Project description:During routine genome duplication, many potential replication origins remain inactive, or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that can be distinguished from baseline (i.e. ordinarily active) origins by their preferential association with MCM2, a component of the replicative helicase, phosphorylated on serine 108 (pS108-MCM2). pS108-MCM2 is a substrate of the ATR kinase, which is recruited to chromatin via an interaction with hyperacetylated TOPBP1 in cells undergoing replication stress or in cells devoid of SIRT1 deacetylase activity. In turn, S108-MCM2 phosphorylation enhances a second, DDK-dependent, S139-MCM2 phosphorylation, which triggers initiation of DNA replication at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational modifications on the MCM helicase that reflect a balance between SIRT1 activity and ATR signaling.
Project description:During routine genome duplication, many potential replication origins remain inactive, or dormant. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that can be distinguished from baseline (i.e. ordinarily active) origins by their preferential association with MCM2, a component of the replicative helicase, phosphorylated on serine 108 (pS108-MCM2). pS108-MCM2 is a substrate of the ATR kinase, which is recruited to chromatin via an interaction with hyperacetylated TOPBP1 in cells undergoing replication stress or in cells devoid of SIRT1 deacetylase activity. In turn, S108-MCM2 phosphorylation enhances a second, DDK-dependent, S139-MCM2 phosphorylation, which triggers initiation of DNA replication at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational modifications on the MCM helicase that reflect a balance between SIRT1 activity and ATR signaling.
Project description:Microcephaly and medulloblastoma result from mutations that compromise genomic stability. We report that Atr, which is mutated in the microcephalic disorder Seckel syndrome, is required to maintain chromosomal integrity during postnatal cerebellar neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis, and cerebellar hypoplasia. Co-deletions of either Bax and Bak or p53 prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. Atr-deficient CGNPs showed impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to Atr-deficient proliferation was p53-driven. Acute Atr inhibition in vivo by nanoparticle-formulated VE-822 reproduced the disruptions seen with Atr deletion. Our data show that p53-driven apoptosis and senescence, and non-apoptotic cell death redundantly limit growth in Atr-deficient progenitors. These overlapping mechanisms that suppress growth in Atr-disrupted CGNPs may be exploited for treatment of CGNP-derived medulloblastoma using Atr inhibition.
Project description:Sirtuin-1 (Sirt1), a class III histone/protein deacetylase is central to cellular metabolism, stress responses and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T-cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation and cytokine production largely unaltered. However, Sirt1 targeting promoted the expression and acetylation of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4+ T-cells or Foxp3+ Treg cells exhibited prolonged survival of MHC-mismatched cardiac allografts. Allografts in Sirt1 targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3+ Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions and its targeting may have therapeutic value in autoimmunity and transplantation. RNA from three independent samples from magnetically separated CD4+CD25+ Treg of Sirt1 knock out, compared to wild type (C57BL6) control
Project description:Sirtuin-1 (Sirt1), a class III histone/protein deacetylase is central to cellular metabolism, stress responses and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T-cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation and cytokine production largely unaltered. However, Sirt1 targeting promoted the expression and acetylation of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4+ T-cells or Foxp3+ Treg cells exhibited prolonged survival of MHC-mismatched cardiac allografts. Allografts in Sirt1 targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3+ Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions and its targeting may have therapeutic value in autoimmunity and transplantation.
Project description:Microcephaly and medulloblastoma result from mutations that compromise genomic stability. We report that Atr, which is mutated in the microcephalic disorder Seckel syndrome, is required to maintain chromosomal integrity during postnatal cerebellar neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis, and cerebellar hypoplasia. Co-deletions of either Bax and Bak or p53 prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. Atr-deficient CGNPs showed impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to Atr-deficient proliferation was p53-driven. Acute Atr inhibition in vivo by nanoparticle-formulated VE-822 reproduced the disruptions seen with Atr deletion. Our data show that p53-driven apoptosis and senescence, and non-apoptotic cell death redundantly limit growth in Atr-deficient progenitors. These overlapping mechanisms that suppress growth in Atr-disrupted CGNPs may be exploited for treatment of CGNP-derived medulloblastoma using Atr inhibition.
Project description:Sirt1, a NAD-dependent deacetylase, has been shown involved in many complex biological functions. We previously demonstrated that Sirt-1 gain of function is neuroprotective in a model of neurodegenenration. We recently found that Sirt1 loss of function (Sirt1Δ mouse) impairs learning, memory, and synaptic plasticity. We used microarray screening to identify potential miRNAs that may be regulated by Sirt1 and ivolved in learning and memory. Total RNA from the hippocampus of SIRT1Δ and control mice was extracted and used for microRNA expression level evaluation.
Project description:Mammalian SIRT1 is a central regulator of metabolism and aging. This project is to analyze global phosphorylation levels of mammalian SIRT1 in proliferating and senescence states using human lung fibroblast IMR90, in order to explore the post-translational regulation of SIRT1 protein upon cellular senescence and its potential roles in the regulatory mechanisms of SIRT1 homeostasis.