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Jane E. Johnson - One of the best experts on this subject based on the ideXlab platform.
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Positive autofeedback regulation of PTF1A transcription generates the levels of PTF1A required to generate itch circuit neurons
Genes & development, 2020Co-Authors: Bishakha Mona, Trisha K. Savage, Rahul K. Kollipara, Juan Villarreal, Brooke E. Boisvert, Jane E. JohnsonAbstract:Peripheral somatosensory input is modulated in the dorsal spinal cord by a network of excitatory and inhibitory interneurons. PTF1A is a transcription factor essential in dorsal neural tube progenitors for specification of these inhibitory neurons. Thus, mechanisms regulating PTF1A expression are key for generating neuronal circuits underlying somatosensory behaviors. Mutations targeted to distinct cis-regulatory elements for PTF1A in mice, tested the in vivo contribution of each element individually and in combination. Mutations in an autoregulatory enhancer resulted in reduced levels of PTF1A, and reduced numbers of specific dorsal spinal cord inhibitory neurons, particularly those expressing Pdyn and Gal Although these mutants survive postnatally, at ∼3-5 wk they elicit a severe scratching phenotype. Behaviorally, the mutants have increased sensitivity to itch, but acute sensitivity to other sensory stimuli such as mechanical or thermal pain is unaffected. We demonstrate a requirement for positive transcriptional autoregulatory feedback to attain the level of the neuronal specification factor PTF1A necessary for generating correctly balanced neuronal circuits.
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Regulating the dorsal neural tube expression of PTF1A through a distal 3' enhancer.
Developmental biology, 2016Co-Authors: Bishakha Mona, David Meredith, Rahul K. Kollipara, John M. Avila, Jane E. JohnsonAbstract:Generating the correct balance of inhibitory and excitatory neurons in a neural network is essential for normal functioning of a nervous system. The neural network in the dorsal spinal cord functions in somatosensation where it modulates and relays sensory information from the periphery. PTF1A is a key transcriptional regulator present in a specific subset of neural progenitor cells in the dorsal spinal cord, cerebellum and retina that functions to specify an inhibitory neuronal fate while suppressing excitatory neuronal fates. Thus, the regulation of PTF1A expression is critical for determining mechanisms controlling neuronal diversity in these regions of the nervous system. Here we identify a sequence conserved, tissue-specific enhancer located 10.8kb 3' of the PTF1A coding region that is sufficient to direct expression to dorsal neural tube progenitors that give rise to neurons in the dorsal spinal cord in chick and mouse. DNA binding motifs for Paired homeodomain (Pd-HD) and zinc finger (ZF) transcription factors are required for enhancer activity. Mutations in these sequences implicate the Pd-HD motif for activator function and the ZF motif for repressor function. Although no repressor transcription factor was identified, both PAX6 and SOX3 can increase enhancer activity in reporter assays. Thus, PTF1A is regulated by active and repressive inputs integrated through multiple sequence elements within a highly conserved sequence downstream of the PTF1A gene.
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Misexpression of PTF1A in cortical pyramidal cells in vivo promotes an inhibitory peptidergic identity.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2015Co-Authors: Jeffrey B. Russ, Jane E. Johnson, Mark D. Borromeo, Rahul K. Kollipara, Praveen K. Bommareddy, Julia A. KaltschmidtAbstract:The intracellular transcriptional milieu wields considerable influence over the induction of neuronal identity. The transcription factor PTF1A has been proposed to act as an identity “switch” between developmentally related precursors in the spinal cord (Glasgow et al., 2005; Huang et al., 2008), retina (Fujitani et al., 2006; Dullin et al., 2007; Nakhai et al., 2007; Lelievre et al., 2011), and cerebellum (Hoshino et al., 2005; Pascual et al., 2007; Yamada et al., 2014), where it promotes an inhibitory over an excitatory neuronal identity. In this study, we investigate the potency of PTF1A to cell autonomously confer a specific neuronal identity outside of its endogenous environment, using mouse in utero electroporation and a conditional genetic strategy to misexpress PTF1A exclusively in developing cortical pyramidal cells. Transcriptome profiling of PTF1A-misexpressing cells using RNA-seq reveals that PTF1A significantly alters pyramidal cell gene expression, upregulating numerous PTF1A-dependent inhibitory interneuron markers and ultimately generating a gene expression profile that resembles the transcriptomes of both PTF1A-expressing spinal interneurons and endogenous cortical interneurons. Using RNA-seq and in situ hybridization analyses, we also show that PTF1A induces expression of the peptidergic neurotransmitter nociceptin, while minimally affecting the expression of genes linked to other neurotransmitter systems. Moreover, PTF1A alters neuronal morphology, inducing the radial redistribution and branching of neurites in cortical pyramidal cells. Thus PTF1A is sufficient, even in a dramatically different neuronal precursor, to cell autonomously promote characteristics of an inhibitory peptidergic identity, providing the first example of a single transcription factor that can direct an inhibitory peptidergic fate.
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A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord
Development (Cambridge England), 2014Co-Authors: Mark D. Borromeo, David Meredith, Kuang-chi Tung, Diogo S. Castro, Joshua C. Chang, François Guillemot, Jane E. JohnsonAbstract:The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and PTF1A, have contrasting functions in specifying these neurons. To understand how Ascl1 and PTF1A function in this process, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and PTF1A directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, PTF1A directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, PTF1A represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and PTF1A bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. PTF1A also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of PTF1A binding. The direct transcriptional targets identified for Ascl1 and PTF1A provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification.
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Prdm13 Mediates the Balance of Inhibitory and Excitatory Neurons in Somatosensory Circuits
Developmental cell, 2013Co-Authors: Joshua C. Chang, David Meredith, Mark D. Borromeo, Paul R. Mayer, Helen C. Lai, Jane E. JohnsonAbstract:Generating a balanced network of inhibitory and excitatory neurons during development requires precise transcriptional control. In the dorsal spinal cord, PTF1A, a basic helix-loop-helix (bHLH) transcription activator, maintains this delicate balance by inducing homeodomain (HD) transcription factors such as Pax2 to specify the inhibitory lineage while suppressing HD factors such as Tlx1/3 that specify the excitatory lineage. We uncover the mechanism by which PTF1A represses excitatory cell fate in the inhibitory lineage. We identify Prdm13 as a direct target of PTF1A and reveal that Prdm13 actively represses excitatory cell fate by binding to regulatory sequences near the Tlx1 and Tlx3 genes to silence their expression. Prdm13 acts through multiple mechanisms, including interactions with the bHLH factor Ascl1, to repress Ascl1 activation of Tlx3. Thus, Prdm13 is a key component of a highly coordinated transcriptional network that determines the balance of inhibitory versus excitatory neurons in the dorsal spinal cord.
Christopher V.e. Wright - One of the best experts on this subject based on the ideXlab platform.
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Prevention and Reversion of Pancreatic Tumorigenesis through a Differentiation-Based Mechanism.
Developmental cell, 2019Co-Authors: Nathan M Krah, Christopher V.e. Wright, Raymond J. Macdonald, Shuba M. Narayanan, Deanne E. Yugawa, Julie A. Straley, L. Charles MurtaughAbstract:Activating mutations in Kras are nearly ubiquitous in human pancreatic cancer and initiate precancerous pancreatic intraepithelial neoplasia (PanINs) when induced in mouse acinar cells. PanINs normally take months to form but are accelerated by deletion of acinar cell differentiation factors such as PTF1A, suggesting that loss of cell identity is rate limiting for pancreatic tumor initiation. Using a genetic mouse model that allows for independent control of oncogenic Kras and PTF1A expression, we demonstrate that sustained PTF1A is sufficient to prevent Kras-driven tumorigenesis, even in the presence of tumor-promoting inflammation. Furthermore, reintroducing PTF1A into established PanINs reverts them to quiescent acinar cells in vivo. Similarly, PTF1A re-expression in human pancreatic cancer cells inhibits their growth and colony-forming ability. Our results suggest that reactivation of an endogenous differentiation program can prevent and reverse oncogene-driven transformation in cells harboring tumor-driving mutations, introducing a potential paradigm for solid tumor prevention and treatment.
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Prevention and reversion of pancreatic tumorigenesis through a differentiation-based mechanism
2017Co-Authors: Nathan M Krah, Christopher V.e. Wright, Raymond J. Macdonald, Deanne E. Yugawa, Julie A. Straley, L. Charles MurtaughAbstract:SUMMARYActivating mutations in Kras are nearly ubiquitous in human pancreatic cancer and initiate precancerous pancreatic intraepithelial neoplasia (PanINs) when induced in adult murine acinar cells. PanINs normally take months to form, but can be rapidly induced by genetic deletion of acinar cell differentiation factors such as PTF1A, suggesting that loss of mature cell identity is a rate-limiting step in pancreatic tumor initiation. Using a novel genetic mouse model that allows for independent control of oncogenic Kras and PTF1A expression, we demonstrate that maintained activity of PTF1A is sufficient to eliminate Kras-driven tumorigenesis, even in the presence of tumor-promoting inflammation. Furthermore, reintroduction of PTF1A into established PanINs reverts their phenotype in vivo. Our results suggest that reactivation of an endogenous differentiation program can prevent and reverse oncogenesis in cells harboring tumor driving mutations, thus introducing a novel paradigm for solid tumor prevention and treatment.
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Transcriptional Maintenance of Pancreatic Acinar Identity, Differentiation and Homeostasis by PTF1A
Molecular and cellular biology, 2016Co-Authors: Chinh Q. Hoang, Christopher V.e. Wright, Tye G. Deering, Mark A. Magnuson, Michael A. Hale, Spencer G. Willet, Fong Cheng Pan, Ana C. Azevedo-pouly, Hans Peter Elsässer, Galvin H. SwiftAbstract:Maintenance of cell type identity is crucial for health, yet little is known of the regulation that sustains the long-term stability of differentiated phenotypes. To investigate the roles that key transcriptional regulators play in adult differentiated cells, we examined the effects of depletion of the developmental master regulator PTF1A on the specialized phenotype of the adult pancreatic acinar cell in vivo Transcriptome sequencing and chromatin immunoprecipitation sequencing results showed that PTF1A maintains the expression of genes for all cellular processes dedicated to the production of the secretory digestive enzymes, a highly attuned surveillance of unfolded proteins, and a heightened unfolded protein response (UPR). Control by PTF1A is direct on target genes and indirect through a ten-member transcription factor network. Depletion of PTF1A causes an imbalance that overwhelms the UPR, induces cellular injury, and provokes acinar metaplasia. Compromised cellular identity occurs by derepression of characteristic stomach genes, some of which are also associated with pancreatic ductal cells. The loss of acinar cell homeostasis, differentiation, and identity is directly relevant to the pathologies of pancreatitis and pancreatic adenocarcinoma.
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The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma
eLife, 2015Co-Authors: Nathan M Krah, Christopher V.e. Wright, Galvin H. Swift, Mary P Bronner, Spencer G. Willet, Chinh Q. Hoang, Jean-paul De La O, Fong Chen Pan, Gabriela M Cash, Raymond J. MacdonaldAbstract:Pancreatic cancer is one of the most lethal forms of cancer, with fewer than 20% of people surviving for longer than twelve months after diagnosis. Two types of genetic mutation play important roles in pancreatic cancer. First, genes called oncogenes can be activated by mutations to drive unscheduled cell division. Second, the genes for tumor suppressors—proteins that prevent cells from dividing when they should not—can be switched off due to other mutations. Together, these mutations cause cells to over-proliferate and disrupt the structure of the pancreas. In a healthy pancreas, several different cell types perform various roles: acinar cells produce proteins that digest food, ductal cells carry these proteins to the intestine, and β cells produce insulin. Certain proteins are responsible for telling each of these cells what tasks to perform, which defines their so-called differentiation state. The protein PTF1A is crucial for establishing the differentiation state of acinar cells. In the most common form of pancreatic cancer, acinar cells are reprogrammed to become ductal cells. Moreover, pancreatic cancer cells contain much lower levels of PTF1A than normal pancreatic cells. To explore the connection between PTF1A and pancreatic cancer, Krah et al. deleted the gene for PTF1A in mice. This led to acinar cells being reprogrammed to become ductal cells. Additionally, when an oncogene mutation was activated at the same time as the gene for PTF1A was deleted, Krah et al. observed the rapid formation of large numbers of malignant pancreatic tumors in the mice. PTF1A therefore protects against pancreatic cancer by acting as a tumor suppressor and keeping acinar cells in their healthy, differentiated state. Unlike other tumor suppressors, however, PTF1A levels are reduced in cancer cells by a mechanism that does not involve a genetic mutation. Therefore, a future challenge is to determine how the amount of PTF1A protein is reduced, and in the longer term, to explore if it is possible to reverse cancer progression by forcing cancer cells back into their original differentiation state.
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abstract pr01 downregulation of PTF1A is a crucial and rate limiting step in pancreatic cancer initiation
Cancer Research, 2015Co-Authors: Nathan M Krah, Christopher V.e. Wright, Mary P Bronner, Charles L MurtaughAbstract:The goal of this study is to understand the endogenous mechanisms limiting the ability of oncogenic KRAS to initiate pancreatic tumorigenesis. Recently, our lab and others discovered that although pancreatic ductal adenocarcinoma (PDAC) shares phenotypic characteristics with normal pancreatic duct cells, it actually originates from mature exocrine acinar cells. In analyzing the initiating events of PDAC, we have identified the acinar cell transcription factor, PTF1A, as a critical factor inhibiting KRAS from reprogramming acinar cells to ductal tumor precursors. Binding sites for PTF1A are found upstream of essentially all acinar differentiation products and, importantly, PTF1A maintains its own expression through a positive autoregulatory loop. Given the central role of PTF1A in regulating acinar-specific gene expression, we wanted to determine if loss of this transcription factor plays a role in pancreatic intraepithelial neoplasia (PanIN) formation. Importantly, we find that PTF1A is downregulated in early (stage 1) PanINs of humans and mouse models, suggesting that loss of PTF1A expression could be a rate-limiting step in pancreatic cancer initiation. To test whether PTF1A loss is a functionally important step in PanIN development, we deleted the PTF1A gene in mice using an acinar-specific inducible Cre (PTF1A cKO). These studies confirm that PTF1A downregulation is an essential step in PanIN formation, as Cre-mediated deletion of acinar cell PTF1A in the presence of oncogenic KRAS (KrasG12D) acutely and dramatically accelerates PanIN formation. Widespread PanINs are observed within two weeks of combined KRAS activation/PTF1A deletion, by which time KRAS alone has induced few or no PanINs. At six weeks post-recombination, the combination of KRAS/PTF1A cKO induces >15-fold more PanINs than KRAS alone. Additionally, we have found that PTF1A heterozygosity sensitizes pancreatic cells to KRAS-mediated PanIN formation, indicating that transformation requires reduction of PTF1A activity below a critical autoregulatory threshold needed to maintain acinar identity. Based on these current studies, we hypothesize the existence of mutual antagonism between acinar differentiation, which is maintained through PTF1A, and transformation driven by KRAS. KRAS normally transforms acinar cells only weakly, but both endogenous and mutant KRAS activity can be increased by inflammation, such as that stimulated by caerulein-induced pancreatitis. As KRAS is potentiated by PTF1A deletion, we asked whether inflammation would be sufficient to transform PTF1A-deficient acinar cells even without mutant KRAS. In the absence of pancreatitis, PTF1A deletion alone has relatively little short-term effect: PTF1A-deficient acinar cells eventually lose their differentiated phenotype and express ductal markers, but they do not undergo hyperplasia or dysplasia. After subjecting PTF1A cKO mice to caerulein-induced pancreatitis, however, we observed widespread acinar-to-ductal metaplasia, histological structures that resemble PanINs and stain positively with Alcian blue, and loss of amylase staining throughout the pancreas. Our current data indicate that PTF1A has a critical role in guarding against genetic (oncogenic KRAS) and environmental (pancreatitis) insults, and demonstrate that loss of PTF1A expression is the critical event of acinar cell transformation. Going forward, we will use gene expression profiling and ChIP-seq analysis to identify PTF1A target genes responsible for inhibiting KRAS, and to characterize the epigenetic processes by which KRAS and inflammation reprogram acinar cells to a PanIN phenotype. This abstract is also presented as Poster A3. Citation Format: Nathan Michael Krah, Mary P. Bronner, Christopher V. Wright, L. Charles Murtaugh. Downregulation of PTF1A is a crucial and rate-limiting step in pancreatic cancer initiation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR01.
Francisco X. Real - One of the best experts on this subject based on the ideXlab platform.
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ICAT is a novel PTF1A interactor that regulates pancreatic acinar differentiation and displays altered expression in tumours
The Biochemical journal, 2013Co-Authors: M. Luisa Campos, Victor J. Sanchez-arévalo Lobo, Annie Rodolosse, Cara J. Gottardi, Andrea Mafficini, Stefania Beghelli, Maria Scardoni, Claudio Bassi, Aldo Scarpa, Francisco X. RealAbstract:The PTF1 (pancreas transcription factor 1) complex is a master regulator of differentiation of acinar cells, responsible for the production of digestive enzymes. In the adult pancreas, PTF1 contains two pancreas-restricted transcription factors: PTF1A and Rbpjl. PTF1 recruits P/CAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] which acetylates PTF1A and enhances its transcriptional activity. Using yeast two-hybrid screening, we identified ICAT (inhibitor of β-catenin and Tcf4) as a novel PTF1A interactor. ICAT regulates the Wnt pathway and cell proliferation. We validated and mapped the ICAT–PTF1A interaction in vitro and in vivo . We demonstrated that, following its overexpression in acinar tumour cells, ICAT regulates negatively PTF1 activity in vitro and in vivo . This effect was independent of β-catenin and was mediated by direct binding to PTF1A and displacement of P/CAF. ICAT also modulated the expression of Pdx1 and Sox9 in acinar tumour cells. ICAT overexpression reduced the interaction of PTF1A with Rbpjl and P/CAF and impaired PTF1A acetylation by P/CAF. ICAT did not affect the subcellular localization of PTF1A. In human pancreas, ICAT displayed a cell-type-specific distribution; in acinar and endocrine cells, it was nuclear, whereas in ductal cells, it was cytoplasmic. In ductal adenocarcinomas, ICAT displayed mainly a nuclear or mixed distribution and the former was an independent marker of survival. ICAT regulates acinar differentiation and it does so through a novel Wnt pathway-independent mechanism that may contribute to pancreatic disease.
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p caf modulates the activity of the transcription factor p48 PTF1A involved in pancreatic acinar differentiation
Biochemical Journal, 2009Co-Authors: Annie Rodolosse, Maria-luisa Campos, Ilse Rooman, Mathieu Lichtenstein, Francisco X. RealAbstract:p48, also called PTF1A (pancreas-specific transcription factor 1a), is a tissue-restricted bHLH (basic helix loop helix) transcription factor which is critical for pancreatic commitment during development and for the activation and maintenance of the acinar differentiation programme in the exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1, formed by p48, RBP-L (recombination signal-binding protein 1-like) and a class A bHLH protein. In addition, p48 induces cell-cycle exit by controlling G 1 /S-phase progression. However, the mechanisms that mediate PTF1-dependent gene activation are poorly understood. In the present study, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional co-activators. The histone acetyltransferase cofactor p/CAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} interacts with p48 in acinar cells in vivo and is associated with the promoter region of acinar genes targeted by the PTF1 complex. p/CAF potentiates PTF1 transcriptional activity by enhancing selectively the p48 transactivation activity. p/CAF promotes the nuclear accumulation of p48 and its in vivo acetylation in Lys 200 . The K200R mutation abolishes the transcriptional activity of p48, as well as its capacity to functionally co-operate with RBP-L to ensure effective PTF1-driven transcription, indicating that p/CAF-mediated acetylation of p48 is required for the full transcriptional activity of PTF1. In contrast, p/CAF did not co-operate with p48 in its growth regulatory effects. These results support a critical and selective role of p/CAF in PTF1-dependent gene activation during acinar differentiation.
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P/CAF modulates the activity of the transcription factor p48/PTF1A involved in pancreatic acinar differentiation
Biochemical Journal, 2009Co-Authors: Annie Rodolosse, Maria-luisa Campos, Ilse Rooman, Mathieu Lichtenstein, Francisco X. RealAbstract:p48, also called PTF1A, is a tissue-restricted basic helix-loop-helix transcription factor critical for pancreatic commitment during development and for the activation/maintenance of the acinar differentiation program in exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1 (pancreas transcription factor 1), formed by p48, RBP-L and a class A bHLH protein. In addition, p48 induces cell cycle exit by controlling G1-S progression. However, the mechanisms that mediate the PTF1-dependent gene activation are poorly understood. Here, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional coactivators. The histone acetyltransferase cofactor p/CAF interacts in vivo with p48 in acinar cells and is associated with the promoter region of acinar genes targeted by the PTF1 complex. P/CAF potentiates the PTF1 transcriptional activity by selectively enhancing the p48 transactivation activity. P/CAF promotes the nuclear accumulation of p48 and its in vivo acetylation in Lys residue K200. The K200R mutation abolishes the transcriptional activity of p48 as well as its capacity to functionally cooperate with RBP-L to ensure an effective PTF1-driven transcription, indicating that p/CAF-mediated acetylation of p48 is required for PTF1 full transcriptional activity. By contrast, p/CAF did not cooperate with p48 in its growth regulatory effects. These results support a critical and selective role of p/CAF in the PTF1-dependent gene activation during acinar differentiation.
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p/CAF modulates the activity of the transcription factor p48/PTF1A involved in pancreatic acinar differentiation.
The Biochemical journal, 2009Co-Authors: Annie Rodolosse, Maria-luisa Campos, Ilse Rooman, Mathieu Lichtenstein, Francisco X. RealAbstract:p48, also called PTF1A (pancreas-specific transcription factor 1a), is a tissue-restricted bHLH (basic helix loop helix) transcription factor which is critical for pancreatic commitment during development and for the activation and maintenance of the acinar differentiation programme in the exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1, formed by p48, RBP-L (recombination signal-binding protein 1-like) and a class A bHLH protein. In addition, p48 induces cell-cycle exit by controlling G(1)/S-phase progression. However, the mechanisms that mediate PTF1-dependent gene activation are poorly understood. In the present study, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional co-activators. The histone acetyltransferase cofactor p/CAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} interacts with p48 in acinar cells in vivo and is associated with the promoter region of acinar genes targeted by the PTF1 complex. p/CAF potentiates PTF1 transcriptional activity by enhancing selectively the p48 transactivation activity. p/CAF promotes the nuclear accumulation of p48 and its in vivo acetylation in Lys(200). The K200R mutation abolishes the transcriptional activity of p48, as well as its capacity to functionally co-operate with RBP-L to ensure effective PTF1-driven transcription, indicating that p/CAF-mediated acetylation of p48 is required for the full transcriptional activity of PTF1. In contrast, p/CAF did not co-operate with p48 in its growth regulatory effects. These results support a critical and selective role of p/CAF in PTF1-dependent gene activation during acinar differentiation.
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Cerebellar GABAergic progenitors adopt an external granule cell-like phenotype in the absence of PTF1A transcription factor expression
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Marta Pascual, Christopher V.e. Wright, Francisco X. Real, Ibane Abasolo, Ana Mingorance-le Meur, Albert Martínez, José Antonio Del Río, Eduardo SorianoAbstract:We report in this study that, in the cerebellum, the pancreatic transcription factor PTF1A is required for the specific generation of Purkinje cells (PCs) and interneurons. Moreover, granule cell progenitors in the external GCL (EGL) appear to be unaffected by deletion of PTF1A. Cell lineage analysis in PTF1ACre/Cre mice was used to establish that, in the absence of PTF1A expression, ventricular zone progenitors, normally fated to produce PCs and interneurons, aberrantly migrate to the EGL and express typical markers of these cells, such as Math1, Reelin, and Zic1/2. Furthermore, these cells have a fine structure typical of EGL progenitors, indicating that they adopt an EGL-like cell phenotype. These findings indicate that PTF1A is necessary for the specification and normal production of PCs and cerebellar interneurons. Moreover, our results suggest that PTF1A is also required for the suppression of the granule cell specification program in cerebellar ventricular zone precursors.
Raymond J. Macdonald - One of the best experts on this subject based on the ideXlab platform.
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Prevention and Reversion of Pancreatic Tumorigenesis through a Differentiation-Based Mechanism.
Developmental cell, 2019Co-Authors: Nathan M Krah, Christopher V.e. Wright, Raymond J. Macdonald, Shuba M. Narayanan, Deanne E. Yugawa, Julie A. Straley, L. Charles MurtaughAbstract:Activating mutations in Kras are nearly ubiquitous in human pancreatic cancer and initiate precancerous pancreatic intraepithelial neoplasia (PanINs) when induced in mouse acinar cells. PanINs normally take months to form but are accelerated by deletion of acinar cell differentiation factors such as PTF1A, suggesting that loss of cell identity is rate limiting for pancreatic tumor initiation. Using a genetic mouse model that allows for independent control of oncogenic Kras and PTF1A expression, we demonstrate that sustained PTF1A is sufficient to prevent Kras-driven tumorigenesis, even in the presence of tumor-promoting inflammation. Furthermore, reintroducing PTF1A into established PanINs reverts them to quiescent acinar cells in vivo. Similarly, PTF1A re-expression in human pancreatic cancer cells inhibits their growth and colony-forming ability. Our results suggest that reactivation of an endogenous differentiation program can prevent and reverse oncogene-driven transformation in cells harboring tumor-driving mutations, introducing a potential paradigm for solid tumor prevention and treatment.
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Prevention and reversion of pancreatic tumorigenesis through a differentiation-based mechanism
2017Co-Authors: Nathan M Krah, Christopher V.e. Wright, Raymond J. Macdonald, Deanne E. Yugawa, Julie A. Straley, L. Charles MurtaughAbstract:SUMMARYActivating mutations in Kras are nearly ubiquitous in human pancreatic cancer and initiate precancerous pancreatic intraepithelial neoplasia (PanINs) when induced in adult murine acinar cells. PanINs normally take months to form, but can be rapidly induced by genetic deletion of acinar cell differentiation factors such as PTF1A, suggesting that loss of mature cell identity is a rate-limiting step in pancreatic tumor initiation. Using a novel genetic mouse model that allows for independent control of oncogenic Kras and PTF1A expression, we demonstrate that maintained activity of PTF1A is sufficient to eliminate Kras-driven tumorigenesis, even in the presence of tumor-promoting inflammation. Furthermore, reintroduction of PTF1A into established PanINs reverts their phenotype in vivo. Our results suggest that reactivation of an endogenous differentiation program can prevent and reverse oncogenesis in cells harboring tumor driving mutations, thus introducing a novel paradigm for solid tumor prevention and treatment.
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The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma
eLife, 2015Co-Authors: Nathan M Krah, Christopher V.e. Wright, Galvin H. Swift, Mary P Bronner, Spencer G. Willet, Chinh Q. Hoang, Jean-paul De La O, Fong Chen Pan, Gabriela M Cash, Raymond J. MacdonaldAbstract:Pancreatic cancer is one of the most lethal forms of cancer, with fewer than 20% of people surviving for longer than twelve months after diagnosis. Two types of genetic mutation play important roles in pancreatic cancer. First, genes called oncogenes can be activated by mutations to drive unscheduled cell division. Second, the genes for tumor suppressors—proteins that prevent cells from dividing when they should not—can be switched off due to other mutations. Together, these mutations cause cells to over-proliferate and disrupt the structure of the pancreas. In a healthy pancreas, several different cell types perform various roles: acinar cells produce proteins that digest food, ductal cells carry these proteins to the intestine, and β cells produce insulin. Certain proteins are responsible for telling each of these cells what tasks to perform, which defines their so-called differentiation state. The protein PTF1A is crucial for establishing the differentiation state of acinar cells. In the most common form of pancreatic cancer, acinar cells are reprogrammed to become ductal cells. Moreover, pancreatic cancer cells contain much lower levels of PTF1A than normal pancreatic cells. To explore the connection between PTF1A and pancreatic cancer, Krah et al. deleted the gene for PTF1A in mice. This led to acinar cells being reprogrammed to become ductal cells. Additionally, when an oncogene mutation was activated at the same time as the gene for PTF1A was deleted, Krah et al. observed the rapid formation of large numbers of malignant pancreatic tumors in the mice. PTF1A therefore protects against pancreatic cancer by acting as a tumor suppressor and keeping acinar cells in their healthy, differentiated state. Unlike other tumor suppressors, however, PTF1A levels are reduced in cancer cells by a mechanism that does not involve a genetic mutation. Therefore, a future challenge is to determine how the amount of PTF1A protein is reduced, and in the longer term, to explore if it is possible to reverse cancer progression by forcing cancer cells back into their original differentiation state.
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Dominant and context-specific control of endodermal organ allocation by PTF1A
Development (Cambridge England), 2014Co-Authors: Spencer G. Willet, Raymond J. Macdonald, Mark A. Magnuson, Michael A. Hale, Anne Grapin-botton, Christopher V.e. WrightAbstract:The timing and gene regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, PTF1A, in embryonic mouse endoderm (PTF1A(EDD)) dramatically expanded the pancreatic gene regulatory network within the foregut. PTF1A(EDD) temporarily suppressed Sox2 broadly over the anterior endoderm. Pancreas-proximal organ territories underwent full tissue conversion. Early-stage PTF1A(EDD) rapidly expanded the endogenous endodermal Pdx1-positive domain and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early PTF1A(EDD) converted essentially the entire glandular stomach, rostral duodenum and extrahepatic biliary system to pancreas, with formation of many endocrine cell clusters of the type found in normal islets of Langerhans. Sliding the PTF1A(EDD) expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage PTF1A(EDD) changed radically towards unipotent, acinar-restricted conversion. We provide strong evidence, beyond previous PTF1A inactivation or misexpression experiments in frog embryos, for spatiotemporally context-dependent activity of PTF1A as a potent gain-of-function trigger of pro-pancreatic commitment.
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PTF1A-mediated control of Dll1 reveals an alternative to the lateral inhibition mechanism
Development, 2011Co-Authors: Jonas Ahnfelt-rønne, Ole D. Madsen, Christopher V.e. Wright, Mette C. Jørgensen, Rasmus Klinck, Jan N. Jensen, Ernst-martin Füchtbauer, Tye G. Deering, Raymond J. Macdonald, Palle SerupAbstract:Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, PTF1A activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain PTF1A expression, and in turn Dll1 expression in early MPCs. Our results show that PTF1A-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and PTF1A protein levels.
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Regulating the dorsal neural tube expression of PTF1A through a distal 3' enhancer.
Developmental biology, 2016Co-Authors: Bishakha Mona, David Meredith, Rahul K. Kollipara, John M. Avila, Jane E. JohnsonAbstract:Generating the correct balance of inhibitory and excitatory neurons in a neural network is essential for normal functioning of a nervous system. The neural network in the dorsal spinal cord functions in somatosensation where it modulates and relays sensory information from the periphery. PTF1A is a key transcriptional regulator present in a specific subset of neural progenitor cells in the dorsal spinal cord, cerebellum and retina that functions to specify an inhibitory neuronal fate while suppressing excitatory neuronal fates. Thus, the regulation of PTF1A expression is critical for determining mechanisms controlling neuronal diversity in these regions of the nervous system. Here we identify a sequence conserved, tissue-specific enhancer located 10.8kb 3' of the PTF1A coding region that is sufficient to direct expression to dorsal neural tube progenitors that give rise to neurons in the dorsal spinal cord in chick and mouse. DNA binding motifs for Paired homeodomain (Pd-HD) and zinc finger (ZF) transcription factors are required for enhancer activity. Mutations in these sequences implicate the Pd-HD motif for activator function and the ZF motif for repressor function. Although no repressor transcription factor was identified, both PAX6 and SOX3 can increase enhancer activity in reporter assays. Thus, PTF1A is regulated by active and repressive inputs integrated through multiple sequence elements within a highly conserved sequence downstream of the PTF1A gene.
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A transcription factor network specifying inhibitory versus excitatory neurons in the dorsal spinal cord
Development (Cambridge England), 2014Co-Authors: Mark D. Borromeo, David Meredith, Kuang-chi Tung, Diogo S. Castro, Joshua C. Chang, François Guillemot, Jane E. JohnsonAbstract:The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and PTF1A, have contrasting functions in specifying these neurons. To understand how Ascl1 and PTF1A function in this process, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and PTF1A directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, PTF1A directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, PTF1A represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and PTF1A bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. PTF1A also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of PTF1A binding. The direct transcriptional targets identified for Ascl1 and PTF1A provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification.
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Program specificity for PTF1A in pancreas versus neural tube development correlates with distinct collaborating cofactors and chromatin accessibility.
Molecular and cellular biology, 2013Co-Authors: David Meredith, Tye G. Deering, Trisha K. Savage, Mark D. Borromeo, Bradford Casey, Paul R. Mayer, Chinh Q. Hoang, Kuang-chi Tung, Manonmani Kumar, Chengcheng ShenAbstract:The lineage-specific basic helix-loop-helix transcription factor PTF1A is a critical driver for development of both the pancreas and nervous system. How one transcription factor controls diverse programs of gene expression is a fundamental question in developmental biology. To uncover molecular strategies for the program-specific functions of PTF1A, we identified bound genomic regions in vivo during development of both tissues. Most regions bound by PTF1A are specific to each tissue, lie near genes needed for proper formation of each tissue, and coincide with regions of open chromatin. The specificity of PTF1A binding is encoded in the DNA surrounding the PTF1A-bound sites, because these regions are sufficient to direct tissue-restricted reporter expression in transgenic mice. Fox and Sox factors were identified as potential lineage-specific modifiers of PTF1A binding, since binding motifs for these factors are enriched in PTF1A-bound regions in pancreas and neural tube, respectively. Of the Fox factors expressed during pancreatic development, Foxa2 plays a major role. Indeed, PTF1A and Foxa2 colocalize in embryonic pancreatic chromatin and can act synergistically in cell transfection assays. Together, these findings indicate that lineage-specific chromatin landscapes likely constrain the DNA binding of PTF1A, and they identify Fox and Sox gene families as part of this process.
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Prdm13 Mediates the Balance of Inhibitory and Excitatory Neurons in Somatosensory Circuits
Developmental cell, 2013Co-Authors: Joshua C. Chang, David Meredith, Mark D. Borromeo, Paul R. Mayer, Helen C. Lai, Jane E. JohnsonAbstract:Generating a balanced network of inhibitory and excitatory neurons during development requires precise transcriptional control. In the dorsal spinal cord, PTF1A, a basic helix-loop-helix (bHLH) transcription activator, maintains this delicate balance by inducing homeodomain (HD) transcription factors such as Pax2 to specify the inhibitory lineage while suppressing HD factors such as Tlx1/3 that specify the excitatory lineage. We uncover the mechanism by which PTF1A represses excitatory cell fate in the inhibitory lineage. We identify Prdm13 as a direct target of PTF1A and reveal that Prdm13 actively represses excitatory cell fate by binding to regulatory sequences near the Tlx1 and Tlx3 genes to silence their expression. Prdm13 acts through multiple mechanisms, including interactions with the bHLH factor Ascl1, to repress Ascl1 activation of Tlx3. Thus, Prdm13 is a key component of a highly coordinated transcriptional network that determines the balance of inhibitory versus excitatory neurons in the dorsal spinal cord.
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Multiple Transcriptional Mechanisms Control PTF1A Levels during Neural Development Including Autoregulation by the PTF1-J Complex
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2009Co-Authors: David Meredith, Raymond J. Macdonald, Toshihiko Masui, Galvin H. Swift, Jane E. JohnsonAbstract:PTF1A, along with an E protein and Rbpj, forms the transcription factor complex PTF1-J that is essential for proper specification of inhibitory neurons in the spinal cord, retina, and cerebellum. Here we show that two highly conserved noncoding genomic regions, a distal 2.3 kb sequence located 13.4 kb 5′ and a 12.4 kb sequence located immediately 3′ of the PTF1A coding region, have distinct activity in controlling PTF1A expression in all of these domains. The 5′ 2.3 kb sequence functions as an autoregulatory element and directs reporter gene expression to all PTF1A domains in the developing nervous system. The autoregulatory activity of this element was demonstrated by binding of the PTF1-J complex in vitro, PTF1A localization to this genomic region in vivo, and the in vivo requirement of PTF1A for the activity of the regulatory element in transgenic mice. In contrast, the 12.4 kb 3′ regulatory region does not contain any conserved PTF1 sites, and its expression in transgenic mice is independent of PTF1A. Thus, regulatory information for initiation of PTF1A expression in the developing nervous system is located within the 12.4 kb sequence 3′ of the PTF1A gene. Together, these results identify multiple transcriptional mechanisms that control PTF1A levels, one modulating levels by autoregulation through the PTF1-J complex, and the other a PTF1A-independent mechanism for initial activation.