In all cases tested, TFIIIB is responsible for recruiting pol III to its genetic templates. In mammalian cells, RB binds TFIIIB and prevents its interactions with both promoter DNA and pol III, thereby suppressing transcription. As TFIIIB is not recruited to its target genes when bound by RB, the mechanism predicts that pol III-dependent templates will not be occupied by RB; this contrasts with the situation at most genes controlled by RB, where it can be tethered by promoter-bound sequence-specific DNA-binding factors such as E2F. Contrary to this prediction, however, ChIP-seq data reveal the presence of RB in multiple cell types and the related protein p130 at many loci that rely on pol III for their expression, including RMRP, RN7SL, and a variety of tRNA genes. The sets of genes targeted varies according to cell type and growth state. In such cases, recruitment of RB and p130 can be explained by binding of E2F1, E2F4 and/or E2F5. Genes transcribed by pol III had not previously been identified as common targets of E2F family members. The data provide evidence that E2F may allow for the selective regulation of specific non-coding RNAs by RB, in addition to its influence on overall pol III output through its interaction with TFIIIB.

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, β, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

Low differentiation rates of mesenchymal stem cells (MSCs) limit their therapeutic effects on patients in clinical studies. Our previous study demonstrated that overexpressing p130 or E2F4 affected the multipotential differentiation of MSCs, and the underlying mechanism was attributed to the regulation of the G1 phase. Improving the efficiency of MSC differentiation into epithelial cells is considered to be a new method. Therefore, this study was conducted to evaluate the effects of overexpressing p130 or E2F4 in MSCs on improving re-epithelization in lipopolysaccharide (LPS)-induced ARDS animals.
Mouse MSCs (mMSCs) stably transfected with p130 and E2F4 were transplanted intratracheally into LPS-induced ARDS mice. After 7 and 14 days, the mice were sacrificed, and the histopathology of the lungs was assessed by haematoxylin-eosin staining and lung injury scoring. Homing and differentiation of mMSCs were analysed by labelling and tracking mMSCs with NIR815 dye and immunofluorescent staining. Surfactant proteins A and C and occludin in the lungs were assessed by western blot. Permeability was evaluated by analysing the protein concentration of BALF using ELISA. Alveolar fluid clearance was assessed by absorbance measurements of BALF. Lung fibrosis was assessed by Masson\'s trichrome staining and Ashcroft scoring.
The engraftment of mMSCs overexpressing p130 or E2F4 led to attenuated histopathological impairment of the lung tissue, and the lung injury scores of the LPS+mBM-MSC-p130 and LPS+mBM-MSC-E2F4 groups were also decreased (p < 0.05). Overexpression of p130 or E2F4 also increased the retention of mMSCs in the lung (p < 0.05), increased differentiation into type II alveolar epithelial cells (p < 0.05), and improved alveolar epithelial permeability (p < 0.05). Additionally, mMSCs overexpressing p130 or E2F4 inhibited lung fibrosis according to the deposition of collagen and the fibrosis score in the lungs (p < 0.05).
Overexpressing p130 or E2F4 in mMSCs could further improve the injured structure and function of epithelial cells in the lungs of ARDS mice as a result of improved differentiation of mMSCs into epithelial cells.

Bone-marrow-derived mesenchymal stem cells (MSCs) have great potential in transplantation medicine due to their multiple advantages. However, the controlled differentiation of MSCs is one of the key aspects of effective clinical transplantation. Growing evidence suggests that the cell cycle plays an important role in regulating differentiation, while p130 and E2F4 are key to cell cycle checkpoints. The aim of the study is to evaluate the effects and mechanism of p130/E2F4 on the multidifferentiation of MSCs. Our data showed that the transduction efficiencies of p130 or E2F4 mediated by lentiviral vectors were 80.3%-84.4%. p130 and E2F4 mRNA expression was significantly higher in MSC-p130 and MSC-E2F4 cells than in MSC normal control (NC) cells. Similar results were also observed for p130 and E2F4 protein expression. After osteogenic or adipogenic differentiation, the G1 phase was significantly delayed in the MSC-p130 and MSC-E2F4 groups compared with that in the MSC-NC group. However, the G1 phase in the MSC-p130 and MSC-E2F4 groups did the opposite after chondrogenic differentiation. Moreover, overexpressing p130 or E2F4 significantly improved osteogenic differentiation while inhibiting adipogenic and chondrogenic differentiation of mouse MSCs (mMSCs). Moreover, overexpressing p130 or E2F4 significantly improved migration but not proliferation of mMSCs. Our data suggest that cell cycle regulation may be involved in p130/E2F4-mediated changes in the multipotential abilities of bone-marrow-derived mMSCs.

Cancer cells are characterized by uncontrolled proliferation, whereas the ability to enter quiescence or dormancy is important for cancer cell survival and disease recurrence. Therefore, understanding the mechanisms regulating cell cycle progression and exit is essential for improving patient outcomes. The MuvB complex of five proteins (LIN9, LIN37, LIN52, RBBP4, and LIN54), also known as LINC (LIN complex), is important for coordinated cell cycle gene expression. By participating in the formation of three distinct transcriptional regulatory complexes, including DREAM (DP, RB-like, E2F, and MuvB), MMB (Myb-MuvB), and FoxM1-MuvB, MuvB represents a unique regulator mediating either transcriptional activation (during S-G2 phases) or repression (during quiescence). With no known enzymatic activities in any of the MuvB-associated complexes, studies have focused on the therapeutic potential of protein kinases responsible for initiating DREAM assembly or downstream enzymatic targets of MMB. Furthermore, the mechanisms governing the formation and activity of each complex (DREAM, MMB, or FoxM1-MuvB) may have important consequences for therapeutic response. The MMB complex is associated with prognostic markers of aggressiveness in several cancers, whereas the DREAM complex is tied to disease recurrence through its role in maintaining quiescence. Here, we review recent developments in our understanding of MuvB function in the context of cancer. We specifically highlight the rationale for additional investigation of MuvB in high-grade serous ovarian cancer and the need for further translational research.

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The retinoblastoma tumor suppressor (RB) and its related family members p107 and p130 regulate cell proliferation through the transcriptional repression of genes involved in cellular G1 to S phase transition. However, RB proteins are functionally versatile, and numerous genetic and biochemical studies point to expansive roles in cellular growth control, pluripotency, and apoptotic response. For the vast majority of genes, RB family members target the E2F family of transcriptional activators as an integral component of its gene regulatory mechanism. These interactions are regulated via reversible phosphorylation by Cyclin/Cyclin-dependent kinase (Cdk) complexes, a major molecular mechanism that regulates transcriptional output of RB/E2F target genes. Recent studies indicate an additional level of regulation involving the ubiquitin-proteasome system that renders pervasive control over each component of the RB pathway. Disruption of the genetic circuitry for proteasome-mediated targeting of the RB pathway has serious consequences on development and cellular transformation, and is associated with several forms of human cancer. In this review, we discuss the role of the ubiquitin-proteasome system in proteolytic control of RB-E2F pathway components, and recent data that points to surprising non-proteolytic roles for the ubiquitin-proteasome system in novel transcriptional regulatory mechanisms.

Chronic arsenite exposure induces immunosuppression, but the precise mechanisms remain elusive. Our previous studies demonstrated that arsenite exposure for 24 h induces G0/G1 arrest in mouse B lymphoma A20 cells and the arrest is caused through induction of cyclin-dependent kinase inhibitor p16(INK4a) followed by accumulation of an Rb family protein, p130. In this study, we further investigated the consequences of long-term arsenite exposure of A20 cells. The results demonstrated that exposure to 10 μM sodium arsenite up to 14 days induces a great increase in G0/G1 arrest, irreversible cell growth suppression, cellular morphological changes and positive staining for senescence-associated β-galactosidase. The long-term arsenite exposure also induced up-regulation of p16(INK4a) followed by robust accumulation of p130 and activation of the p53 pathway. Knockdown experiments with siRNA showed that p130 accumulation is essential for cell cycle arrest by long-term arsenite exposure. Since p16(INK4a) and the p53 pathway are known to be activated by DNA damage, we investigated the involvement of DNA damage formation by long-term arsenite exposure. We found that a variety of DNA repair-related genes were significantly down-regulated from 24 h of arsenite exposure and activation-induced cytidine deaminase was greatly up-regulated after long-term arsenite exposure. Consistent with these findings, long-term arsenite exposure increased a DNA double-strand break marker, γ-H2AX and increased mutation frequency in a Bcl6 gene region. These results revealed that long-term arsenite exposure induces premature senescence through DNA damage increase and p130 accumulation in lymphoid cells.

In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.

OBJECTIVE: It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells.
RESULTS: The association of B-myb with both LIN-54 and LIN-9 was equally decreased by depleting LIN-54 in CaSki cells. Flow cytometry analysis showed that LIN-54 depletion caused an increased proportion of G2/M cells in T98G, SiHa and CaSki cells. The mRNA levels of certain S/G2 genes such as cyclin B, aurora kinase A and Polo-like kinase 1 have demonstrated a marginal increased in CaSki-Lin-54-depleted cells when compared to SiHa- and T98G-Lin-54-depleted cells. We further confirmed this experiment by depleting the B-myb itself in CaSki cells and the results showed the same pattern of cell cycle and mRNA levels for S/G2 genes when compared to LIN-54- and LIN-9-depleted cells.
CONCLUSIONS: The B-myb-DREAM complex might not be vital for progression through mitosis in cells lacking a G1/S checkpoint and not as crucial as the p130-DREAM complex for the survival of the HPV virus.