Here we report the identification of the LIN complex (LINC), a human multiprotein complex that is required for transcriptional activation of G 2 /M genes. LINC is related to the recently identified dREAM and DRM complexes of Drosophila and C. elegans that contain homologs of the mammalian retinoblastoma tumor suppressor protein. The LINC core complex consists of at least five subunits including the chromatin-associated LIN-9 and RbAp48 proteins. LINC dynamically associates with pocket proteins, E2F and B-MYB during the cell cycle. In quiescent cells, LINC binds to p130 and E2F4. During cell cycle entry, E2F4 and p130 dissociate and LINC switches to B-MYB and p107. Chromatin Immunoprecipitation experiments demonstrate that LINC associates with a large number of E2F-regulated promoters in quiescent cells. However, RNAi experiments reveal that LINC is not required for repression. In S-phase, LINC selectively binds to the promoters of G 2 /M genes whose products are required for mitosis and plays an important role in their cell cycle dependent activation.

The retinoblastoma tumor suppressor protein (pRB) and related p107 and p130 "pocket proteins" function together with the E2F transcription factors to repress gene expression during the cell cycle and development. Recent biochemical studies have identified the multisubunit DREAM pocket protein complexes in Drosophila melanogaster and Caenorhabditis elegans in regulating developmental gene repression. Although a conserved DREAM complex has also been identified in mammalian cells, its physiological function in vivo has not been determined. Here we addressed this question by targeting Lin9, a conserved core subunit of DREAM. We found that LIN9 is essential for early embryonic development and for viability of adult mice. Loss of Lin9 abolishes proliferation and leads to multiple defects in mitosis and cytokinesis because of its requirement for the expression of a large set of mitotic genes, such as Plk1, Aurora A, and Kif20a. While Lin9 heterozygous mice are healthy and normal, they are more susceptible to lung tumorigenesis induced by oncogenic c-Raf than wild-type mice. Together these experiments provide the first direct genetic evidence for the role of LIN9 in development and mitotic gene regulation and they suggest that it may function as a haploinsufficient tumor suppressor.The retinoblastoma tumor suppressor protein (pRB) and related p107 and p130 "pocket proteins" function together with the E2F transcription factors to regulate gene expression during the cell cycle (7). The identification of evolutionary conserved pocket protein/E2F complexes in Drosophila melanogaster has provided new insights into E2F-mediated gene regulation (21,24). These multisubunit complexes, alternatively called dREAM or Myb-MuvB (MMB), consist of at least eight subunits, including the repressor dE2F2 and one of the two retinoblastoma-related proteins, RBF1 or RBF2. In addition, the complex also contains Drosophila dMYB and three Myb-interacting proteins. RNA interference (RNAi)-mediated depletion of several subunits of the complex demonstrated a role in stable repression of developmental genes, although more recent genome-wide studies have found that dREAM/ MMB also functions in activation of genes involved in G 2 and mitosis (2,13,21,24).Remarkably, all subunits of dREAM/MMB, except for dMYB, are related to the Caenorhabditis elegans synMuv class B genes that antagonize RAS-induced vulva differentiation (3,9). Indeed, several synMuv proteins form a multisubunit complex that is highly related to dREAM/MMB (14). Therefore, in analogy to dREAM/MMB, it has been suggested that DRM mainly functions in gene repression during development (14).We and others recently identified a complex in human cells that is closely related to dREAM and DRM (20,25,31,40). The human complex, alternatively called LINC or human DREAM, consists of a five-protein core module that binds in quiescent cells to the repressors p130 and E2F4. In S phase this binding is lost and B-MYB associates with the complex. The high degree of conservation of the DREAM-like complex...

SummaryThe mammalian DREAM complex is a key regulator of cell-cycle-regulated gene transcription and drives the expression of many gene products required for mitosis and cytokinesis. In this study, we characterized GAS2L3, which belongs to the GAS2 family of proteins with putative actin-and microtubule-binding domains as a target gene of DREAM. We found that GAS2L3 localizes to the spindle midzone and the midbody during anaphase and cytokinesis, respectively. Biochemical studies show that GAS2L3 binds to and bundles microtubules as well as F-actin in vitro. Strikingly, the RNAi-mediated knockdown of GAS2L3 results in chromosome segregation defects in multinucleated cells and in cells with multi-lobed nuclei. Likewise, chronic downregulation of GAS2L3 causes chromosome loss and aneuploidy. Time-lapse videomicroscopy experiments in GAS2L3-knockdown cells reveal abnormal oscillation of chromatin and the spindle during cytokinesis. Taken together, our data reveal novel, important roles of GAS2L3 for faithful cell division. Our work thus contributes to the understanding of how DREAM regulates cytokinesis.

The DREAM complex is an important regulator of mitotic gene expression during the cell cycle. Here we report that inactivation of LIN9, a subunit of DREAM, results in premature senescence, which can be overcome by the SV40 large T (LT) antigen. Together with the observation that p16 INK4a and p21 Waf1 are upregulated upon loss of LIN9, these results indicate that senescence is triggered by the pRB and p53 tumor suppressor pathways. We also find that LIN9-null cells that escape senescence are chromosomally instable because of compromised mitotic fidelity. SV40 LT-expressing cells that adapt to the loss of LIN9 can grow anchorage-independently in soft agar, a hallmark of oncogenic transformation. Taken together, these results suggest an important role of mitotic gene regulation in the maintenance of genomic stability and tumor suppression.

Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.