ABSTRACT
The accurate segregation of chromosomes to daughter cells is essential for healthy development to occur. Imbalances in chromosome number have long been associated with cancers amongst other medical disorders. Little is known whether abnormal chromosome numbers are an early contributor to the cancer progression pathway. Centromere DNA and protein defects are known to impact on the fidelity of chromosome segregation in cell and model systems. In this chapter we discuss recent developments in understanding the contribution of centromere abnormalities at the protein and DNA level and their role in cancer in human and mouse systems.
Subject(s)
Centromere/genetics , Centromere/pathology , Chromosome Segregation , DNA-Binding Proteins/metabolism , DNA/metabolism , Neoplasms/genetics , Neoplasms/pathology , Animals , Centromere/metabolism , Humans , Neoplasms/metabolismABSTRACT
Molecular and cellular signaling pathways are involved in the process of neural differentiation from human embryonic stem cells (hESC) to terminally differentiated neurons. The Sonic hedgehog (SHH) morphogen is required to direct the differentiation of hESC to several neural subtypes, for example, dopaminergic (DA) or motor neurons. However, the roles of SHH signaling and the pathway target genes that regulate the diversity of cellular responses arising from SHH activation during neurogenesis of hESC have yet to be elucidated. In this study, we report that overexpression of SHH in hESC promotes the derivation of neuroprogenitors (NP), increases proliferation of NP, and subsequently increases the yield of DA neurons. Next, gene expression changes resulting from the overexpression of SHH in hESC-derived NP were examined by genome-wide transcriptional profiling. Categorizing the differentially expressed genes according to the Gene Ontology biological processes showed that they are involved in numerous cellular processes, including neural development, NP proliferation, and neural specification. In silico GLI-binding sites analysis of the differentially expressed genes also identified a set of putative novel direct target genes of SHH in hESC-derived NP, which are involved in nervous system development. Electrophoretic mobility shift assays and promoter-luciferase assays confirmed that GLI1 binds to the promoter region and activates transcription of HEY2, a NOTCH signaling target gene. Taken together, our data provide evidence for the first time that there is cross-talk between the NOTCH and SHH signaling pathways in hESC-derived NP and also provide significant new insights into transcriptional targets in SHH-mediated neural differentiation of hESC.
Subject(s)
Dopaminergic Neurons/metabolism , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Hedgehog Proteins/genetics , Neural Stem Cells/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Blotting, Western , Cell Differentiation/genetics , Cell Line , Dopaminergic Neurons/cytology , Dopaminergic Neurons/physiology , Embryonic Stem Cells/cytology , Eye Proteins/genetics , Eye Proteins/metabolism , Fluorescent Antibody Technique , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Hedgehog Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Membrane Potentials , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin , Neural Stem Cells/cytology , Neural Stem Cells/physiology , PAX6 Transcription Factor , Paired Box Transcription Factors/genetics , Paired Box Transcription Factors/metabolism , Patch-Clamp Techniques , Promoter Regions, Genetic/genetics , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Finger Protein GLI1ABSTRACT
Rapid cellular growth and multiplication, limited replicative senescence, calibrated sensitivity to apoptosis, and a capacity to differentiate into almost any cell type are major properties that underline the self-renewal capabilities of human pluripotent stem cells (hPSCs). We developed an integrated bioinformatics pipeline to understand the gene regulation and functions involved in maintaining such self-renewal properties of hPSCs compared to matched fibroblasts. An initial genome-wide screening of transcription factor activity using in silico binding-site and gene expression microarray data newly identified E2F as one of major candidate factors, revealing their significant regulation of the transcriptome. This is underscored by an elevated level of its transcription factor activity and expression in all tested pluripotent stem cell lines. Subsequent analysis of functional gene groups demonstrated the importance of the TFs to self-renewal in the pluripotency-coupled context; E2F directly targets the global signaling (e.g. self-renewal associated WNT and FGF pathways) and metabolic network (e.g. energy generation pathways, molecular transports and fatty acid metabolism) to promote its canonical functions that are driving the self-renewal of hPSCs. In addition, we proposed a core self-renewal module of regulatory interplay between E2F and, WNT and FGF pathways in these cells. Thus, we conclude that E2F plays a significant role in influencing the self-renewal capabilities of hPSCs.
