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Author: Terra Kuhn
Publisher:
ISBN:
Category :
Languages : en
Pages : 250
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Book Description
Nuclear pore complexes are best known for their regulation of nucleocytoplasmic transport as integral components of the eukaryotic nuclear envelope. Over the years, their importance in regulation of genome function has become apparent. Many of the 30 individual nuclear pore proteins, Nups, have been found to play distinct roles interacting with and regulating various genomic targets, especially in a cell-type specific manner. The mechanism behind this regulation is often unknown. We have developed a method by which to study the roles of Nups on chromatin using an ectopic-tethering system. Drosophila melanogaster provide a powerful tool with which to combine many genetic elements of interest together in individual organisms quickly and efficiently, and additionally has allowed for powerful high-resolution visualization of chromatin structure perturbations through the imaging of their larval salivary gland polytene chromosomes. Using this system we observed that tethering Nups to chromatin was sufficient to induce chromatin decondensation, visualized by robust and reproducible loss of DNA and histone fluorescene signal associated with Nup binding. Additionally we observed recruitment of chromatin-remodeling complex PBAP, and reliance on PBAP for the observed Nup-induced decondensation, suggesting an important functional relationship between these proteins. We then took our findings and hypotheses generated from this ectopic-tethering imaging system to next conduct functional biochemical analysis of these proteins in Drosophila S2 cell culture. We found that nucleoporin Elys has a robust biochemical interaction with components of PBAP in an endogenous context, supporting the recruitment of these proteins we observed via immunofluorescence. Additionally, MNase experiments determined that Elys was critical for facilitating the formation and/or maintenance of open chromatin, both genome-wide and on a local nucleosomal level at Elys target genes. Together these results demonstrate the importance of nucleoporins in regulation of chromatin structure, and provide one mechanism to explain this phenomenon. These findings are of particular interest in the fields of chromatin biology and the study of nuclear pore protein function, demonstrating a possible explanation for not only associations of NPCs with decondensed chromatin at the nuclear periphery, but also regulation of Nup target gene expression, through regulation of chromatin accessibility.
Author: Terra Kuhn
Publisher:
ISBN:
Category :
Languages : en
Pages : 250
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Book Description
Nuclear pore complexes are best known for their regulation of nucleocytoplasmic transport as integral components of the eukaryotic nuclear envelope. Over the years, their importance in regulation of genome function has become apparent. Many of the 30 individual nuclear pore proteins, Nups, have been found to play distinct roles interacting with and regulating various genomic targets, especially in a cell-type specific manner. The mechanism behind this regulation is often unknown. We have developed a method by which to study the roles of Nups on chromatin using an ectopic-tethering system. Drosophila melanogaster provide a powerful tool with which to combine many genetic elements of interest together in individual organisms quickly and efficiently, and additionally has allowed for powerful high-resolution visualization of chromatin structure perturbations through the imaging of their larval salivary gland polytene chromosomes. Using this system we observed that tethering Nups to chromatin was sufficient to induce chromatin decondensation, visualized by robust and reproducible loss of DNA and histone fluorescene signal associated with Nup binding. Additionally we observed recruitment of chromatin-remodeling complex PBAP, and reliance on PBAP for the observed Nup-induced decondensation, suggesting an important functional relationship between these proteins. We then took our findings and hypotheses generated from this ectopic-tethering imaging system to next conduct functional biochemical analysis of these proteins in Drosophila S2 cell culture. We found that nucleoporin Elys has a robust biochemical interaction with components of PBAP in an endogenous context, supporting the recruitment of these proteins we observed via immunofluorescence. Additionally, MNase experiments determined that Elys was critical for facilitating the formation and/or maintenance of open chromatin, both genome-wide and on a local nucleosomal level at Elys target genes. Together these results demonstrate the importance of nucleoporins in regulation of chromatin structure, and provide one mechanism to explain this phenomenon. These findings are of particular interest in the fields of chromatin biology and the study of nuclear pore protein function, demonstrating a possible explanation for not only associations of NPCs with decondensed chromatin at the nuclear periphery, but also regulation of Nup target gene expression, through regulation of chromatin accessibility.
Author: Guilia Jale Gurun Ruben
Publisher:
ISBN:
Category :
Languages : en
Pages : 288
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Author: Maximiliano D’Angelo
Publisher: Springer
ISBN: 331971614X
Category : Medical
Languages : en
Pages : 245
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Book Description
The three-dimensional organization of the DNA inside the eukaryotic cell nucleus has emerged a critical regulator of genome integrity and function. Increasing evidence indicates that nuclear pore complexes (NPCs), the large protein channels that connect the nucleus to the cytoplasm, play a critical role in the establishment and maintenance of chromatin organization and in the regulation of gene activity. These findings, which oppose the traditional view of NPCs as channels with only one: the facilitation of nucleocytoplasmic molecule exchange, have completely transformed our understanding of these structures. This book describes our current knowledge of the role of NPCs in genome organization and gene expression regulation. It starts by providing an overview of the different compartments and structures of the nucleus and how they contribute to organizing the genome, then moves to examine the direct roles of NPCs and their components in gene expression regulation in different organisms, and ends by describing the function of nuclear pores in the infection and genome integration of HIV, in DNA repair and telomere maintenance, and in the regulation of chromosome segregation and mitosis. This book provides an intellectual backdrop for anyone interested in understanding how the gatekeepers of the nucleus contribute to safeguarding the integrity and function of the eukaryotic genome.
Author: David Wallace Van de Vosse
Publisher:
ISBN:
Category : Genetic regulation
Languages : en
Pages :
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Author: R. Curtis Bird
Publisher: Elsevier
ISBN: 0080537812
Category : Science
Languages : en
Pages : 317
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Book Description
Nuclear Structure and Gene Expression assimilates the contributions of genome organization and of the components of the nuclear matrix to the control of DNA and RNA synthesis. Nuclear domains which accommodate DNA replication and gene expression are considered in relation to short-term developmental and homeostatic requirements as well as to long-term commitments to phenotypic gene expression in differentiated cells. Consideration is given to the involvement of nuclear structure in gene localization as well as to the targeting and concentration of transcription factors. Aberrations in nuclear architecture associated with and potentially functionally related to pathologies are evaluated. Tumor cells are described from the perspective of the striking modifications in both the composition and organization of nuclear components. Nuclear Structure and Gene Expression presents concepts as well as experimental approaches, which define functionality of nuclear morphology. * Mechanisms of interaction between nuclear structure and genes * Gene expression regulation by elements of the nuclear matrix * How nuclear structure exerts a regulatory effect on other aspects of cell function/physiology
Author: Dennis Jacob Bua
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
The physiological state of the genome is chromatin. Chromatin is a dynamic polymer composed of genomic DNA, histone proteins, and other factors. Dysregulation of chromatin homeostasis can lead to diverse human pathologies including cancer. This work focuses on chromatin-based mechanisms of gene regulation; more specifically the (1) targeting and (2) stabilization of gene-regulatory proteins on the chromatin template. In regard to chromatin targeting, not much is known about how gene-regulatory proteins selectively associate with their targets. Herein we detail the discovery of a new targeting factor, the nuclear phospholipid phosphatidylinositol-5-phosphate (PtdIns(5)P). PtdIns(5)P directly interacts with the tumor suppressor ING2 (inhibitor of growth family member 2) in the nucleus to coordinate gene expression of select ING2 targets. In regard to chromatin stabilization, lysine methylation is a principle mechanism for retaining chromatin-effector modules at discrete chromatin zones. Numerous lysine methylation events have been discovered on the major protein component of chromatin, histones, but relatively few specific modules have been characterized to sense these modifications. We screened through a library of putative methyl-lysine binding domains, which resulted in the identification of three novel chromatin binding modules. Taken together these data shed insight into the molecular modes of action of gene regulation by chromatin and phosphoinositide pathways.
Author: Christophe Lavelle
Publisher: Academic Press
ISBN: 012803503X
Category : Science
Languages : en
Pages : 620
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Book Description
Nuclear Architecture and Dynamics provides a definitive resource for (bio)physicists and molecular and cellular biologists whose research involves an understanding of the organization of the genome and the mechanisms of its proper reading, maintenance, and replication by the cell. This book brings together the biochemical and physical characteristics of genome organization, providing a relevant framework in which to interpret the control of gene expression and cell differentiation. It includes work from a group of international experts, including biologists, physicists, mathematicians, and bioinformaticians who have come together for a comprehensive presentation of the current developments in the nuclear dynamics and architecture field. The book provides the uninitiated with an entry point to a highly dynamic, but complex issue, and the expert with an opportunity to have a fresh look at the viewpoints advocated by researchers from different disciplines. Highlights the link between the (bio)chemistry and the (bio)physics of chromatin Deciphers the complex interplay between numerous biochemical factors at task in the nucleus and the physical state of chromatin Provides a collective view of the field by a large, diverse group of authors with both physics and biology backgrounds
Author: Min Kim
Publisher:
ISBN:
Category :
Languages : en
Pages : 99
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Book Description
Chromatin is a mixture of DNA and DNA binding proteins that control transcription. Dynamic chromatin structure modulates gene expression and is responsible for an extraordinary spectrum of developmental processes. An intricate interplay of DNA methylation, histone modifications, histone variants, small RNA accumulation, and ATPase chromatin remodelers defines chromatin re-configuration in a precise manner, locally within a cell and globally across different cell types. The development of high-throughput screening methods such as microarray and whole-genome sequencing has led to an explosion of chromatin studies in the past decade. Moreover, genetic and molecular studies resulted in identification of a number of proteins that may influence chromatin structure. However, the exact functions of individual proteins as well as their functional relationships to each other are less understood. Also, the role of chromatin components in establishing cell- and tissue-specific chromatin structure is largely unknown. To address these open questions in chromatin biology, I focused my dissertation work on 1) studying tissue-specific DNA demethylation in seed, and 2) determining the role of a ubiquitous DNA binding protein, linker histone H1, in regulating chromatin structure. Tissue specific DNA methylation in seed. In endosperm, the nutritive tissue that nourishes the embryo, parent-of-origin specific gene expression is regulated by DNA demethylation. However, the extent to which DNA demethylation occurs in a tissue-specific manner and regulates transcription in the endosperm of crop plants like rice remains unknown. To address these questions, my colleagues and I examined the DNA methylation patterns of two rice seed tissues, embryo and endosperm. We found that endosperm genome is globally hypomethylated at non-CG sites and locally hypomethylated at CG-sites compared to embryo. We also identified that small transposons near genes (euchromatic regions) are the primary targets of DNA demethylation. The loci near the genes preferentially expressed in endosperm (e.g. storage protein and starch synthesizing enzymes) are subjected to local hypomethylation, suggesting that DNA methylation plays a role in inducing tissue-specific genes in endosperm. The role of H1 in regulating chromatin structure. H1 is proposed to facilitate higher order chromatin structure, but its effects on individual chromatin components and transcription are less understood. To resolve this issue, we investigated the role of H1 in regulating DNA methylation, nucleosome positioning, and transcription. We identified that H1 was most enriched in transposons. H1 was also found in genes at a lower level compared to transposons, and the abundance of H1 was anticorrelated with gene expression. Moreover, H1 influences nucleosome positioning by increasing the distance between two nucleosomes. Lack of H1 resulted in increased DNA methylation of transposons with heterochromatic features. In contrast, an h1 mutant showed a reduction of DNA methylation in genes and transposons with euchromatic features. Our finding suggests that H1 has a dual function in regulating DNA methylation. That is, H1 inhibits both DNA methyltransferases and DNA demethylation-associated enzymes from binding heterochromatin and euchromatin, respectively. In addition, the hypermethylated loci in our h1 mutant almost perfectly overlapped with the hypomethylated loci in a ddm1 mutant in heterochromatin, suggesting a link between these two proteins. DDM1 is an Snf2 chromatin remodeler that can slide nucleosomes along DNA and has been proposed to provide DNA methyltransferase access to target sequences. We further determined their functional relationship by crossing h1 and ddm1 mutants, and generated a map of DNA methylation of the cross. We identified that loss of DNA methylation from ddm1 was partially recovered by removing H1. Also the mutant phenotype observed in ddm1 disappeared in h1ddm1. Based on our results, we proposed a model where DDM1-mediated chromatin destabilization releases H1 binding, which in turn increases DNA accessibility. It is noteworthy that DNA demethylation preferentially occurred in euchromatin in both the rice seed DNA methylation study and the H1 study. Based on this result, we proposed that the apparent target preference of DNA demethylation-associated proteins depends on the underlying chromatin structure. We think that this chromatin structure-mediated specificity also dictates other nucleoproteins to determine/recognize their targets. My dissertation work tackled multiple aspects of chromatin biology: tissue-specific chromatin regulation, and the interplay between chromatin components in chromatin organization. Together, the results from my work enhanced our knowledge of how chromatin components influence overall chromatin structure.
Author: Pierre Bensidoun
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
The export of mRNAs from the nucleus to the cytoplasm is one of many steps along the gene expression pathway and is fundamental for mRNAs to meet with ribosomes for translation in the cytoplasm. Exchanges between nucleus and cytoplasm occur through the nuclear pore complex (NPC), which is a large multi-protein complex embedded in the nuclear membrane and assembled by 30 different proteins the nucleoporins. The nucleoplasmic side of the pore is believed to orchestrate many fundamental nuclear processes. Indeed, a growing body of evidence suggests that the nuclear pore is involved in a broad range of activities including modulation of DNA topology, DNA repair, epigenetic regulation of gene expression, and selective access to exporting molecules. The structural component required for orchestrating those nucleoplasmic functions is the basket, a ∼60- to 80-nm-long structure protruding into the nucleoplasm. The consensus view depicts the basket as a structure assembled by filamentous proteins, TPR (Translocated Promoter Region protein) in humans and by its two paralogues Mlp1 and Mlp2 (myosin-like proteins) in yeast, converging into a distal ring. In the first part of this thesis, we characterized the motion of specific mRNAs at the vicinity of the nuclear periphery. We observed that transcripts scan along the nuclear envelope, likely to find a nuclear pore to be exported. We also showed the scanning behavior was affected upon Mlp1 deletion or truncation as well as upon mutation of the nuclear poly(A) binding protein Nab2. These observations indicated that Mlp1 and hence baskets, as well as specific RNA binding proteins, facilitate the interaction of mRNA with the nuclear periphery. While the canonical structure of the NPC is well established, our understanding of the conditions and factors contributing to the assembly of a basket, as well as the stoichiometry of its components, remains incomplete. Although basket proteins have been implicated in the regulation of gene expression through gene anchoring to the nuclear periphery and in mRNA scanning before export, how this is mediated by Mlp1/2 is poorly understood. Moreover, the dynamics of basket proteins in yeast seem to obey different rules than those of other nucleoporins as their turnover at the pore is faster than any other NPC components. Furthermore, it has been observed that during heat shock Mlp1 and Mlp2 dissociate from nuclear pores and form intra-nuclear granules, sequestering mRNAs and RNA export factors. Yet the mechanism for the formation of these granules or their role during heat shock is poorly understood. In yeast, the nuclear baskets are not associated with all NPCs, as no baskets assemble on the pores adjacent to the nucleolus. Yet, how cells establish these basket-less pores and whether they represent specialized nuclear pores with different functions from basket-containing pores is still unknown. To understand the dynamics of basket assembly and the biological relevance of establishing distinct sets of pores, we dissected the biological processes leading to the formation of baskets. In addition, to highlight potential functional differences between the two types of pores, we identified the interactors of nuclear basket-containing and nucleolar basket-less pores. We showed that assembling a basket is not a default mode for a pore in the nucleoplasm and that active mRNA processing is required to maintain baskets integrity. While mRNA can be found associated with both types of pores, our results suggest that export kinetics may be different on basket-containing and basket-less pores. The eukaryotes organize their nucleus in discrete functional regions and the nuclear envelope has been envisioned as an organelle by and of itself. Our analyzes indicate that mRNAs and Mlp1 participate in an additional degree of nuclear compartmentalization by enabling the formation of a dynamic structure: the basket. Overall my project sheds new light on the nuclear organization and highlights the surprising entanglement between mRNA export and NPC plasticity.
Author: Jeanae Kaneshiro
Publisher:
ISBN:
Category :
Languages : en
Pages : 119
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Book Description
The nuclear envelope (NE) is associated with two major protein complexes: nuclear pore complexes (NPCs) and the nuclear lamina. NPCs are ~120 MDa structures that perforate the NE and mediate nucleocytoplasmic transport, and the nuclear lamina is a network of type V intermediate filaments that forms a meshwork lining the nucleoplasmic side of the inner nuclear membrane (INM) and provides structure to the nucleus. Aside from these canonical functions of NPCs and the nuclear lamina, it has been discovered that these complexes and their subcomponents have additional roles, such as those in chromatin organization and gene regulation. This dissertation aims to expand the known functions of proteins within these complexes, and focuses on two NPC components, Nup98 and Nup96 (Nup98/96), and the lamin isoform, Lamin B1 (LmnB1). Chapter 2 suggests a novel function for Nup98/96 in maintaining the integrity of the nuclear lamina. Small interfering RNA (siRNA) mediated knockdown (KD) of Nup98/96 enlarges the lamina meshwork across the nuclear surface and increases the frequency of transient nuclear envelope rupture. This weakening of the nuclear lamina was further determined to be a consequence of reduced LmnB1 expression, and Chapter 3 shows that LmnB1 is post-transcriptionally regulated through its 3' untranslated region upon Nup98/96 KD. However, further analysis indicated that the siRNA oligonucleotides designed against Nup98/96 also target LmnB1 for degradation. These siRNAs mimicked microRNAs, hsa-miR-218 and hsa-miR-636, through sequence similarities within their seed regions, allowing these siRNAs to directly target LmnB1. Therefore, Nup98/96 does not regulate the nuclear lamina through LmnB1, and the ability of siRNAs to mimic miRNAs should be carefully considered when designing and utilizing siRNAs in future studies. Chapter 4 describes the effects of LmnB1 overexpression (OE) on chromatin organization and senescence induction. LmnB1 OE induces the formation of heterochromatic DNA foci within the nucleoplasm, coinciding with a reduction of heterochromatin at the nuclear periphery. This leads to changes in gene expression, which may be a consequence of altered chromatin accessibility or changes in histone modifications. The release of heterochromatin from the nuclear periphery is not a consequence of reducing other NE proteins, such as Lamin A or lamin B receptor, that are important for tethering heterochromatin at the nuclear periphery. This suggests that LmnB1 OE might increase the thickness of the nuclear lamina and disrupt the binding of heterochromatin tethers at the nuclear periphery. Finally, although the induced heterochromatin foci are reminiscent of the DNA organization described in senescent cells, LmnB1 OE slows cell proliferation but does not induce senescence. Overall, this dissertation demonstrates the importance of tightly regulating LmnB1 expression to maintain nuclear integrity and chromatin organization. Several human diseases show misregulation of LmnB1, and an interesting observation is the increased expression of LmnB1 observed in various types of cancer, some of which also correlate with higher tumor grade and poor prognosis. This suggests that LmnB1 OE might promote tumorigenesis, which may be mediated through changes in chromatin organization and gene expression, as shown in Chapter 4. However, given the reduction in cell proliferation that was also observed upon LmnB1 OE, further mechanistic studies will be required to reconcile whether LmnB1 OE may be pro-tumorigenic or tumor suppressive.
