2001 — 2005 |
Panning, Barbara |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Xist Rna and Regulation of Chromatin Structure @ University of California San Francisco
DESCRIPTION (APPLICANT'S ABSTRACT): The long-term goal of this work is to understand how RNA containing complexes modulate chromatin structure and gene expression. X chromosome inactivation is a particularly dramatic example of a change in chromatin structure that is regulated by RNA. The Xist gene regulates X-inactivation. Xist encodes a non-coding RNA that is transcribed exclusively from the Xi in female somatic cells. During initiation of X-inactivation Xist RNA spreads in cis to coat the chromosome that will become the Xi, and the spread of transcriptional silencing correlates tightly with the cis-spread of Xist RNA. My genetic, developmental and cytological analysis showed that Xist RNA is stabilized at the initiation of X-inactivation. These results lead to the hypothesis that a developmentally regulated Xist RNA - protein interaction facilitates stabilization and spread of Xist RNA on the Xi. As an independent investigator I have demonstrated that Xist RNA is part of an RNA-protein complex and that antisense affinity chromatography can be employed to purify this complex. Using a combination of powerful and complementary approaches, including biochemical purification, genetic manipulation of embryonic stem cells, and molecular cytogenetics, we propose to identify proteins that interact with Xist RNA. The mechanisms Xist uses to regulate X-inactivation are poorly understood and the identification of Xist-interacting proteins is the first step in analysis of the molecular basis of Xist RNA function. It is highly likely that RNA molecules are more generally used for chromatin remodeling and regulation of gene expression in eukaryotes since RNA is crucial for the functional organization of the nucleus and is an integral part of the dosage compensation apparatus in flies and mammals. Our understanding of the mechanisms by which RNA-protein complexes affect chromatin structure would be greatly facilitated by identification of additional Xist-like regulatory RNA molecules. We have developed a method to isolate chromatin associated RNAs and used this method to isolate an RNA that accumulates on mouse centromeres. We propose to characterize this novel RNA. The characterization of both the RNA and the protein components of Xist and other RNA-protein complexes that affect chromatin structure will provide insight into the role nuclear RNA plays in regulating gene expression.
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1 |
2006 — 2011 |
Panning, Barbara |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Posttranslational Modifications On Histones and Histone Variants @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Multicellular organisms consist of various cell types with same genetic information but high degree of differentiation, characterized by a unique pattern of gene expression for each cell type. Establishment and maintenance of these diverse expression patterns is fundamentally important for cell identity and organism survival, and aberrant gene expression in a single cell can lead to developmental abnormalities or cancer. Epigenetic regulatory mechanisms employ dynamic modifications in chromatin structure, such as histone methylation, acetylation, phosphorylation and ubiquitination, to regulate gene expression. These posttranslational modifications are integrated in a combinatorial fashion to provide cells with transcriptional memory to stably maintain gene expression patterns throughout many divisions, and developmental flexibility to facilitate programmed alterations in gene expression. Equipped with the unprecedented sensitivity and structural specificity offered by tandem mass spectrometry, we are hoping to elucidate the nature and functional role of the rich epigenetic information in mammalian chromatin.
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1 |
2009 — 2012 |
Panning, Barbara |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Investigation of the Tip60-P400 Complex Role in Embryonic Stem Cell Self-Ren @ University of California, San Francisco
DESCRIPTION (provided by applicant): Multicellular organisms consist of hundreds of different cell types, each of which is characterized by a unique pattern of gene expression. Different cell types must stably maintain the transcriptional patterns that specify that cell type, yet have sufficient plasticity to allow the alterations in gene expression that are necessary for differentiation or responses to environmental changes. Establishment and maintenance of these diverse expression patterns is fundamentally important for cell identity and organismal survival, since aberrant gene expression in a single cell can lead to developmental abnormalities or cancer. We use mouse embryonic stem cells (ESCs) to study the molecular mechanisms that are used to regulate gene expression in mammalian cells. ESCs are pluripotent, with the ability to differentiate into every cell type that makes up the developing and adult organism. ESCs continue to self-renew, or indefinitely proliferate in the pluripotent state, when they are cultured under appropriate conditions. Several transcription factors, such as Oct4, Nanog, Sox2, Foxd3, and Stat3, are essential for self-renewal and pluripotency. While it is thought that regulation at the level of chromatin structure is also important in ESC self-renewal and pluripotency, the role of chromatin regulatory proteins in these processes remains more poorly understood. Here we propose to investigate the role of the Tip60-p400 histone acetyltransferase-chromatin remodeling complex in ESC self-renewal. Our preliminary results indicate that tip60-p400 cooperates with the pluripotency transcription factor Nanog to maintain the ESC-specific expression profile that promotes self-renewal and pluripotency. Our studies will focus on understanding the molecular nature of the functional interaction between the Tip60-p400 complex and Nanog. Finally, our preliminary results implicate a second chromatin remodeling complex, the Brg1-containing Swi/Snf complex in ESC self-renewal. We will also investigate the mechanisms by which this complex regulates the ESC-specific gene expression profile. PUBLIC HEALTH RELEVANCE: Embryonic stem cells can grow indefinitely as an established cell line, a process referred to as self-renewal. ESCs are highly proliferative in their undifferentiated state. In contrast, somatic stem cells, such as hematopoietic stem cells, grow more slowly than ESCs and do not expand without significant accompanying differentiation. ESCs are also pluripotent - they have the ability to differentiate into all cell types. Because of these properties, ESCs are regarded as a major potential source of material for stem cell therapies. However, the molecular mechanisms governing self-renewal and pluripotency are largely unknown. A better molecular understanding at the factors that induce and maintain the stem cell state ESCs is essential for moving towards the implementation of ESC-based therapies. We employ mouse ESCs to understand the role of chromatin regulatory proteins, which regulate gene expression by affecting the accessibility of the DNA template, in ESC self-renewal and pluripotency. These studies will have important impacts on the development of ESC lines for stem-cell based therapies.
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1 |
2010 — 2013 |
Panning, Barbara |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Xist Rna Processing in Embryonic Stem Cells @ University of California, San Francisco
DESCRIPTION (provided by applicant): ABSTRACT Non-coding RNAs with gene regulatory functions are starting to be seen as a common feature of mammalian gene regulation with the discovery that most of the transcriptome is non-coding RNA. Given that a significant proportion of the genome encodes non-coding RNAs, relatively little is known about the regulatory mechanisms and functions of these RNAs. Thus, new insights into how non-coding RNAs are regulated and how they regulate gene expression are essential for a complete understanding of mammalian gene expression. We employ X chromosome inactivation as a model system to study the regulation and function of non-coding RNAs. X-inactivation is the developmentally regulated transcriptional silencing of one X chromosome in female cells, used to equalize X-linked gene dosage with male cells. An antisense pair of non-coding RNAs, Xist and Tsix RNA, are central in the regulation of X-inactivation. X-inactivation is random - the X chromosome from each parent is silenced with equal frequency. Xist and Tsix RNA are expressed before the onset of X chromosome silencing and are necessary for X-inactivation to occur randomly. In Xist or Tsix mutant cells, X-inactivation is non-random and the fates of the mutant and the wild-type X chromosomes are fixed. Xist and Tsix mutations have opposite effects on X chromosome fate: an Xist mutant chromosome is always chosen as the active X and a Tsix mutant X chromosome is always chosen as the inactive X. Xist and Tsix also negatively regulate each other's expression, forming a feedback loop. In this proposal we dissect the Xist/Tsix feedback loop and explore how it is used to ensure that each X chromosome has an equal frequency of being silenced. PUBLIC HEALTH RELEVANCE: Project Narrative (Public Health Relevance) Non-coding RNAs play critical roles in regulating DNA structure, RNA expression, and translation, and thus affect normal development. While non-coding RNAs are already being identified as markers for cancer and associated with other complex diseases such as coronary disease and diabetes, little is understood about their regulation and function. A better understanding of non-coding RNAs will undoubtedly be important in the diagnosis and treatment of these conditions. We use X-inactivation as a model system to study the regulation and function of non-coding RNAs. In mammalian female cells, one X chromosome is silenced. This silencing ensures that X-linked gene dosage in XX female cells is equivalent to that in XY male cells. This process is essential for the survival of females. A pair of non- coding RNAs, Xist and Tsix RNA, is central in the regulation of X-inactivation. In this submission, we propose to study the regulation and function of Xist and Tsix RNA. What we learn will undoubtedly contribute to our understanding of X-inactivation, and more generally to the function of non-coding RNAs.
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1 |
2011 |
Panning, Barbara |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Regulation of 3-D Chromatin Architecture of Embryonic Stem Cells @ University of California, San Francisco
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Embryonic stem cells (ESCs) provide an experimentally tractable system to study developmentally regulated alterations in chromatin structure. ESCs are regarded as a potential source of material for stem cell therapies because they are pluripotent, or have the ability to differentiate into all cell types, and can grow indefinitely in the pluripotent state[14]. However, the molecular mechanisms governing self-renewal and pluripotency are not well understood. Work from many labs, including our own, shows that chromatin regulatory proteins play an important role in ESC self-renewal and pluripotency. ESCs are characterized by higher order chromatin structure that is generally dynamic and permissive to the transcriptional machinery. Genome-wide mapping of DNA methylation profiles and of post-translational histone modifications showed that specialized chromatin states characterize promoters of active, repressed and potentially active genes in ESCs. In addition, the same chromatin regulatory complex can have a very different role in ESCs than in somatic cells. In some instances, complexes that are essential in somatic cells are dispensable in ESCs: the Polycomb Repressive Complexes (PRCs) provide such an example.
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1 |
2012 — 2015 |
Panning, Barbara |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Investigation of X Chromosome Organization Before the Onset of X-Inactivation @ University of California, San Francisco
DESCRIPTION (provided by applicant): The position of substructures and DNA sequences within the nucleus is highly organized, and this organization plays a role in regulating chromatin-dependent processes, such as gene expression. X chromosome inactivation is an experimentally amenable example of a phenomenon in which regulated changes in the location of specific DNA sequences is implicated in control of gene expression. In mammalian female cells, one X chromosome is silenced for dosage compensation of X-linked genes between males and females, and this process is essential for female survival. Here, we propose to investigate the molecular basis of the regulated changes in X chromosome nuclear organization that accompany X-inactivation. The insights gained are likely to be more generally applicable, since many of the factors that regulate X-inactivation also regulate gene expression in other contexts.
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1 |
2015 — 2019 |
Panning, Barbara Olshen, Adam Fung, Jennifer (co-PI) [⬀] Marshall, Wallace [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Quantitative Cell Geometry - Defining Cell State At the Organelle Level @ University of California-San Francisco
Cells are complex machines filled with molecules that can perform simple logic functions like the circuits inside a computer. Many cells can perform complex behaviors such as engulfing other cells, movement towards a source of food, and remembering past events. Even though most cellular components are believed to be discovered, it is still unclear how the molecules in a cell work together to perform relatively complex actions. This project seeks to solve the problem by applying concepts from theoretical computer science to understand how the cell switches from one activity to another. This will open up new possibilities for re-engineering cells by reprogramming their internal controls. This research program will create novel educational and outreach opportunities based on exposing students from a variety of scientific backgrounds, as well as members of the public with interests in electronics and computer hobbies, to the idea that cells can be viewed as computing machines; therefore awareness of quantitative cell biology among the next generation of engineering students will increase.
While general understanding of the molecular biology of cells is constantly increasing, it has proven difficult to integrate this molecular scale information into a global view of how cells make decisions and perform complex behaviors like migration, phagocytosis, and division. The goal of this project is to connect the huge gap in complexity and detail from the molecular scale to the level of cell behavior by using concepts from computer science. In computer science, the highly complex details of electronic circuitry can often be understood, analyzed, and designed by using abstract models in which a complex system is represented by a finite set of states, allowing behavior to be represented by transitions between states. Such models are called finite state automata and they are the most fundamental representation of a computing device. In this project cells are described as finite state automata, by using organelle size measurements to identify and define distinct states. It is hypothesized that an organelle-level state description will allow for the reduction of the dimensionality of the state space from millions of dimensions corresponding to individual molecules in the cell down to a much smaller number of dimensions based on organelle morphological measurements that are readily observable in living cells. Large numbers of cells will be imaged at high resolution, numerical descriptors of each organelle will be described, and a state space for cellular organization will be defined using statistically rigorous methods to define states and state transitions. The investigators will explore how chemical and mechanical inputs to the cell drive transitions within this state space, thus providing a way to view the cell as a type of finite-state automaton. Such a representation will also provide a framework for synthetic biology applications in which the regulatory pathways that determine state transitions could be re-wired to produce different behaviors, essentially turning a single cell into a programmable microdevice. The conceptual framework of the cell as a decision-making computational device will be harnessed to present outreach exhibits at Maker Faires, which are an ongoing series of events that bring together electronics, computer, and crafts hobbyists.
This project is co-funded by the program in Cellular Dynamics and Function in the division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences and the programs in Statistics and Mathematical Biology in the division of Mathematical Sciences in the Directorate of Mathematics and Physical Sciences.
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1 |
2016 — 2019 |
Panning, Barbara |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Sox2 O-Gicnacylation in Pluripotency @ University of California, San Francisco
? DESCRIPTION (provided by applicant): Transcription factors are central for establishing the cell type-specific gene expression patterns that characterize each of the hundreds of different cell types in developing and adult mammals. Transcription factors are often regulated by signaling pathways, which affect the activity of these factors and ensure appropriate transcriptional responses to developmental and environmental cues. Our preliminary studies indicate that a transcription factor central to embryonic stem cell self-renewal, SOX2, is modified by addition of an O-linked N-acetylglucosamine sugar to serine residue 248 and threonine residue 213. This modification is mediated by the enzyme OGT, which is a central player in the nutrient sensing pathway that detects levels of glucose and other nutrients. We propose to investigate the role of this post-translational modification of SOX2 in self-renewal, to better understand how metabolic pathways impact gene expression in pluripotent cells.
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1 |
2019 — 2021 |
Burlingame, Alma L (co-PI) [⬀] Panning, Barbara |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ogt as a Dosage Sensor @ University of California, San Francisco
Project Summary Epigenetic regulators often lie downstream of signaling pathways that culminate in post-translational modifications, which affect their activity and ensure appropriate transcriptional responses to developmental and environmental cues. Our preliminary studies indicate that an interaction between epigenetic regulator, TET3, and an X-linked, post-translational modification enzyme, OGT, may be central in detecting X chromosome dosage and poising cells for X-inactivation. We propose to investigate the role of the TET3-OGT interaction in regulation of X chromosome inactivation, to obtain molecular insight into how cells count X chromosomes.
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