ABSTRACT

Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. We demonstrated a direct role for histone modifications in alternative splicing. We found distinctive histone modification signatures that correlate with the splicing outcome in a set of human genes, and modulation of histone modifications causes splice site switching. Histone marks affect splicing outcome by influencing the recruitment of splicing regulators via a chromatin-binding protein. These results outline an adaptor system for the reading of histone marks by the pre-mRNA splicing machinery.

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Normal cells have limited proliferative potential in culture, a fact that has been the basis of their use as a model for replicative senescence for many years. Recent molecular analyses have identified numerous changes in gene expression that occur as cells become senescent, and the results indicate that multiple levels of control contribute to the irreversible growth arrest. These include repression of growth stimulatory genes, overexpression of growth inhibitory genes, and interference with downstream pathways. Studies with cell types other than fibroblasts will better define the role of cell senescence in the aging process and in tumorigenesis.

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in Gl. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (MI) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed MI mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active MI mechanism.Normal diploid cells exhibit a finite proliferative capacity in culture (13-16, 25, 32, 43), characterized by a decreasing mitogen responsiveness (33) and an eventual arrest in GI. The frequency with which cells from different species escape cellular senescence and give rise to immortal cell lines varies extensively (23). Whereas mouse cells immortalize with a high probability (6,19,24,29) (2, 3,18,26,34,36,39,41). The role of T antigen in the maintenance of immortality of human cells has not yet been determined. Because the mechanisms regulating senescence in rodent and human cells are different, this study was undertaken to examine this question. We report that the continued expression of T antigen is required for the proliferation of T-antigen-immortalized human fibroblasts and discuss a model suggesting why it is misleading to say that T antigen is necessary for the maintenance of immortality. A similar observation for a temperature-sensitive T antigen is presented in the accompanying paper (40). METHODSCells and media. IMR-90 normal human fibroblasts (ATCC 186) were maintained in a 4:1 mixture of Dulbecco modified Eagle medium-medium 199 supplemented with 10% defined supplemented bovine calf serum (Hyclone Laboratories, Logan, Utah). When necessary, serum was depleted of steroids by being stirred overnight at 4°C with 5 mg of sterile activated charcoal (Norit A) per 100 ml of serum (10). After removal of the charcoal by spinning at 25,000 x g for 60 min, the serum was decanted and stored at -10°C. Plasmids (MCDB 202 [11]) supplemented with 400 ,ug of G418 per ml and 10-6 M dexamethasone and placed in modular incubators (gassed with 1% oxygen-5% carbon dioxide-94% nitrogen to increase ...

Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the "strings and binders switch" model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed "fractal-globule" model, but only as one of many possible transient conformations.genome organization | genome architecture | long-range chromatin interactions | fluorescence in situ hybridization | Monte Carlo simulations U nderstanding the interplay between genome architecture and gene regulation is one of the most challenging problems in biology. During mitosis, chromosomes are found in a condensed state, but decondense during interphase, when highly coordinated cellular processes such as transcription, DNA repair, and replication take place, creating cell-type-specific chromatin folding (1-3).Chromosome organization occurs at different scales of genomic length to yield variable degrees of compaction (4). Linear nucleosome arrays fold into higher-order structures, first through local chromatin interactions, such as between promoters and enhancers, and then eventually giving rise to discrete chromosome territories (1).Spatial genome organization is guided by intra-and interchromosomal interactions mediated by nuclear components that include transcription factors, transcription and replication factories, Polycomb bodies, and contacts with the lamina (5-8). However, how binding of diffusible factors to specific genomic regions drives chromatin folding remains poorly understood.Imaging of single loci by FISH and genome-wide mapping of chromatin interactions by chromosome conformation capture (3C) approaches revealed a variety of chromatin architectures across genomic regions and cell types, and upon environmental cues (9-14) (Fig. S1A). In FISH experiments, chromatin folding is often measured by the mean-square spatial distance, R 2 ðsÞ, between two genomic regions as a function of their linear genomic distance, s (Fig. S1B), which usually exhibits scaling properties R 2 ðsÞ ∼ s 2v . Although the behavior of R 2 ðsÞ appears to depend on the genomic regions and cell types assessed (Fig. S1A), in general, at large genomic distances, R 2 ðsÞ reaches a plateau (i.e., v ¼ 0) that reflects the folding of chromosomes into territories (15).A global analysis of genome-wide 3C (Hi-C) ligation products in human cells averaged across all chromosomes has been used to estimate the "contact probability," P c ðsÞ (13). Th...

Mortalin, also known as mthsp70/GRP75/PBP74, interacts with the tumor suppressor protein p53 and inactivates its transcriptional activation and apoptotic functions. Here, we examined the level of mortalin expression in a large variety of tumor tissues, tumor-derived and in vitro immortalized human cells. It was elevated in many human tumors, and in all of the tumor-derived and in vitro immortalized cells. In human embryonic fibroblasts immortalized with an expression plasmid for hTERT, the telomerase catalytic subunit, with or without human papillomavirus E6 and E7 genes, we found that subclones with spontaneously increased mortalin expression levels became anchorage-independent and acquired the ability to form tumors in nude mice. Furthermore, overexpression of mortalin was sufficient to increase the malignancy of breast carcinoma cells. The study demonstrates that upregulation of mortalin contributes significantly to tumorigenesis, and thus is a good candidate target for cancer therapy. ' 2006 Wiley-Liss, Inc.