Project description:RNAi pathways are prevalent throughout the eukaryotic kingdom and are well known to regulate gene expression on a post-transcriptional level in the cytoplasm. Less is known about possible functions of RNAi in the nucleus. In the fission yeast Schizosaccharomyces pombe, RNAi is crucial to establish and maintain centromeric heterochromatin and functions to repress genome activity by a chromatin silencing mechanism referred to as cotranscriptional gene silencing (CTGS). Mechanistic details and the physiological relevance of CTGS are unknown. Here we show that RNAi components interact with chromatin at nuclear pores to keep stress response genes in check. We demonstrate that RNAi-mediated CTGS represses stress-inducible genes by degrading mRNAs under noninduced conditions. Under chronic heat stress conditions, a Dicer thermoswitch deports Dicer to the cytoplasm, thereby disrupting CTGS and enabling expression of genes implicated in the acquisition of thermotolerance. Taken together, our work highlights a role for nuclear pores and the stress response transcription factor Atf1 in coordinating the interplay between the RNAi machinery and the S. pombe genome and uncovers a novel mode of RNAi regulation in response to an environmental cue.
Project description:Analysis of the mutational landscape of various hematological malignancies has shown that key regulators implicated in lineage commitment and differentiation are frequently implicated in leukemic transformation. In T-cell acute lymphoblastic leukemia, the role of various oncogenic and tumor suppressing transcription factors has been investigated. More recently, an exceptional high prevalence of mutations affecting epigenetic modifiers in T-ALL has also been noted. In order to understand the exact contribution of such lesions to normal differentiation and oncogenic transformation, further functional characterization of this subclass of proteins is warranted. PHF6 is one of these key epigenetic players, which was described as a frequently mutated and deleted novel tumor suppressor in T-ALL. In a first step towards unraveling the role of PHF6 in normal and malignant hematopoiesis, we characterized the function of PHF6 in both normal T-cell differentiation and other hematopoietic lineages as a prelude to understand its contribution to leukemogenesis. Here, we show for the first time that PHF6 controlled epigenetic regulation positively controls a NOTCH1 gene signature and the effects observed on T-lineage differentiation seem to be additive with Notch1. In this study, we show for the first time that PHF6 is a master regulator of early hematopoietic differentiation along the T-cell, B-cell, myeloid and NK-cell lineages.
Project description:Analysis of the mutational landscape of various hematological malignancies has shown that key regulators implicated in lineage commitment and differentiation are frequently implicated in leukemic transformation. In T-cell acute lymphoblastic leukemia, the role of various oncogenic and tumor suppressing transcription factors has been investigated. More recently, an exceptional high prevalence of mutations affecting epigenetic modifiers in T-ALL has also been noted. In order to understand the exact contribution of such lesions to normal differentiation and oncogenic transformation, further functional characterization of this subclass of proteins is warranted. PHF6 is one of these key epigenetic players, which was described as a frequently mutated and deleted novel tumor suppressor in T-ALL. In a first step towards unraveling the role of PHF6 in normal and malignant hematopoiesis, we characterized the function of PHF6 in both normal T-cell differentiation and other hematopoietic lineages as a prelude to understand its contribution to leukemogenesis. Here, we show for the first time that PHF6 controlled epigenetic regulation positively controls a NOTCH1 gene signature and the effects observed on T-lineage differentiation seem to be additive with Notch1. In this study, we show for the first time that PHF6 is a master regulator of early hematopoietic differentiation along the T-cell, B-cell, myeloid and NK-cell lineages.
Project description:Polycomb-repressive complex 2 (PRC2) facilitates the maintenance and inheritance of chromatin domains repressive to transcription through catalysis of methylation of histone H3 at Lys27 (H3K27me2/3). However, through its EZH2 subunit, PRC2 also binds to nascent transcripts from active genes that are devoid of H3K27me2/3 in embryonic stem cells. Here, biochemical analyses indicated that RNA interaction inhibits SET domain-containing proteins, such as PRC2, nonspecifically in vitro. However, CRISPR-mediated truncation of a PRC2-interacting nascent RNA rescued PRC2-mediated deposition of H3K27me2/3. That PRC2 activity is inhibited by interactions with nascent transcripts supports a model in which PRC2 can only mark for repression those genes silenced by transcriptional repressors.
Project description:Analysis of the mutational landscape of various hematological malignancies has shown that key regulators implicated in lineage commitment and differentiation are frequently implicated in leukemic transformation. In T-cell acute lymphoblastic leukemia, the role of various oncogenic and tumor suppressing transcription factors has been investigated. More recently, an exceptional high prevalence of mutations affecting epigenetic modifiers in T-ALL has also been noted. In order to understand the exact contribution of such lesions to normal differentiation and oncogenic transformation, further functional characterization of this subclass of proteins is warranted. PHF6 is one of these key epigenetic players, which was described as a frequently mutated and deleted novel tumor suppressor in T-ALL. In a first step towards unraveling the role of PHF6 in normal and malignant hematopoiesis, we characterized the function of PHF6 in both normal T-cell differentiation and other hematopoietic lineages as a prelude to understand its contribution to leukemogenesis. Here, we show for the first time that PHF6 controlled epigenetic regulation positively controls a NOTCH1 gene signature and the effects observed on T-lineage differentiation seem to be additive with Notch1. In this study, we show for the first time that PHF6 is a master regulator of early hematopoietic differentiation along the T-cell, B-cell, myeloid and NK-cell lineages.
Project description:Analysis of the mutational landscape of various hematological malignancies has shown that key regulators implicated in lineage commitment and differentiation are frequently implicated in leukemic transformation. In T-cell acute lymphoblastic leukemia, the role of various oncogenic and tumor suppressing transcription factors has been investigated. More recently, an exceptional high prevalence of mutations affecting epigenetic modifiers in T-ALL has also been noted. In order to understand the exact contribution of such lesions to normal differentiation and oncogenic transformation, further functional characterization of this subclass of proteins is warranted. PHF6 is one of these key epigenetic players, which was described as a frequently mutated and deleted novel tumor suppressor in T-ALL. In a first step towards unraveling the role of PHF6 in normal and malignant hematopoiesis, we characterized the function of PHF6 in both normal T-cell differentiation and other hematopoietic lineages as a prelude to understand its contribution to leukemogenesis. Here, we show for the first time that PHF6 controlled epigenetic regulation positively controls a NOTCH1 gene signature and the effects observed on T-lineage differentiation seem to be additive with Notch1. In this study, we show for the first time that PHF6 is a master regulator of early hematopoietic differentiation along the T-cell, B-cell, myeloid and NK-cell lineages.
Project description:In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.
Project description:Tumor necrosis factor receptor-1 (TNFR1) signaling, apart from its pleiotropic functions in inflammation, plays a role in embryogenesis as deficiency of varieties of its downstream molecules leads to embryonic lethality in mice. Caspase-8 noncleavable receptor interacting serine/threonine kinase 1 (RIPK1) mutations occur naturally in humans, and the corresponding D325A mutation in murine RIPK1 leads to death at early midgestation. It is known that both the demise of Ripk1D325A/D325A embryos and the death of Casp8-/- mice are initiated by TNFR1, but they are mediated by apoptosis and necroptosis, respectively. Here, we show that the defects in Ripk1D325A/D325A embryos occur at embryonic day 10.5 (E10.5), earlier than that caused by Casp8 knockout. By analyzing a series of genetically mutated mice, we elucidated a mechanism that leads to the lethality of Ripk1D325A/D325A embryos and compared it with that underlies Casp8 deletion-mediated lethality. We revealed that the apoptosis in Ripk1D325A/D325A embryos requires a scaffold function of RIPK3 and enzymatically active caspase-8. Unexpectedly, caspase-1 and caspase-11 are downstream of activated caspase-8, and concurrent depletion of Casp1 and Casp11 postpones the E10.5 lethality to embryonic day 13.5 (E13.5). Moreover, caspase-3 is an executioner of apoptosis at E10.5 in Ripk1D325A/D325A mice as its deletion extends life of Ripk1D325A/D325A mice to embryonic day 11.5 (E11.5). Hence, an unexpected death pathway of TNFR1 controls RIPK1 D325A mutation-induced lethality at E10.5.
Project description:Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
Project description:The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central "triskelion" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass.