Emerin modulates spatial organization of chromosome territories in cells on softer matrices.

Cells perceive and relay external mechanical forces into the nucleus through the nuclear envelope. Here we examined the effect of lowering substrate stiffness as a paradigm to address the impact of altered mechanical forces on nuclear structure-function relationships. RNA sequencing of cells on softer matrices revealed significant transcriptional imbalances, predominantly in chromatin associated processes and transcriptional deregulation of human Chromosome 1. Furthermore, 3-Dimensional fluorescence in situ hybridization (3D-FISH) analyses showed a significant mislocalization of Chromosome 1 and 19 Territories (CT) into the nuclear interior, consistent with their transcriptional deregulation. However, CT18 with relatively lower transcriptional dysregulation, also mislocalized into the nuclear interior. Furthermore, nuclear Lamins that regulate chromosome positioning, were mislocalized into the nuclear interior in response to lowered matrix stiffness. Notably, Lamin B2 overexpression retained CT18 near the nuclear periphery in cells on softer matrices. While, cells on softer matrices also activated emerin phosphorylation at a novel Tyr99 residue, the inhibition of which in a phospho-deficient mutant (emerinY99F), selectively retained chromosome 18 and 19 but not chromosome 1 territories at their conserved nuclear locations. Taken together, emerin functions as a key mechanosensor, that modulates the spatial organization of chromosome territories in the interphase nucleus

Lamin A/C modulates spatial organization and function of the Hsp70 gene locus via nuclear myosin I.

The structure-function relationship of the nucleus is tightly regulated, especially during heat shock. Typically, heat shock activates molecular chaperones that prevent protein misfolding and preserve genome integrity. However, the molecular mechanisms that regulate nuclear structure-function relationships during heat shock remain unclear. Here, we show that lamin A and C (hereafter lamin A/C; both lamin A and C are encoded by LMNA) are required for heat-shock-mediated transcriptional induction of the Hsp70 gene locus (HSPA genes). Interestingly, lamin A/C regulates redistribution of nuclear myosin I (NM1) into the nucleus upon heat shock, and depletion of either lamin A/C or NM1 abrogates heat-shock-induced repositioning of Hsp70 gene locus away from the nuclear envelope. Lamins and NM1 also regulate spatial positioning of the SC35 (also known as SRSF2) speckles - important nuclear landmarks that modulates Hsp70 gene locus expression upon heat shock. This suggests an intricate crosstalk between nuclear lamins, NM1 and SC35 organization in modulating transcriptional responses of the Hsp70 gene locus during heat shock. Taken together, this study unravels a novel role for lamin A/C in the regulation of the spatial dynamics and function of the Hsp70 gene locus upon heat shock, via the nuclear motor protein NM1.This article has an associated First Person interview with the first author of the paper

Lamin A/C and Emerin depletion impacts chromatin organization and dynamics in the interphase nucleus.

BACKGROUND: Nuclear lamins are type V intermediate filament proteins that maintain nuclear structure and function. Furthermore, Emerin - an interactor of Lamin A/C, facilitates crosstalk between the cytoskeleton and the nucleus as it also interacts with actin and Nuclear Myosin 1 (NM1). RESULTS: Here we show that the depletion of Lamin A/C or Emerin, alters the localization of the nuclear motor protein - Nuclear Myosin 1 (NM1) that manifests as an increase in NM1 foci in the nucleus and are rescued to basal levels upon the combined knockdown of Lamin A/C and Emerin. Furthermore, Lamin A/C-Emerin co-depletion destabilizes cytoskeletal organization as it increases actin stress fibers. This further impinges on nuclear organization, as it enhances chromatin mobility more toward the nuclear interior in Lamin A/C-Emerin co-depleted cells. This enhanced chromatin mobility was restored to basal levels either upon inhibition of Nuclear Myosin 1 (NM1) activity or actin depolymerization. In addition, the combined loss of Lamin A/C and Emerin alters the otherwise highly conserved spatial positions of chromosome territories. Furthermore, knockdown of Lamin A/C or Lamin A/C-Emerin combined, deregulates expression levels of a candidate subset of genes. Amongst these genes, both KLK10 (Chr.19, Lamina Associated Domain (LAD+)) and MADH2 (Chr.18, LAD-) were significantly repressed, while BCL2L12 (Chr.19, LAD-) is de-repressed. These genes differentially reposition with respect to the nuclear envelope. CONCLUSIONS: Taken together, these studies underscore a remarkable interplay between Lamin A/C and Emerin in modulating cytoskeletal organization of actin and NM1 that impinges on chromatin dynamics and function in the interphase nucleus

Evaluating annotations of an Agilent expression chip suggests that many features cannot be interpreted

<p>Abstract</p> <p>Background</p> <p>While attempting to reanalyze published data from Agilent 4 × 44 human expression chips, we found that some of the 60-mer olignucleotide features could not be interpreted as representing single human genes. For example, some of the oligonucleotides align with the transcripts of more than one gene. We decided to check the annotations for all autosomes and the X chromosome systematically using bioinformatics methods.</p> <p>Results</p> <p>Out of 42683 reporters, we found that 25505 (60%) passed all our tests and are considered "fully valid". 9964 (23%) reporters did not have a meaningful identifier, mapped to the wrong chromosome, or did not pass basic alignment tests preventing us from correlating the expression values of these reporters with a unique annotated human gene. The remaining 7214 (17%) reporters could be associated with either a unique gene or a unique intergenic location, but could not be mapped to a transcript in RefSeq. The 7214 reporters are further partitioned into three different levels of validity.</p> <p>Conclusion</p> <p>Expression array studies should evaluate the annotations of reporters and remove those reporters that have suspect annotations. This evaluation can be done systematically and semi-automatically, but one must recognize that data sources are frequently updated leading to slightly changing validation results over time.</p

Nuclear envelope, chromatin organizers, histones, and DNA: The many achilles heels exploited across cancers

In eukaryotic cells, the genome is organized in the form of chromatin composed of DNA and histones that organize and regulate gene expression. The dysregulation of chromatin remodeling, including the aberrant incorporation of histone variants and their consequent post-translational modifications, is prevalent across cancers. Additionally, nuclear envelope proteins are often deregulated in cancers, which impacts the 3D organization of the genome. Altered nuclear morphology, genome organization, and gene expression are defining features of cancers. With advances in single-cell sequencing, imaging technologies, and high-end data mining approaches, we are now at the forefront of designing appropriate small molecules to selectively inhibit the growth and proliferation of cancer cells in a genome- and epigenome-specific manner. Here, we review recent advances and the emerging significance of aberrations in nuclear envelope proteins, histone variants, and oncohistones in deregulating chromatin organization and gene expression in oncogenesis

Artificially Introduced Aneuploid Chromosomes Assume a Conserved Position in Colon Cancer Cells

BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density) that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained

Translin binding to DNA: recruitment through DNA ends and consequent conformational transitions

The human translin protein binds a variety of sequences (chromosomal breakpoint consensus sequences, their sequence variants, as well as nonbreakpoint sequences such as simple AT and GC repeats) at nanomolar protein concentration when short single strands (&#8764;20−30mers) are used as DNA targets. The protein, which is known to exist as an octamer in its free state, undergoes a conformational transition upon binding to short single strands leading either to a compaction or to the dissociation of the oligomer. Moreover, the protein oligomers tend to aggregate into complexes that get progressively larger as the length of the single-stranded DNA target increases. The protein loads onto duplexes via the free ends of DNA, generating higher oligomeric complexes as a function of protein concentration. Interestingly, the conformation of DNA targets encased by translin oligomer is significantly altered such that the single strand is rendered hypersensitive to DNase I. Furthermore, the loading of translin oligomers leads to tighter clamping of duplex ends. All of these observations, taken together, suggest that translin is a bona fide binder of DNA ends, thereby subjecting the DNA to a conformation conducive for repair steps during translocation events. We discuss the results in the perspective of translin biology

Trapping an Elusive Fe(IV)-Superoxo Intermediate Inside a Self-Assembled Nanocage at Room Temperature

Natural metalloenzymes stabilize reactive intermediates through specific metal-substrate interactions in protein confinement. Using the structural blueprint of enzyme pockets it is possible to trap elusive intermediates inside molecular cavities. Here we demonstrate room temperature trapping of a rare yet stable Fe(IV)-superoxo [FeIV(O2)-bTAML] intermediate subsequent to dioxygen binding at the Fe(III) site of a (Et4N)2[FeIII(Cl)(bTAML)] catalyst confined inside the hydrophobic interior of a water-soluble Pd6L412+ nanocage. <br /

MOESM5 of HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

Additional file 5: Table S1. List of ChIP-qPCR primers. Table S2. List of RT-PCR primers. Table S3. Antibodies and their dilutions used in this study