Transcriptional and Epigenetic Regulation of Human CD4+ T Helper Lineage Specification

Activated T helper (Th) cells have ability to differentiate into functionally distinct Th1, Th2 and Th17 subsets through a series of overlapping networks that include signaling and transcriptional control and the epigenetic mechanisms to direct immune responses. However, inappropriate execution in the differentiation process and abnormal function of these Th cells can lead to the development of several immune mediated diseases. Therefore, the thesis aimed at identifying genes and gene regulatory mechanisms responsible for Th17 differentiation and to study epigenetic changes associated with early stage of Th1/Th2 cell differentiation. Genome wide transcriptional profiling during early stages of human Th17 cell differentiation demonstrated differential regulation of several novel and currently known genes associated with Th17 differentiation. Selected candidate genes were further validated at protein level and their specificity for Th17 as compared to other T helper subsets was analyzed. Moreover, combination of RNA interference-mediated downregulation of gene expression, genome-wide transcriptome profiling and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq), combined with computational data integration lead to the identification of direct and indirect target genes of STAT3, which is a pivotal upstream transcription factor for Th17 cell polarization. Results indicated that STAT3 directly regulates the expression of several genes that are known to play a role in activation, differentiation, proliferation, and survival of Th17 cells. These results provide a basis for constructing a network regulating gene expression during early human Th17 differentiation. Th1 and Th2 lineage specific enhancers were identified from genome-wide maps of histone modifications generated from the cells differentiating towards Th1 and Th2 lineages at 72h. Further analysis of lineage-specific enhancers revealed known and novel transcription factors that potentially control lineage-specific gene expression. Finally, we found an overlap of a subset of enhancers with SNPs associated with autoimmune diseases through GWASs suggesting a potential role for enhancer elements in the disease development. In conclusion, the results obtained have extended our knowledge of Th differentiation and provided new mechanistic insights into dysregulation of Th cell differentiation in human immune mediated diseases.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Lineage specification of neuronal precursors in the mouse spinal cord.

We have investigated the differentiation potential of precursor cells within the developing spinal cord of mice and have shown that spinal cord cells from embryonic day 10 specifically give rise to neurons when plated onto an astrocytic monolayer, Ast-1. These neurons had the morphology of motor neurons and > 83% expressed the motor neuron markers choline acetyltransferase, peripherin, calcitonin gene-related peptide, and L-14. By comparison, < 10% of the neurons arising on monolayers of other neural cell lines or 3T3 fibroblasts had motor neuron characteristics. Cells derived from dorsal, intermediate, and ventral regions of the spinal cord all behaved similarly and gave rise to motor neuron-like cells when plated onto Ast-1. By using cells that expressed the lacZ reporter gene, it was shown that > 93% of cells present on the Ast-1 monolayers were motor neuron-like. Time-lapse analysis revealed that the precursors on the Ast-1 monolayers gave rise to neurons either directly or following a single cell division. Together, these results indicate that precursors in the murine spinal cord can be induced to differentiate into the motor neuron phenotype by factors produced by Ast-1 cells, suggesting that a similar factor(s) produced by cells akin to Ast-1 may regulate motor neuron differentiation in vivo.

In vitro fertilization, embryo development, and cell lineage segregation after pre- and/or postnatal exposure of female mice to ambient fine particulate matter

Objective: To evaluate effects of pre- and/or postnatal exposure to ambient fine particulate matter on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. Design: Animal model. Setting: Academic institution. Animal(S): Six-week-old, superovulated mice. Intervention(s): Pre- and postnatal exposure to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24 hours a day for 9 weeks. Main Outcome Measure(S): Gestation length, litter size, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to inner-cell mass (ICM) and trophectoderm (TE). Result(S): Gestation length, litter size and birth weight, live-birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in the FA-AA and AA-AA protocols. No difference in total cell count was observed among groups. Conclusion(S): Our study suggests that exposure to ambient fine particulate matter may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage without interfering in early development of the mouse embryo. (Fertil Steril (R) 2009;92:1725-35. (C) 2009 by American Society for Reproductive Medicine.)

Mouse Sox8 is located between, not within, the t-complex deletions t(w18) and t(h20) on chromosome 17

The SOX family of developmental transcription factors is known to play critical roles in cell lineage specification, fate determination and differentiation during development in diverse phyla. Their importance is underscored by their involvement in a number of human diseases and mouse mutants, and by targeted mutation in mice. SOX8 is broadly expressed during development and is located on human chromosome 16p and within the t-complex on mouse chromosome 17, in the vicinity of two mutations t(w18) and t(h20). Here we analyse mutant genomic DNA to show that the Sox8 gene locus lies outside the deletion regions of both t(w18) and t(h20) and between these deletions. These data exclude Sox8 from contributing to the t(w18) and t(h20) phenotypes, and provide an additional marker for structural characterization of this complex genomic region. Copyright (C) 2001 S. Karger AG, Basel.

Paradigms of notch signaling in mammals.

Notch proteins regulate a broad spectrum of cell fate decisions and differentiation processes during fetal and postnatal life. These proteins are involved in organogenesis during embryonic development as well as in the maintenance of homeostasis of self-renewing systems. The paradigms of Notch function, such as stem and progenitor cell maintenance, lineage specification mediated by binary cell fate decisions, and induction of terminal differentiation, were initially established in invertebrates and subsequently confirmed in mammals. Moreover, aberrant Notch signaling is linked to tumorigenesis. In this review, we discuss the origin of postulated Notch functions, give examples from different mammalian organ systems, and try to relate them to the hematopoietic system.

Specific fibroblastic niches in secondary lymphoid organs orchestrate distinct Notch-regulated immune responses.

Fibroblast-like cells of secondary lymphoid organs (SLO) are important for tissue architecture. In addition, they regulate lymphocyte compartmentalization through the secretion of chemokines, and participate in the orchestration of appropriate cell-cell interactions required for adaptive immunity. Here, we provide data demonstrating the functional importance of SLO fibroblasts during Notch-mediated lineage specification and immune response. Genetic ablation of the Notch ligand Delta-like (DL)1 identified splenic fibroblasts rather than hematopoietic or endothelial cells as niche cells, allowing Notch 2-driven differentiation of marginal zone B cells and of Esam(+) dendritic cells. Moreover, conditional inactivation of DL4 in lymph node fibroblasts resulted in impaired follicular helper T cell differentiation and, consequently, in reduced numbers of germinal center B cells and absence of high-affinity antibodies. Our data demonstrate previously unknown roles for DL ligand-expressing fibroblasts in SLO niches as drivers of multiple Notch-mediated immune differentiation processes.

Globin Gene Expression: Role of Transcription Factors

La dérégulation de l'expression génétique est une base pathophysiologique de plusieurs maladies. On a utilisé le locus du gène β-globine humain comme modèle pour élucider le mécanisme de régulation de la transcription génétique et évaluer son expression génétique durant l'érythropoïèse. La famille des protéines 'E' est composée de facteurs de transcription qui possèdent plusieurs sites de liaison au sein de locus du gène β-globine, suggérant leur rôle potentiel dans la régulation de l’expression de ces gènes. Nous avons montré que les facteurs HEB, E2A et ETO2 interagissent d’une manière significative avec la région contrôle du Locus (LCR) et avec les promoteurs des gènes de la famille β-globine. Le recrutement de ces facteurs au locus est modifié lors de l'érythropoïèse dans les cellules souches hematopoitiques et les cellules erythroides de souris transgéniques pour le locus de la β-globine humain, ainsi que dans les cellules progénitrices hématopoïétiques humaines. De plus par cette étude, nous démontrons pour la première fois que le gène β-globine humain est dans une chromatine active et qu’il interagit avec des facteurs de transcriptions de type suppresseurs dans les cellules progénitrices lymphoïdes (voie de différentiation alternative). Cette étude a aussi été faite dans des souris ayant une génétique mutante caractérisée par l'absence des facteurs de transcription E2A ou HEB.

DOWNREGULATION OF PAX2 SUPPRESSES OVARIAN CANCER CELL GROWTH

PAX2 is one of nine PAX genes regulating tissue development and cellular differentiation in embryos. PAX2 promotes cell proliferation, oncogenic transformation, cell-lineage specification, migration, and survival. Unattenuated PAX2 has been found in several cancer types. We therefore sought to elucidate the role of PAX2 in ovarian carcinomas. We found that PAX2 was expressed in low-grade serous, clear cell, endometrioid and mucinous cell ovarian carcinomas, which are relatively chemoresistant compared to high grade serous ovarian carcinomas. Four ovarian cancer cell lines, RMUGL (mucinous), TOV21G (clear cell), MDAH-2774 (endometrioid) and IGROV1 (endometrioid), which express high-levels of PAX2, were used to study the function of PAX2. Lentiviral shRNAs targeting PAX2 were used to knock down PAX2 expression in these cell lines. Cellular proliferation and motility assays subsequently showed that PAX2 stable knockdown had slower growth and migration rates. Microarray gene expression profile analysis further identified genes that were affected by PAX2 including the tumor suppressor gene G0S2. Reverse phase protein array (RPPA) data showed that PAX2 knockdown affected several genes that are involved in apoptosis, which supports the fact that downregulation of PAX2 in PAX2-expressing ovarian cancer cells inhibits cell growth. We hypothesize that this growth inhibition is due to upregulation of the tumor suppressor gene G0S2 via induction of apoptosis. PAX2 represents a potential therapeutic target for chemoresistant PAX2-expressing ovarian carcinomas.

The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle

The mammalian Cutl1 gene codes for the CCAAT displacement protein (CDP), which has been implicated as a transcriptional repressor in diverse processes such as terminal differentiation, cell cycle progression, and the control of nuclear matrix attachment regions. To investigate the in vivo function of Cutl1, we have replaced the C-terminal Cut repeat 3 and homeodomain exons with an in-frame lacZ gene by targeted mutagenesis in the mouse. The CDP-lacZ fusion protein is retained in the cytoplasm and fails to repress gene transcription, indicating that the Cutl1(lacZ) allele corresponds to a null mutation. Cutl1 mutant mice on inbred genetic backgrounds are born at Mendelian frequency, but die shortly after birth because of retarded differentiation of the lung epithelia, which indicates an essential role of CDP in lung maturation. A less pronounced delay in lung development allows Cutl1 mutant mice on an outbred background to survive beyond birth. These mice are growth-retarded and develop an abnormal pelage because of disrupted hair follicle morphogenesis. The inner root sheath (IRS) is reduced, and the transcription of Sonic hedgehog and IRS-specific genes is deregulated in Cutl1 mutant hair follicles, consistent with the specific expression of Cutl1 in the progenitors and cell lineages of the IRS. These data implicate CDP in cell-lineage specification during hair follicle morphogenesis, which resembles the role of the related Cut protein in specifying cell fates during Drosophila development.

TRANSCRIPTIONAL AND POST-TRANSLATIONAL MECHANISMS CONTRIBUTE TO MAINTENANCE OF REST IN NEURAL TUMORS

The RE-1 silencing transcription factor (REST) is an important regulator of normal nervous system development. It negatively regulates neuronal lineage specification in neural progenitors by binding to its consensus RE-1 element(s) located in the regulatory region of its target neuronal differentiation genes. The developmentally coordinated down-regulation of REST mRNA and protein in neural progenitors triggers terminal neurogenesis. REST is overexpressed in pediatric neural tumors such as medulloblastoma and neuroblastoma and is associated with poor neuronal differentiation. High REST protein correlate with poor prognosis for patients with medulloblastoma, however similar studies have not been done with neuroblastoma patients. Mechanism(s) underlying elevated REST levels medulloblastoma and neuroblastoma are unclear, and is the focus of this thesis project. We discovered that transcriptional and post-translational mechanisms govern REST mis-regulation in medulloblastoma and neuroblastoma. In medulloblastoma, REST transcript is aberrantly elevated in a subset of patient samples. Using loss of function and gain of function experiments, we provide evidence that the Hairy Enhancer of Split (HES1) protein represses REST transcription in medulloblastoma cell lines, modulates the expression of neuronal differentiation genes, and alters the survival potential of these cells in vitro. We also show that REST directly represses its own expression in an auto-regulatory feedback loop. Interestingly, our studies identified a novel interaction between REST and HES1. We also observed their co-occupancy at the RE-1 sites, thereby suggesting potential for co-regulation of REST expression. Our pharmacological studies in neuroblastoma using retinoic acid revealed that REST levels are controlled by transcriptional and post-transcriptional mechanisms. Post-transcriptional mechanisms are mediated by modulation of E3 ligase or REST, SCFβ-TRCP, and contribute to resistance of some cells to retinoic acid treatment.

Identification and characterization of the vegetally localized Hermes mRNA

One way developing embryos regulate the expression of their genes is by localizing mRNAs to specific subcellular regions. In the oocyte of the frog, Xenopus laevis, many RNAs are localized specifically to the animal or the vegetal halves of the oocyte. The localization of these RNAs contributes to the primary polarity of the oocyte, the asymmetry that is the basis for patterning and lineage specification in the embryo. I have screened a cDNA library for clones containing the Xlsirt repeat, an element known to target RNAs to the vegetal cortex of the oocyte. I have identified seventeen cDNA clones that contain this element. One of these cDNAs encodes the RNA binding protein Hermes. The Hermes mRNA is localized to the vegetal cortex of the oocyte. Additionally, Hermes protein is also vegetally localized in the oocyte and is found in subcellular structures known to contain localized mRNAs. This suggests that Hermes might interact with localized RNAs. While Hermes protein is present in oocytes, it disappears at germinal vesicle breakdown during maturation. We therefore believe that the time period during which Hermes functions is during oogenesis or maturation prior to the time of Hermes degradation. To determine Hermes function, an antisense depletion strategy was used that involved injecting morpholino oligos (HE-MO) into oocytes. Injection of these morpholinos causes the level of Hennes protein to drop prematurely during maturation. Embryos produced from these oocytes exhibit cleavage defects that are most prevalent in the vegetal blastomeres. The phenotype can be partially rescued by injection of a heterologous Hermes mRNA and is therefore specific to Hermes. The Hermes expression and depletion results are consistent with a model in which Hermes interacts with one or more vegetally localized mRNAs in the oocyte and during the early stages of maturation. The interaction is required for cleavage of the vegetal blastomeres. Therefore, it is likely that at least one mRNA that interacts with Hermes is a cell cycle regulator. ^

TRIB2 in normal and malignant hematopoiesis - a transcription factor network

Hematopoiesis is the tightly controlled and complex process in which the entire blood system is formed and maintained by a rare pool of hematopoietic stem cells (HSCs), and its dysregulation results in the formation of leukaemia. TRIB2, a member of the Tribbles family of serine/threonine pseudokinases, has been implicated in a variety of cancers and is a potent murine oncogene that induces acute myeloid leukaemia (AML) in vivo via modulation of the essential myeloid transcription factor CCAAT-enhancer binding protein α (C/EBPα). C/EBPα, which is crucial for myeloid cell differentiation, is commonly dysregulated in a variety of cancers, including AML. Two isoforms of C/EBPα exist - the full-length p42 isoform, and the truncated oncogenic p30 isoform. TRIB2 has been shown to selectively degrade the p42 isoform of C/EBPα and induce p30 expression in AML. In this study, overexpression of the p30 isoform in a bone marrow transplant (BMT) leads to perturbation of myelopoiesis, and in the presence of physiological levels of p42, this oncogene exhibited weak transformative ability. It was also shown by BMT that despite their degradative relationship, expression of C/EBPα was essential for TRIB2 mediated leukaemia. A conditional mouse model was used to demonstrate that oncogenic p30 cooperates with TRIB2 to reduce disease latency, only in the presence of p42. At the molecular level, a ubiquitination assay was used to show that TRIB2 degrades p42 by K48-mediated proteasomal ubiquitination and was unable to ubiquitinate p30. Mutation of a critical lysine residue in the C-terminus of C/EBPα abrogated TRIB2 mediated C/EBPα ubiquitination suggesting that this site, which is frequently mutated in AML, is the site at which TRIB2 mediates its degradative effects. The TRIB2-C/EBPα axis was effectively targeted by proteasome inhibition. AML is a very difficult disease to target therapeutically due to the extensive array of chromosomal translocations and genetic aberrations that contribute to the disease. The cell from which a specific leukaemia arises, or leukaemia initiating cell (LIC), can affect the phenotype and chemotherapeutic response of the resultant disease. The LIC has been elucidated for some common oncogenes but it is unknown for TRIB2. The data presented in this thesis investigate the ability of the oncogene TRIB2 to transform hematopoietic stem and progenitor cells in vitro and in vivo. TRIB2 overexpression conferred in vitro serially replating ability to all stem and progenitor cells studied. Upon transplantation, only TRIB2 overexpressing HSCs and granulocyte/macrophage progenitors (GMPs) resulted in the generation of leukaemia in vivo. TRIB2 induced a mature myeloid leukaemia from the GMP, and a mixed lineage leukaemia from the HSC. As such the role of TRIB2 in steady state hematopoiesis was also explored using a Trib2-/- mouse and it was determined that loss of Trib2 had no effect on lineage distribution in the hematopoietic compartment under steady-state conditions. The process of hematopoiesis is controlled by a host of lineage restricted transcription factors. Recently members of the Nuclear Factor 1 family of transcription factors (NFIA, NFIB, NFIC and NFIX) have been implicated in hematopoiesis. Little is known about the role of NFIX in lineage determination. Here we describe a novel role for NFIX in lineage fate determination. In human and murine datasets the expression of Nfix was shown to decrease as cells differentiated along the lymphoid pathway. NFIX overexpression resulted in enhanced myelopoiesis in vivo and in vitro and a block in B cell development at the pre-pro-B cell stage. Loss of NFIX resulted in disruption of myeloid and lymphoid differentiation in vivo. These effects on stem and progenitor cell fate correlated with changes in the expression levels of key transcription factors involved in hematopoietic differentiation including a 15-fold increase in Cebpa expression in Nfix overexpressing cells. The data presented support a role for NFIX as an important transcription factor influencing hematopoietic lineage specification. The identification of NFIX as a novel transcription factor influencing lineage determination will lead to further study of its role in hematopoiesis, and contribute to a better understanding of the process of differentiation. Elucidating the relationship between TRIB2 and C/EBPα not only impacts on our understanding of the pathophysiology of AML but is also relevant in other cancer types including lung and liver cancer. Thus in summary, the data presented in this thesis provide important insights into key areas which will facilitate the development of future therapeutic approaches in cancer treatment.

T cell fate specification and alphabeta/gammadelta lineage commitment.

The development of T cells from pluripotent stem cells involves a coordinated series of lineage-commitment steps. Common lymphoid precursors in the fetal liver or adult bone marrow must first choose between a T, B or NK cell fate. Committed T cell precursors in the thymus then differentiate into cells committed to the alphabeta or gammadelta lineages. Recent advances have been made in our understanding of the mechanisms underlying T cell fate specification and alphabeta/gammadelta lineage divergence.

Deficient T cell fate specification in mice with an induced inactivation of Notch1.

Notch proteins are cell surface receptors that mediate developmental cell specification events. To explore the function of murine Notch1, an essential portion of the gene was flanked with loxP sites and inactivation induced via interferon-regulated Cre recombinase. Mice with a neonatally induced loss of Notch1 function were transiently growth retarded and had a severe deficiency in thymocyte development. Competitive repopulation of lethally irradiated wild-type hosts with wild-type- and Notch1-deficient bone marrow revealed a cell autonomous blockage in T cell development at an early stage, before expression of T cell lineage markers. Notch1-deficient bone marrow did, however, contribute normally to all other hematopoietic lineages. These findings suggest that Notch1 plays an obligatory and selective role in T cell lineage induction.