Autophagy is a biological process that is crucial to maintain cellular homeostasis and is regulated by several metabolic pathways, including the p53 tumor suppressor pathway. In this issue of Genes & Development, Kenzelmann Broz and colleagues (pp. 1016–1031) show how the p53 family as a whole, including p63 and p73, collaborate in controlling autophagy to support tumor suppression.

The retinoblastoma tumor suppressor RB is well known for its capacity to restrict cell cycle progression at the G1/S transition of the cell cycle by controlling the transcription of cell cycle genes. In this issue of Genes & Development, Hilgendorf and colleagues (pp. 1003–1015) have identified a novel tumor suppressor function for RB independent of its role as a transcriptional regulator, in which RB directly activates the apoptosis regulator Bax at the mitochondria to promote cell death.

Remodeling of RNA–protein complexes (mRNPs) plays a critical role in mRNA biogenesis and metabolism. However, relatively little is known about the underlying mechanism and regulation of the mRNP remodeling. In this issue of Genes & Development, Zhou and colleagues (pp. 1046–1058) report that a protein remodeling machine, the p97–UBXD8 complex, disassembles mRNPs containing the AU-rich elements (AREs) bound by HuR proteins in a nondegradative, ubiquitin signaling-dependent manner, revealing a novel mechanism to regulate mRNA turnover.

Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The median survival of this group of patients after diagnosis is ~1 yr. Here we show that the levels of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, we also observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and Ezh2 at gene promoters alters the expression of genes that are associated with various cancer pathways. These results indicate that H3.3K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis.

Specific microRNAs (miRNAs), including miR-134, localize to neuronal dendrites, where they control synaptic protein synthesis and plasticity. However, the mechanism of miRNA transport is unknown. We found that the neuronal precursor-miRNA-134 (pre-miR-134) accumulates in dendrites of hippocampal neurons and at synapses in vivo. Dendritic localization of pre-miR-134 is mediated by the DEAH-box helicase DHX36, which directly associates with the pre-miR-134 terminal loop. DHX36 function is required for miR-134-dependent inhibition of target gene expression and the control of dendritic spine size. Dendritically localized pre-miR-134 could provide a local source of miR-134 that can be mobilized in an activity-dependent manner during plasticity.

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior–posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb^–/–;p53^–/– tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive.

The mechanisms by which the p53 tumor suppressor acts remain incompletely understood. To gain new insights into p53 biology, we used high-throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. Chromatin immunoprecipitation sequencing reveals 4785 p53-bound sites in the genome located near 3193 genes involved in diverse biological processes. RNA sequencing analysis shows that only a subset of p53-bound genes is transcriptionally regulated, yielding a list of 432 p53-bound and regulated genes. Interestingly, we identify a host of autophagy genes as direct p53 target genes. While the autophagy program is regulated predominantly by p53, the p53 family members p63 and p73 contribute to activation of this autophagy gene network. Induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity. While p53-induced autophagy does not affect cell cycle arrest in response to DNA damage, it is important for both robust p53-dependent apoptosis triggered by DNA damage and transformation suppression by p53. Together, our data highlight an intimate connection between p53 and autophagy through a vast transcriptional network and indicate that autophagy contributes to p53-dependent apoptosis and cancer suppression.

To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies.

The assembly and disassembly of ribonucleoproteins (RNPs) are dynamic processes that control every step of RNA metabolism, including mRNA stability. However, our knowledge of how RNP remodeling is achieved is largely limited to RNA helicase functions. Here, we report a previously unknown mechanism that implicates the ATPase p97, a protein-remodeling machine, in the dynamic regulation of mRNP disassembly. We found that p97 and its cofactor, UBXD8, destabilize p21, MKP-1, and SIRT1, three established mRNA targets of the RNA-binding protein HuR, by promoting release of HuR from mRNA. Importantly, ubiquitination of HuR with a short K29 chain serves as the signal for release. When cells are subjected to stress conditions, the steady-state levels of HuR ubiquitination change, suggesting a new mechanism through which HuR mediates the stress response. Our studies reveal a new paradigm in RNA biology: nondegradative ubiquitin signaling-dependent disassembly of mRNP promoted by the p97–UBXD8 complex to control mRNA stability.

Notch signaling plays crucial roles in mediating cell fate choices in all metazoans largely by specifying the transcriptional output of one cell in response to a neighboring cell. The DNA-binding protein RBPJ is the principle effector of this pathway in mammals and, together with the transcription factor moiety of Notch (NICD), regulates the expression of target genes. The prevalent view presumes that RBPJ statically occupies consensus binding sites while exchanging repressors for activators in response to NICD. We present the first specific RBPJ chromatin immunoprecipitation and high-throughput sequencing study in mammalian cells. To dissect the mode of transcriptional regulation by RBPJ and identify its direct targets, whole-genome binding profiles were generated for RBPJ; its coactivator, p300; NICD; and the histone H3 modifications H3 Lys 4 trimethylation (H3K4me3), H3 Lys 4 monomethylation (H3K4me1), and histone H3 Lys 27 acetylation (H3K27ac) in myogenic cells under active or inhibitory Notch signaling conditions. Our results demonstrate dynamic binding of RBPJ in response to Notch activation at essentially all sites co-occupied by NICD. Additionally, we identify a distinct set of sites where RBPJ recruits neither NICD nor p300 and binds DNA statically, irrespective of Notch activity. These findings significantly modify our views on how RBPJ and Notch signaling mediate their activities and consequently impact on cell fate decisions.