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E2f transcription factors The in vivo function of E2f transcription factors has been a matter of debate. Here it is shown that E2f1–3 contribute to the activation of cell cycle genes in dividing progenitor cells during mouse development but are dispensable for cell division. However, in differentiating cells, E2f1–3 act as repressors to facilitate cell cycle exit. The in vivo function of E2f transcription factors has been a matter of debate. The effects of E2f1 , E2f2 and E2f3 triple deficiency are now examined in murine embryonic stem cells, embryos and small intestines. E2f1–3 are shown to function as transcriptional activators in normal dividing progenitor cells; however, contrary to the current view, they are dispensable for cell division but are necessary for cell survival. In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators ( E2f1 , E2f2 and E2f3 ), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase 1 , 2 . Using a panel of tissue-specific cre -transgenic mice and conditional E2f alleles we examined the effects of E2f1 , E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1–3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1–3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1–3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1–3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1–3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.

E2f transcription factors The in vivo function of E2f transcription factors has been a matter of debate. They are generally thought to be essential for cell division, but in vivo experiments using the mouse retina as a model tissue now show, surprisingly, that cells can still divide in the absence of E2f1–3, due to functional redundancy with Myc. The activating E2f transcription factors induce transcription and drive cells out of quiescence, but whether activating E2fs are necessary for normal division is an area of debate. Here, the mouse retina is genetically manipulated to address E2f function in normal cells in vivo . Cells in the mouse retina can still divide in the absence of E2f1–3, although loss of activating E2fs leads to elevated apoptosis; thus, E2fs are not universally required for normal mammalian cell division. The activating E2f transcription factors (E2f1, E2f2 and E2f3) induce transcription and are widely viewed as essential positive cell cycle regulators. Indeed, they drive cells out of quiescence, and the ‘cancer cell cycle’ in Rb1 null cells is E2f-dependent 1 , 2 . Absence of activating E2fs in flies or mammalian fibroblasts causes cell cycle arrest 3 , 4 , but this block is alleviated by removing repressive E2f or the tumour suppressor p53, respectively 5 , 6 , 7 . Thus, whether activating E2fs are indispensable for normal division is an area of debate 1 . Activating E2fs are also well known pro-apoptotic factors, providing a defence against oncogenesis 8 , yet E2f1 can limit irradiation-induced apoptosis 9 , 10 . In flies this occurs through repression of hid (also called Wrinkled ; Smac/Diablo in mammals). However, in mammals the mechanism is unclear because Smac/Diablo is induced, not repressed, by E2f1 11 , and in keratinocytes survival is promoted indirectly through induction of DNA repair targets 12 . Thus, a direct pro-survival function for E2f1–3 and/or its relevance beyond irradiation has not been established. To address E2f1–3 function in normal cells in vivo we focused on the mouse retina, which is a relatively simple central nervous system component that can be manipulated genetically without compromising viability and has provided considerable insight into development and cancer 2 , 13 . Here we show that unlike fibroblasts, E2f1–3 null retinal progenitor cells or activated Müller glia can divide. We attribute this effect to functional interchangeability with Mycn. However, loss of activating E2fs caused downregulation of the p53 deacetylase Sirt1, p53 hyperacetylation and elevated apoptosis, establishing a novel E2f–Sirt1–p53 survival axis in vivo . Thus, activating E2fs are not universally required for normal mammalian cell division, but have an unexpected pro-survival role in development.

E2F proteins: reasons for diversity The E2F family is a family of proteins, some of which act as transcription activators and others as repressors. Here Shih-Yin Tsai et al . tested why there is such genetic complexity by inactivating the entire subset of activators singly or in combination in mice. They show that E2f3a is sufficient to support mouse embryonic and postnatal development. However, expression of E2f3b or E2f1 from the E2f3a locus suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. They conclude there is functional redundancy among activators and that the requirement for E2f3a during postnatal development is dictated by its regulatory sequences, not by its protein function. These findings provide a molecular basis for the observed specificity among E2F activators during development. The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions 1 , 2 . Whereas C. elegans 3 and Drosophila melanogaster 4 , 5 have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins 1 , 2 , 6 . Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1 , E2f2 , E2f3a and E2f3b , singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus ( E2f3a 3bki or  E2f3a 1ki , respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a . We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.

Background The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development. Results We used the HeliScopeCAGE library sequencing on cerebellar samples over 8 embryonic and 4 early postnatal times. This study showcases temporal expression pattern changes during cerebellar development. Through a bioinformatics analysis that focused on transcription factors, their promoters and binding sites, we identified genes that appear as strong candidates for involvement in cerebellar development. We selected several candidate transcriptional regulators for validation experiments including qRT-PCR and shRNA transcript knockdown. We observed marked and reproducible developmental defects in Atf4, Rfx3, and Scrt2 knockdown embryos, which support the role of these genes in cerebellar development. Conclusions The successful identification of these novel gene regulators in cerebellar development demonstrates that the FANTOM5 cerebellum time series is a high-quality transcriptome database for functional investigation of gene regulatory networks in cerebellar development.

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain 1 – 4 . Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage 5 – 8 . By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9 , 10 . However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3 , 4 . Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2 , CBFA2T3 , PRDM6 , UTX and OTX2 . CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens , and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES + KI67 + unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB. Derailed differentiation of human-specific progenitors of the developing cerebellar rhombic lip is the cause of group 4 medulloblastoma, the most common childhood brain tumour. 

The E2F transcription factors control key elements of development, including mammary gland branching morphogenesis, with several E2Fs playing essential roles. Additional prior data has demonstrated that loss of individual E2Fs can be compensated by other E2F family members, but this has not been tested in a mammary gland developmental context. Here we have explored the role of the E2Fs and their ability to functionally compensate for each other during mammary gland development. Using gene expression from terminal end buds and chromatin immunoprecipitation data for E2F1, E2F2 and E2F3, we noted both overlapping and unique mammary development genes regulated by each of the E2Fs. Based on our computational findings and the fact that E2Fs share a common binding motif, we hypothesized that E2F transcription factors would compensate for each other during mammary development and function. To test this hypothesis, we generated RNA from E2F1-/-, E2F2-/- and E2F3+/- mouse mammary glands. QRT-PCR on mammary glands during pregnancy demonstrated increases in E2F2 and E2F3a in the E2F1-/- mice and an increase in E2F2 levels in E2F3+/- mice. During lactation we noted that E2F3b transcript levels were increased in the E2F2-/- mice. Given that E2Fs have previously been noted to have the most striking effects on development during puberty, we hypothesized that loss of individual E2Fs would be compensated for at that time. Double mutant mice were generated and compared with the single knockouts. Loss of both E2F1 and E2F2 revealed a more striking phenotype than either knockout alone, indicating that E2F2 was compensating for E2F1 loss. Interestingly, while E2F2 was not able to functionally compensate for E2F3+/- during mammary outgrowth, increased E2F2 expression was observed in E2F3+/- mammary glands during pregnancy day 14.5 and lactation day 5. Together, these findings illustrate the specificity of E2F family members to compensate during development of the mammary gland.

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.

The ‘activating’ E2fs (E2f1-3) are transcription factors that potently induce quiescent cells to divide. Work on cultured fibroblasts suggested they were essential for division, but in vivo analysis in the developing retina and other tissues disproved this notion. The retina, therefore, is an ideal location to assess other in vivo adenovirus E2 promoter binding factor (E2f) functions. It is thought that E2f1 directly induces apoptosis, whereas other activating E2fs only induce death indirectly by upregulating E2f1 expression. Indeed, mouse retinoblastoma ( Rb )-null retinal neuron death requires E2f1, but not E2f2 or E2f3. However, we report an entirely distinct mechanism in dying cone photoreceptors. These neurons survive Rb loss, but undergo apoptosis in the cancer-prone retina lacking both Rb and its relative p107 . We show that while E2f1 killed Rb/p107 null rod, bipolar and ganglion neurons, E2f2 was required and sufficient for cone death, independent of E2f1 and E2f3. Moreover, whereas E2f1-dependent apoptosis was p53 and p73-independent, E2f2 caused p53-dependent cone death. Our in vivo analysis of cone photoreceptors provides unequivocal proof that E2f-induces apoptosis independent of E2f1, and reveals distinct E2f1- and E2f2-activated death pathways in response to a single tumorigenic insult.

Insulin from the β-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the β-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes. Insulin production boosted by Rfx6 The transcription factor neurogenin 3 (Neurog3) initiates the differentiation of insulin-producing β-cells and other islet cell types from pancreatic endoderm in the developing embryo, but the genetic program that subsequently completes this differentiation is incompletely understood. German et al . now show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Loss of Rfx6 function in mice leads to specific loss of pancreatic-polypeptide-producing cells, while in human infants mutations in RFX6 underlie a recessive syndrome of neonatal diabetes. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors coordinating pancreatic islet development. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes. Pancreatic β-cells release insulin, which controls energy homeostasis in vertebrates, and its lack causes diabetes mellitus. The transcription factor neurogenin 3 (Neurog3) initiates differentiation of β-cells and other islet cell types from pancreatic endoderm; here, the transcription factor Rfx6 is shown to direct islet cell differentiation downstream of Neurog3 in mice and humans. This may be useful in efforts to generate β-cells for patients with diabetes.

It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle-independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors.