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T. Brucei, Nup-1 Depletion

ABSTRACT: Organization of the genome into compacted chromatin is a eukaryotic innovation facilitating increased sophistication in transcriptional regulation. In metazoa coiled-coil lamin proteins are major components of the chromatin organizer at the nuclear periphery and maintain nuclear integrity. While identifiable lamin homologues are restricted to metazoans, morphologically analogous structures maintaining nuclear organization in other eukaryotic lineages are known, but the molecular constituents remain undefined. Trypanosoma brucei NUP-1 is a large coiled-coil protein associated with fibrils at the inner face of the nuclear envelope. Using transcriptome analysis in combination with RNA interference and various imaging techniques, we demonstrate that NUP-1 forms a stable immobile cage around the nucleus, is required for viability and nuclear structural integrity, directs the positional organization of nuclear pore complexes, and serves to organize chromatin and specifically repress genes located at the nuclear periphery involved in immune evasion. Based on architectural similarity and functionality, we propose that NUP-1 is a novel, highly divergent lamin

ORGANISM(S): Trypanosoma brucei

PROVIDER: GSE26256 | GEO | 2011/12/15

SECONDARY ACCESSION(S): PRJNA135091

REPOSITORIES: GEO

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T. Brucei, Nup-1 Depletion

Project description:Organization of the genome into compacted chromatin is a eukaryotic innovation facilitating increased sophistication in transcriptional regulation. In metazoa coiled-coil lamin proteins are major components of the chromatin organizer at the nuclear periphery and maintain nuclear integrity. While identifiable lamin homologues are restricted to metazoans, morphologically analogous structures maintaining nuclear organization in other eukaryotic lineages are known, but the molecular constituents remain undefined. Trypanosoma brucei NUP-1 is a large coiled-coil protein associated with fibrils at the inner face of the nuclear envelope. Using transcriptome analysis in combination with RNA interference and various imaging techniques, we demonstrate that NUP-1 forms a stable immobile cage around the nucleus, is required for viability and nuclear structural integrity, directs the positional organization of nuclear pore complexes, and serves to organize chromatin and specifically repress genes located at the nuclear periphery involved in immune evasion. Based on architectural similarity and functionality, we propose that NUP-1 is a novel, highly divergent lamin The effect of Nup-1 depletion on the transcriptome was examined in three independent experiments (A, B, & C). T. brucei cultures were either treated with RNAi (plus) or left untreated (minus) and RNA was extracted from each sample at the indicated time point (0h, 6h, 12h, 24h, or 48h). Two color microarrays were performed comparing treated and untreated samples at each time point. Dye swaps were performed and are indicated. Replicates of t=12h and t=24h for sample B were also included.

2011-12-15 | E-GEOD-26256 | biostudies-arrayexpress

Effect of truncation mutants of the trypanosomatid nuclear lamina protein NUP-1 on the whole cell proteome in T. brucei

Project description:The nuclear lamina has multiple functions, including maintaining nuclear structural integrity and differential gene expression. Correct spatial and temporal lamina assembly is essential to meet these and other roles. Recently, it emerged that multiple lamina systems exist that are likely products of independent origins, while all these systems share remarkably analogous functions. Several lamina proteins are known in trypanosomes, two of which, NUP-1 and NUP-2, are essential, coiled-coil proteins with a molecular mass 450 and 250 kDa, respectively. Sequence analysis indicates distinct quaternary structures when compared to the ~60 kDa lamin proteins of multiple lineages, including metazoa. To uncover organisational principles of the trypanosome lamina we generated NUP-1 deletion mutants (N=N-terminal domain; C= C-terminal domain; NC: fusion of the N- and C-terminal domain with entire repeat region deletedd))designed to identify domains of NUP-1 responsible for oligomerisation. We find that both N- and C-termini act as interaction domains and disruption of these interactions impacts additional components of the lamina, the nuclear envelope and nucleoporin TbNup98. By contrast there is remarkably little impact on transcription, crucially including silencing of telomeric variant surface glycoprotein genes. These data indicate that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and suggest an architecture distinct to the lamin system is based on a ‘hub and spoke’ configuration.

2021-06-03 | PXD019978 | Pride

Nuclear pore complex evolution: A trypanosome Mlp analog functions in chromosomal segregation but lacks transcriptional barrier activity

Project description:The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organisation. In many eukaryotes the coiled coil Mlp/Tpr proteins of the NPC nuclear basket have specific roles in interactions with chromatin and defining specialised regions of active transcription, while Mlp2 associates with the mitotic spindle in a cell-cycle dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and low fidelity telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site, but apparently has minimal roles in the control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analog of Mlp/Tpr proteins, and together with the lamina analog NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. Whole transcriptome comparison between parental and TbNup92? cells

2014-04-03 | E-GEOD-56436 | biostudies-arrayexpress

Lamin A dimer and tetramer cross-links help explain disease mutations and polymer plasticity

Project description:Lamins are intermediate filament proteins responsible for nuclear mechanical integrity. Though linked to multiple heritable diseases, lamin structure and that of other intermediate filaments remains elusive. We employed cross-linking mass spectrometry to gain structural insights into lamin A dimer and tetramer structure. While confirming the parallel coiled-coil rod, we report that, contrary to prediction, rod linker regions are highly flexible and compressible. This explains the recently reported rod shortening in the assembled polymer and aspects of intermediate filament stretch properties. We further elucidate an extended interface in lamin A tetramers where both head and tail unstructured regions flanking the rod of each dimer act as polar bridges to stabilize lamin head-to-tail assembly. Furthermore, changes in these regions between dimer and tetramer forms suggest an ordered polymer assembly. Importantly, several residues mutated in laminopathies disrupt this interface and impair assembly, potentially explaining their role in disease.

2019-07-12 | PXD008337 | Pride

Nuclear pore complex evolution: A trypanosome Mlp analog functions in chromosomal segregation but lacks transcriptional barrier activity

2014-04-03 | GSE56436 | GEO

Expression data from HT-1080 cells incubated or not in the presence of laminin111 coiled-coiled recombinant fragment (rLCC111)

Project description:Laminins are large heterotrimeric cross-shaped extracellular matrix (ECM) glycoproteins with terminal globular domains and a coiled-coil region. We have produced for the first time a recombinant laminin coiled-coil domain and studied the transcriptional profile of cells incubated in coiled-coil-coated wells. Cells were incubated in 24w plates, onto coiled-coil-coated wells or control BSA-coated wells (one each), 24h

2011-10-11 | E-GEOD-32896 | biostudies-arrayexpress

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

Project description:Background: The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Results: Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to chromatin redistribution and decompaction. Consequently, lamina-associated domains (LADs) are detached from the nuclear periphery. The inter-chromosomal interactions and overlap between chromosome territories are increased. Moreover, Hi-C data revealed that lamin B1 is required for the integrity and segregation of chromatin compartments but not for the topologically associating domains (TADs). Using live cell single molecule tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Conclusions: Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting its crucial role in chromatin higher-order structure and chromatin dynamics.

2020-11-08 | GSE124409 | GEO

Loss of lamin B1 results in prolongation of S-phase and decondensation of chromosome territories in the interphase nucleus of mammalian cells

Project description:Nuclear lamin B1 constitutes one of the major structural proteins in the lamina mesh. We silenced the expression of lamin B1 by RNA interference in the colon cancer cell line DLD-1 and showed a dramatic redistribution of H3K27me3 from the periphery to a more homogeneous nuclear dispersion; in addition we observed an increased frequency of micronuclei and nuclear blebs. By 3D-FISH analyses, we demonstrate that the volume and surface of chromosome territories were significantly larger in LMNB1-depleted cells, suggesting that lamin B1 is required to maintain chromatin condensation in interphase nuclei. These changes led to a prolonged S-phase due to activation of Chk1 and telomere attrition. Finally, silencing of LMNB1 resulted in extensive changes in alternative splicing of multiple genes and in a higher number of enlarged nuclear speckles. Taken together, our results suggest a mechanistic role of the nuclear lamina in the organization of chromosome territories, maintenance of genome integrity and proper gene splicing. This dataset includes the comparison of gene transcripts between cells with silenced lamin B1 and empty vector negative control transfected cells. Single cell clones were generated after transfection of DLD-1 cells with shRNA against LMNB1. Two different constructs were used. For clone number 2, three replicates of the the same clone were included in the analysis. The rest of the clones correspond to biological replicates. As control, we used three different cones transfected with an empty vector control as well as wild type cells.

2014-07-16 | E-GEOD-53546 | biostudies-arrayexpress

Loss of lamin B1 results in prolongation of S-phase and decondensation of chromosome territories in the interphase nucleus of mammalian cells

2014-07-16 | GSE53546 | GEO

Expression data from HT-1080 cells incubated or not in the presence of laminin111 coiled-coiled recombinant fragment (rLCC111)

2011-10-12 | GSE32896 | GEO