PubMed Health⌕ Search

Biomedical subjects

Tom Misteli

Publications and source records attributed to Tom Misteli.

At least 19 recordsLinked to original sources
PubMed2025

Transcriptional profiling of Hutchinson-Gilford Progeria patients identifies primary target pathways of progerin.

Hutchinson Gilford Progeria Syndrome (HGPS) is an ultra-rare pediatric premature aging disorder. The disease is caused by a point mutation in the LMNA gene leading to the production of the dominant-negative progerin isoform of the nuclear envelope protein lamin A. Disease severity and progression amongst the population of ~140 known patients is variable. Most of the mechanistic insights into the disease have come from studies using cellular or mouse models of HGPS. To probe the clinical relevance of previously implicated cellular pathways and to address the extent of gene expression heterogeneity between patients, we have performed transcriptomic analysis of a comprehensive set of HGPS patients. We find misexpression of several cellular pathways across the patient population, particularly of multiple signaling pathways as well as the Unfolded Protein Response (UPR) and mesodermal cell fate specification. Variability amongst individual patients was limited, with misregulation of the major pathways observed in most patients. Comparing the transcriptome of patients with an inducible HGPS cell model, we distinguished immediate-early cellular response pathways from secondary adaptive pathways and identified mTORC1, the UPR, UV response, apoptosis and TNFα signaling via NF-κB as primary targets of the disease-causing progerin protein.

Hutchinson-Gilford Progeria Syndrome↗
PubMed2006

Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo.

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.

Amino Acid Sequence↗
PubMed2006

Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus.

Determining how genes are epigenetically regulated to ensure their correct spatial and temporal expression during development is key to our understanding of cell lineage commitment. Here we examined epigenetic changes at an important proneural regulator gene Mash1 (Ascl1), as embryonic stem (ES) cells commit to the neural lineage. In ES cells where the Mash1 gene is transcriptionally repressed, the locus replicated late in S phase and was preferentially positioned at the nuclear periphery with other late-replicating genes (Neurod, Sprr2a). This peripheral location was coupled with low levels of histone H3K9 acetylation at the Mash1 promoter and enhanced H3K27 methylation but surprisingly location was not affected by removal of the Ezh2/Eed HMTase complex or several other chromatin-silencing candidates (G9a, SuV39h-1, Dnmt-1, Dnmt-3a and Dnmt-3b). Upon neural induction however, Mash1 transcription was upregulated (>100-fold), switched its time of replication from late to early in S phase and relocated towards the interior of the nucleus. This spatial repositioning was selective for neural commitment because Mash1 was peripheral in ES-derived mesoderm and other non-neural cell types. A bidirectional analysis of replication timing across a 2 Mb region flanking the Mash1 locus showed that chromatin changes were focused at Mash1. These results suggest that Mash1 is regulated by changes in chromatin structure and location and implicate the nuclear periphery as an important environment for maintaining the undifferentiated state of ES cells.

Animals↗
PubMed2006

Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.

Differentiation of embryonic stem (ES) cells from a pluripotent to a committed state involves global changes in genome expression patterns. Gene activity is critically determined by chromatin structure and interactions of chromatin binding proteins. Here, we show that major architectural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells. Upon differentiation, the hyperdynamic proteins become immobilized on chromatin. Hyperdynamic binding is a property of pluripotent cells, but not of undifferentiated cells that are already lineage committed. ES cells lacking the nucleosome assembly factor HirA exhibit elevated levels of unbound histones, and formation of embryoid bodies is accelerated. In contrast, ES cells, in which the dynamic exchange of H1 is restricted, display differentiation arrest. We suggest that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.

Animals↗
PubMed2005

Differential recruitment of pre-mRNA splicing factors to alternatively spliced transcripts in vivo.

Alternative splicing in mammalian cells has been suggested to be largely controlled by combinatorial binding of basal splicing factors to pre-mRNA templates. This model predicts that distinct sets of pre-mRNA splicing factors are associated with alternatively spliced transcripts. However, no experimental evidence for differential recruitment of splicing factors to transcripts with distinct splicing fates is available. Here we have used quantitative single-cell imaging to test this key prediction in vivo. We show that distinct combinations of splicing factors are recruited to sites of alternatively spliced transcripts in intact cells. While a subset of serine/arginine protein splicing factors, including SF2/ASF, SC35, and SRp20, is efficiently recruited to the tau gene when exon 10 is included, these factors are less frequently associated with tau transcription sites when exon 10 is excluded. In contrast, the frequency of recruitment of several other splicing factors is independent of splicing outcome. Mutation analysis of SF2/ASF shows that both protein-protein as well as protein-RNA interactions are required for differential recruitment. The differential behavior of the various splicing factors provides the basis for combinatorial occupancy at pre-mRNAs. These observations represent the first in vivo evidence for differential association of pre-mRNA splicing factors with alternatively spliced transcripts. They confirm a key prediction of a stochastic model of alternative splicing, in which distinct combinatorial sets of generic pre-mRNA splicing factors contribute to splicing outcome.

Alternative Splicing↗
PubMed2005

Systems biology in the cell nucleus.

The mammalian nucleus is arguably the most complex cellular organelle. It houses the vast majority of an organism's genetic material and is the site of all major genome regulatory processes. Reductionist approaches have been spectacularly successful at dissecting at the molecular level many of the key processes that occur within the nucleus, particularly gene expression. At the same time, the limitations of analyzing single nuclear processes in spatial and temporal isolation and the validity of generalizing observations of single gene loci are becoming evident. The next level of understanding of genome function is to integrate our knowledge of their sequences and the molecular mechanisms involved in nuclear processes with our insights into the spatial and temporal organization of the nucleus and to elucidate the interplay between protein and gene networks in regulatory circuits. To do so, catalogues of genomes and proteomes as well as a precise understanding of the behavior of molecules in living cells are required. Converging technological developments in genomics, proteomics, dynamics and computation are now leading towards such an integrated biological understanding of genome biology and nuclear function.

Animals↗
PubMed2005

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.

In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle.

Animals↗
PubMed2005

Splicing misplaced.

Newly synthesized transcripts are usually spliced during transcription or immediately thereafter. So pre-mRNA splicing has been presumed to occur exclusively in the cell nucleus. In this issue of Cell, Denis et al. (2005) now report the presence of functional spliceosomes and signal-dependent pre-mRNA splicing in the cytoplasm of platelets.

Alternative Splicing↗
PubMed2005

Differential in vivo binding dynamics of somatic and oocyte-specific linker histones in oocytes and during ES cell nuclear transfer.

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

Animals↗
PubMed2005

E2F-dependent repression of topoisomerase II regulates heterochromatin formation and apoptosis in cells with melanoma-prone mutation.

RB pathway mutations, especially at the CDK4 and INK4A loci, are hallmarks of melanomagenesis. It is presently unclear what advantages these alterations confer during melanoma progression and how they influence melanoma therapy. Topoisomerase II inhibitors are widely used to treat human malignancies, including melanoma, although their variable success is attributable to a poor understanding of their mechanism of action. Using mouse and human cells harboring the melanoma-prone p16Ink4a-insensitive CDK4R24C mutation, we show here that topoisomerase II proteins are direct targets of E2F-mediated repression. Drug-treated cells fail to load repressor E2Fs on topoisomerase II promoters leading to elevated topoisomerase II levels and an enhanced sensitivity of cells to apoptosis. This is associated with the increased formation of heterochromatin domains enriched in structural heterochromatin proteins, methylated histones H3/H4, and topoisomerase II. We refer to these preapoptotic heterochromatin domains as apoptosis-associated heterochromatic foci. We suggest that cellular apoptosis is preceded by an intermediary chromatin remodeling state that involves alterations of DNA topology by topoisomerase II enzymes and gene silencing via formation of heterochromatin. These observations provide novel insight into the mechanism of drug action that influence treatment outcome: drug sensitivity or drug resistance.

Animals↗
PubMed2005

A non-random walk through the genome.

Recent publications on a wide range of eukaryotes indicate that genes showing particular expression patterns are not randomly distributed in the genome but are clustered into contiguous regions that we call neighborhoods. It seems probable that this organization is related to chromatin and the structure of the nucleus.

Animals↗
PubMed2005

Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a childhood premature aging disease caused by a spontaneous point mutation in lamin A (encoded by LMNA), one of the major architectural elements of the mammalian cell nucleus. The HGPS mutation activates an aberrant cryptic splice site in LMNA pre-mRNA, leading to synthesis of a truncated lamin A protein and concomitant reduction in wild-type lamin A. Fibroblasts from individuals with HGPS have severe morphological abnormalities in nuclear envelope structure. Here we show that the cellular disease phenotype is reversible in cells from individuals with HGPS. Introduction of wild-type lamin A protein does not rescue the cellular disease symptoms. The mutant LMNA mRNA and lamin A protein can be efficiently eliminated by correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site. Upon splicing correction, HGPS fibroblasts assume normal nuclear morphology, the aberrant nuclear distribution and cellular levels of lamina-associated proteins are rescued, defects in heterochromatin-specific histone modifications are corrected and proper expression of several misregulated genes is reestablished. Our results establish proof of principle for the correction of the premature aging phenotype in individuals with HGPS.

Alternative Splicing↗
PubMed2005

Going in GTP cycles in the nucleolus.

Proteins are directed to cellular compartments by specific localization signals. A GTP-driven cycle has now been identified as a mechanism for protein targeting to the nucleolus. The involvement of a GTP switch suggests that nucleolar localization can be regulated and may be responsive to extracellular stimuli via signaling pathways. The uncovered mechanism also implies that localization is determined by increased retention rather than directed targeting.

Carrier Proteins↗
PubMed2005

Concepts in nuclear architecture.

Genomes are defined by their primary sequence. The functional properties of genomes, however, are determined by far more complex mechanisms and depend on multiple layers of regulatory control processes. A key emerging contributor to genome function is the architectural organization of the cell nucleus. The spatial and temporal behavior of genomes and their regulatory proteins are now being recognized as important, yet still poorly understood, control mechanisms in genome function. Combined cell biological, molecular and computational analysis of architectural aspects of genome function has added a further dimension to the investigation of some of the most fundamental cellular processes including transcription and maintenance of genome integrity. The complete elucidation of the contribution that nuclear architecture makes to gene expression will be required to fully understand physiological processes such as differentiation, development and disease at the cellular level. Here I give an overview of some of the emerging concepts in the study of in vivo genome organization and function.

Animals↗
PubMed2005

Chromokinesins: multitalented players in mitosis.

Molecular motors generate cellular forces and act in a multitude of intracellular transport processes. The chromokinesins are a subgroup of kinesin motors. Chromokinesins act in various steps of mitosis, including chromosome condensation, metaphase alignment, chromosome segregation, cytokinesis and they help maintain genome stability. The emerging multifunctional nature of the chromokinesins provides insights into the coordination of distinct mitotic steps, and their role in maintenance of genome stability makes them attractive potential targets for therapeutic intervention.

Animals↗
PubMed2004

Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins.

During mitosis, chromosomes are highly condensed and transcription is silenced globally. One explanation for transcriptional repression is the reduced accessibility of transcription factors. To directly test this hypothesis and to investigate the dynamics of mitotic chromatin, we evaluate the exchange kinetics of several RNA polymerase I transcription factors and nucleosome components on mitotic chromatin in living cells. We demonstrate that these factors rapidly exchange on and off ribosomal DNA clusters and that the kinetics of exchange varies at different phases of mitosis. In addition, the nucleosome component H1c-GFP also shows phase-specific exchange rates with mitotic chromatin. Furthermore, core histone components exchange at detectable levels that are elevated during anaphase and telophase, temporally correlating with H3-K9 acetylation and recruitment of RNA polymerase II before the onset of bulk RNA synthesis at mitotic exit. Our findings indicate that mitotic chromosomes in general and ribosomal genes in particular, although highly condensed, are accessible to transcription factors and chromatin proteins. The phase-specific exchanges of nucleosome components during late mitotic phases are consistent with an emerging model of replication independent core histone replacement.

Acetylation↗