David L. Spector

Rb-Mediated Heterochromatin Formation and Silencing of E2F Target Genes during Cellular Senescence

Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. In this study, we describe a distinct heterochromatic structure that accumulates in senescent human fibroblasts, which we designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor.

Nuclear speckles: a model for nuclear organelles

Speckles are subnuclear structures that are enriched in pre-messenger RNA splicing factors and are located in the interchromatin regions of the nucleoplasm of mammalian cells. At the fluorescence-microscope level they appear as irregular, punctate structures, which vary in size and shape, and when examined by electron microscopy they are seen as clusters of interchromatin granules. Speckles are dynamic structures, and both their protein and RNA–protein components can cycle continuously between speckles and other nuclear locations, including active transcription sites. Studies on the composition, structure and behaviour of speckles have provided a model for understanding the functional compartmentalization of the nucleus and the organization of the gene-expression machinery.

The Noncoding RNA MALAT1 Is a Critical Regulator of the Metastasis Phenotype of Lung Cancer Cells

The long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), also known as MALAT-1 or NEAT2 (nuclear-enriched abundant transcript 2), is a highly conserved nuclear noncoding RNA (ncRNA) and a predictive marker for metastasis development in lung cancer. To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements using zinc finger nucleases. The achieved 1,000-fold MALAT1 silencing provides a unique loss-of-function model. Proposed mechanisms of action include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but actively regulates gene expression including a set of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft. Antisense oligonucleotides (ASO) blocking MALAT1 prevent metastasis formation after tumor implantation. Thus, targeting MALAT1 with ASOs provides a potential therapeutic approach to prevent lung cancer metastasis with this ncRNA serving as both predictive marker and therapeutic target. Finally, regulating gene expression, but not alternative splicing, is the critical function of MALAT1 in lung cancer metastasis. In summary, 10 years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, our loss-of-function model unravels the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis.

From silencing to gene expression: Real-time analysis in single cells

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.

Associations between distinct pre-mRNA splicing components and the cell nucleus

SC‐35 is a non‐snRNP spliceosome component that is specifically recognized by the anti‐spliceosome monoclonal antibody alpha SC‐35. In this paper we provide direct evidence that SC‐35 is an essential splicing factor and we examine the immunolocalization of SC‐35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC‐35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC‐35 speckled staining pattern is lost during prophase and speckles containing SC‐35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC‐35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.

53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility.

Double-strand breaks activate the ataxia telangiectasia mutated (ATM) kinase, which promotes the accumulation of DNA damage factors in the chromatin surrounding the break. The functional significance of the resulting DNA damage foci is poorly understood. Here we show that 53BP1 (also known as TRP53BP1), a component of DNA damage foci, changes the dynamic behaviour of chromatin to promote DNA repair. We used conditional deletion of the shelterin component TRF2 (also known as TERF2) from mouse cells (TRF2fl/-) to deprotect telomeres, which, like double-strand breaks, activate the ATM kinase, accumulate 53BP1 and are processed by non-homologous end joining (NHEJ). Deletion of TRF2 from 53BP1-deficient cells established that NHEJ of dysfunctional telomeres is strongly dependent on the binding of 53BP1 to damaged chromosome ends. To address the mechanism by which 53BP1 promotes NHEJ, we used time-lapse microscopy to measure telomere dynamics before and after their deprotection. Imaging showed that deprotected telomeres are more mobile and sample larger territories within the nucleus. This change in chromatin dynamics was dependent on 53BP1 and ATM but did not require a functional NHEJ pathway. We propose that the binding of 53BP1 near DNA breaks changes the dynamic behaviour of the local chromatin, thereby facilitating NHEJ repair reactions that involve distant sites, including joining of dysfunctional telomeres and AID (also known as AICDA)-induced breaks in immunoglobulin class-switch recombination.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression

A growing number of long nuclear‐retained non‐coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis‐associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II‐dependent transcription is active. Knock‐down studies revealed that Malat1 modulates the recruitment of SR family pre‐mRNA‐splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1‐depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock‐down of Malat1 decreases synaptic density, whereas its over‐expression results in a cell‐autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

Biogenesis and function of nuclear bodies

Nuclear bodies including nucleoli, Cajal bodies, nuclear speckles, Polycomb bodies, and paraspeckles are membraneless subnuclear organelles. They are present at steady-state and dynamically respond to basic physiological processes as well as to various forms of stress, altered metabolic conditions and alterations in cellular signaling. The formation of a specific nuclear body has been suggested to follow a stochastic or ordered assembly model. In addition, a seeding mechanism has been proposed to assemble, maintain, and regulate particular nuclear bodies. In coordination with noncoding RNAs, chromatin modifiers and other machineries, various nuclear bodies have been shown to sequester and modify proteins, process RNAs and assemble ribonucleoprotein complexes, as well as epigenetically regulate gene expression. Understanding the functional relationships between the 3D organization of the genome and nuclear bodies is essential to fully uncover the regulation of gene expression and its implications for human disease.

A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence

The peripheral nuclear lamina, which is largely but not entirely associated with inactive chromatin, is considered to be an important determinant of nuclear structure and gene expression. We present here an inducible system to target a genetic locus to the nuclear lamina in living mammalian cells. Using three-dimensional time-lapse microscopy, we determined that targeting of the locus requires passage through mitosis. Once targeted, the locus remains anchored to the nuclear periphery in interphase as well as in daughter cells after passage through a subsequent mitosis. Upon transcriptional induction, components of the gene expression machinery are recruited to the targeted locus, and we visualized nascent transcripts at the nuclear periphery. The kinetics of transcriptional induction at the nuclear lamina is similar to that observed at an internal nuclear region. This new cell system provides a powerful approach to study the dynamics of gene function at the nuclear periphery in living cells.

RNA Polymerase II Targets Pre-mRNA Splicing Factors to Transcription Sites In Vivo

Biochemical evidence indicates that pre-mRNA splicing factors physically interact with the C-terminal domain of the largest subunit of RNA polymerase II. We have investigated the in vivo function of this interaction. In mammalian cells, truncation of the CTD of RNA pol II LS prevents the targeting of the splicing machinery to a transcription site. In the absence of the CTD, pre-mRNA splicing is severely reduced. The presence of unspliced RNA alone is not sufficient for the accumulation of splicing factors at the transcription site, nor for its efficient splicing. Our results demonstrate a critical role for the CTD of RNA pol II LS in the intranuclear targeting of splicing factors to transcription sites in vivo.