data source to assess health communication, health knowledge, and use of health information technology. We used the data to assess perceived knowledge of palliative care and preferred trusted source for knowledge about palliative care among immigrants in the US. We then used qualitative research approaches, based on grounded theory methodology to conduct 40 one-on-one semi structured interviews of physicians, bedside nurses, and interpreters in the ICU. During these interviews we explored the clinicians’ perceptions of the reasons for differences in decision making and care in the ICU for patients with LEP. All transcripts were audio-recorded, transcribed, and coded independently and in duplicate to consensus. For our third study we performed a systematic review using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to describe and appraise interventions designed to improve end of life decision making for patients with LEP. We developed a search strategy using specific terms such as end of life, LEP, advance care planning, goals of care and searched a variety of traditionally used databases from 1946 to November 2018.Results: In the first study we did not find statistically significant differences in perceived knowledge of palliative care between immigrants and those born in the US following adjustments. Furthermore, in both groups, 80% of respondents declared their preferred trusted source for palliative care information was a health care provider (p=0.53). Over 70% in both native born and immigrant groups responded that they did not know what palliative care was. Low levels of education predicted poor perceived knowledge of palliative care (p<0.001). The second, a qualitative study highlighted that both poor communication as well as variations in preferences for end of life care were responsible for the differences in end of life care noted in the ICU. Following analysis of the coded transcripts, several themes were derived including specific barriers to and facilitators of communication and decision making for patients with LEP in the ICU. The third study, a systematic review revealed that only 8 interventions exist that focus on improving decision making at end of life for patients with LEP. These used trained personnel, videos, web-based modules, and written materials. Seven of eight studies were based in the US and directed towards Hispanic populations. One study was based in Australia and directed towards Greek and Italian populations. Interventions to increase advance care planning and advance directives were generally successful. Interventions aiming to influence other end of life decision making such as hospice use worked less wellConclusions: Based on our HINTS study findings, differences in end of life care among patients with LEP cannot be explained by poorer knowledge of palliative care compared to patients who speak English. However our qualitative study showed that communication is suboptimal for patients with LEP in the ICU and this negatively impacts decision making and subsequent care. Some patients with LEP also have cultural and faith-based preferences that drive some of the noted differences in decision making and care. Few scalable, feasible, and successful interventions exist to address this problem based on our systematic review findings. This research provides the groundwork for developing more effective interventions to improve end of life decision making for patients with LEP that respect and acknowledge preference variation in end of life care but prioritize improved communication.

Aspirin, Other NSAIDs, Statins and Metformin in Biliary Tract Cancer Risk and Survival: A Swedish Population-Based Cohort Study

Marcano Bonilla, Lorena ProQuest Dissertations & Theses College of Medicin 2019 해외박사(DDOD)

Alterations in Centrosome Separation and the Actomyosin Cortex as a Source of Tumor Promoting Instability

Limzerwala, Jazeel Firdos ProQuest Dissertations & Theses College of Medicin 2020 해외박사(DDOD)

Chromosomal Instability (CIN), the condition where cells constantly mis-segregate their chromosomes in mitosis, is a widely recognized hallmark of human cancers. CIN is now beginning to be recognized as an important source of intra-tumor heterogeneity that can drive disease progression, metastasis, relapse and patient death. It is therefore imperative to fully understand how is CIN engendered and how does it impact different stages of tumorigenesis, to better advance patient care and diagnosis. In the work presented in this thesis, involving several genetically engineered mice strains, we uncover a novel tumor suppressive function for the transcription factor FoxM1 that is independent of its transcriptional ability, identify deregulations in the actomyosin cortex as a driver of CIN and provide preliminary proof-of-concept that CIN drives tumor progression and metastasis. Using genetically engineered mice that express low amounts of FoxM1, we found that FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling, resulting in increased cortical actin density that impedes centrosome separation and predisposes cells to increased incidence of CIN and tumor formation. Re-expression of the FoxM1 Nterminal Domain (NTD) in FoxM1 insufficient mice restores proper control over cortical actin nucleation, mitotic chromosome segregation and tumor suppression, providing evidence for a causal role for increased cortical actin density in driving CIN and tumorigenesis. Our work has also identified the mechanism of how overexpression of Cyclin E1 can predispose mice to hepatocellular carcinoma, which can have far reaching impacts for patients infected with Hepatitis B/C virus or Adeno-associated virus 2, which integrate into the CCNE1 gene locus in humans and cause its overexpression. Collectively, the work presented in this thesis has advanced our understanding of the role of CIN in tumor development.

Understanding Succinate-Induced Transcriptional and Epigenetic Reprogramming and Identifying Unique Vulnerabilities Intrinsic to SDH-Deficient Cells

Smestad, John Andrew ProQuest Dissertations & Theses College of Medicin 2019 해외박사(DDOD)

Modeling Gene x Environment Interactions with Glucocorticoids Unmasks Novel Disease Risk Mechanisms

Nguyen, Thanh Thanh Le ProQuest Dissertations & Theses College of Medicin 2022 해외박사(DDOD)

Understanding the function of non-coding genomic sequence variants represents a major challenge for biomedicine. Many diseases are products of gene x environment interactions with complex mechanisms. My thesis addresses these themes by mechanistic characterization of environment-responsive non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, I integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, I identified cis-regulatory elements and 3-dimensional interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants identified had already been associated with clinical phenotypes. However, their affected genes were “unmasked” only after glucocorticoid exposure and those genes often displayed function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or as therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer, demonstrating an interaction between environmental and genetic risk factors in disease predisposition. My subsequent functional studies of genes discovered via this mechanism in bipolar disorder with metabolic comorbidity yielded novel insights into disease pathophysiology. The team then moved beyond the traditional role of the glucocorticoid receptor (GR) as a ligand-activated transcription factor to interrogate its role in post-transcriptional regulation. We discovered that glucocorticoids could also regulate alternative polyadenylation of mRNA for a variety of genes involved in immunity. We also observed evidence that genetic risk variants for certain immune-related diseases could modulate glucocorticoid-dependent alternative polyadenylation. Together, these results provide a mechanistic framework for understanding the function of non-coding genetic variants across chemical environments.