The factors that guide DNA hypermethylation in cancer are poorly understood. We identified the candidate tumor-suppressor gene, RIL, as a frequent methylation target in cancer. Here, we report on a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Methylation analysis showed that, in aging colon, colon cancer, and leukemias, the short allele had 2.1–3.1-fold higher methylation than the long allele (P<0.001). Short and long alleles had similar expression levels in EBV-transformed cell lines. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors. Transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on, but methylation-seeded constructs showed gradually decreasing transcription from the short allele with eventual spreading of de novo methylation. By contrast, the long allele showed stable expression over time as measured by luciferase, and ~2–3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

PLoS Genet 4(8): e1000162. doi:10.1371/journal.pgen.1000162

Brain-derived neurotrophic factor (BDNF) and other neurotrophins have a vital role in the development of the rat and mouse nervous system by influencing the expression of many specific genes that promote differentiation, cell survival, synapse formation and, later, synaptic plasticity1. Although nitric oxide (NO) is known to be an important mediator of BDNF signalling in neurons2, the mechanisms by which neurotrophins influence gene expression during development and plasticity remain largely unknown. Here we show that BDNF triggers NO synthesis and S-nitrosylation of histone deacetylase 2 (HDAC2) in neurons, resulting in changes to histone modifications and gene activation. S-nitrosylation of HDAC2 occurs at Cys 262 and Cys 274 and does not affect deacetylase activity. In contrast, nitrosylation of HDAC2 induces its release from chromatin, which increases acetylation of histones surrounding neurotrophin-dependent gene promoters and promotes transcription. Notably, nitrosylation of HDAC2 in embryonic cortical neurons regulates dendritic growth and branching, possibly by the activation of CREB (cyclic-AMP-responsive-element-binding protein)-dependent genes. Thus, by stimulating NO production and S-nitrosylation of HDAC2, neurotrophic factors promote chromatin remodelling and the activation of genes that are associated with neuronal development.

Nature advance online publication 27 August 2008 | doi:10.1038/nature07238

The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions1, 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 proteins1, 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 (E2f3a3bki or E2f3a1ki, 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.

Nature 454, 1137-1141 (28 August 2008) | doi:10.1038/nature07066

Adult stem cells reside in specialized microenvironments, or niches, that have an important role in regulating stem cell behaviour1. Therefore, tight control of niche number, size and function is necessary to ensure the proper balance between stem cells and progenitor cells available for tissue homeostasis and wound repair. The stem cell niche in the Drosophila male gonad is located at the tip of the testis where germline and somatic stem cells surround the apical hub, a cluster of approximately 10–15 somatic cells that is required for stem cell self-renewal and maintenance2, 3, 4. Here we show that somatic stem cells in the Drosophila testis contribute to both the apical hub and the somatic cyst cell lineage. The Drosophila orthologue of epithelial cadherin (DE-cadherin) is required for somatic stem cell maintenance and, consequently, the apical hub. Furthermore, our data indicate that the transcriptional repressor escargot regulates the ability of somatic cells to assume and/or maintain hub cell identity. These data highlight the dynamic relationship between stem cells and the niche and provide insight into genetic programmes that regulate niche size and function to support normal tissue homeostasis and organ regeneration throughout life.

Nature 454, 1132-1136 (28 August 2008) doi:10.1038/nature07173

Blood and endothelium arise in close association during development, possibly from a common precursor, the hemangioblast [1], [2], [3] and [4]. Genes essential for blood and endothelial development contain functional ETS binding sites, and binding and expression data implicate the transcription factor, friend leukaemia integration 1 (Fli1) [5], [6], [7], [8], [9] and [10]. However, loss-of-function phenotypes in mice, although suffering both blood and endothelial defects, have thus far precluded the conclusion that Fli1 is essential for these two lineages [11] and [12]. By using Xenopus and zebrafish embryos, we show that loss of Fli1 function results in a substantial reduction or absence of hemangioblasts, revealing an absolute requirement. TUNEL assays show that the cells are eventually lost by apoptosis, but only after the regulatory circuit has been disrupted by loss of Fli1. In addition, a constitutively active form of Fli1 is sufficient to induce expression of key hemangioblast genes, such as Scl/Tal1, Lmo2, Gata2, Etsrp, and Flk1. Epistasis assays show that Fli1 expression is induced by Bmp signaling or Cloche, depending on the hemangioblast population, and in both cases Fli1 acts upstream of Gata2, Scl, Lmo2, and Etsrp. Taken together, these results place Fli1 at the top of the transcriptional regulatory hierarchy for hemangioblast specification in vertebrate embryos.

Current Biology, Volume 18, Issue 16, 26 August 2008, Pages 1234-1240

In all of nature, scientists have discovered only six different mechanisms by which organisms sense light, and only one of these mechanisms can detect ultraviolet light (the rhodopsins that sense ultraviolet light in non-mammalian vertebrates). The widely studied model organism Caenorhabditis elegans has none of the known light transduction systems, but we discovered that C. elegans has a robust locomotory response to ultraviolet light. C. elegans may use this light response to escape damaging or lethal doses of sunlight. Ultraviolet and other shortwave light, such as violet and blue wavelengths, drive locomotion by bypassing two critical signals, cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG), that neurons use to shape and control behaviors. C. elegans mutants lacking these signals are paralyzed and unresponsive to harsh physical stimuli in ambient light, but short-wavelength light rapidly rescues their paralysis and restores greater-than-normal levels of coordinated locomotion. This astonishing light response is mediated by a novel ultraviolet light receptor that acts in neurons. Our results reveal a novel molecular solution for ultraviolet light detection and an unusual sensory modality in C. elegans that is unlike any previously described light response in any organism.

PLoS Biol 6(8): e198 doi:10.1371/journal.pbio.0060198

Animal microRNAs (miRNAs) regulate gene expression by inhibiting translation and/or by inducing degradation of target messenger RNAs. It is unknown how much translational control is exerted by miRNAs on a genome-wide scale. We used a new proteomic approach to measure changes in synthesis of several thousand proteins in response to miRNA transfection or endogenous miRNA knockdown. In parallel, we quantified mRNA levels using microarrays. Here we show that a single miRNA can repress the production of hundreds of proteins, but that this repression is typically relatively mild. A number of known features of the miRNA-binding site such as the seed sequence also govern repression of human protein synthesis, and we report additional target sequence characteristics. We demonstrate that, in addition to downregulating mRNA levels, miRNAs also directly repress translation of hundreds of genes. Finally, our data suggest that a miRNA can, by direct or indirect effects, tune protein synthesis from thousands of genes.

Nature advance online publication 30 July 2008 | doi:10.1038/nature07228; Received 8 April 2008; Accepted 3 July 2008; Published online 30 July 2008 Read More...

MicroRNAs are endogenous approx23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output.

Nature advance online publication 30 July 2008 | doi:10.1038/nature07242; Received 17 April 2008; Accepted 10 July 2008; Published online 30 July 2008 Read More...