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Female mammals have two X chromosomes, one of which is almost completely shut down during development. The long noncoding Xist RNA plays a role in this process. To understand how a whole chromosome can be stably inactivated, Minajigi et al. identified many of the proteins that bind to the Xist RNA, which include cohesins. Paradoxically, the interaction between Xist and cohesin subunits resulted in repulsion of cohesin complexes from the inactive X chromosome, changing the three-dimensional shape of the whole chromosome. Science , this issue 10.1126/science.aab2276 A screen for factors that bind directly to RNA reveals the proteins that interact with the long noncoding RNA Xist. The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform “identification of direct RNA interacting proteins” (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors—including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, and modifiers—that synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. Although Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist , the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes.

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, we isolated atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2. We show that the normal activity of Ataxin-2 (Atx2) is critical for SCA3 degeneration and that Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). We show here that PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. We propose that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by “priming” cells with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Key Points More than 40 Drosophila melanogaster genes have been discovered for which recessive, loss-of-function mutations cause adult onset degeneration of the central nervous system (CNS). A table presenting these genes is provided, and an expanded, updated version can be found at the Bonini laboratory homepage . Almost all of these genes have easily identifiable orthologues in the mouse and human. Over half have mouse or human orthologues that are also associated with neurodegeneration. The swiss cheese ( sws ) gene demonstrates the value of unbiased screens in the fly. Since its discovery, two biochemical functions have been characterized for the protein; loss of the mouse orthologue in the brain has been shown to cause neurodegeneration, and loss-of-function mutations in the human orthologue have been discovered as the cause of spastic paraplegia 39. Pink1 and park are associated with Parkinson's disease, and they demonstrate the value of epistasis experiments in the fly, which have shown that these two genes function together in a pathway that regulates mitochondrial fusion and fission. Fly neurodegeneration genes can be grouped into the following cellular processes: mitochondrial function, signal transduction, lipid homeostasis, protein homeostasis and the cytoskeleton. Many of the genes have roles in more than one of these processes. Some glial-specific genes have been shown to be required for maintaining neurons in the adult. Mutations in other, more widely expressed genes have defective glia, underscoring the importance of glia in CNS integrity. Many genetic tricks are possible in the fly, such as: the precise control in space and time of the expression of transgenes, including through RNAi constructs; and the possibility of making marked homozygous mutant clones as small as a single neuron in otherwise heterozygous animals. These techniques, and the ease of forward genetics screens for identifying new neurodegeneration mutants, ensure that D. melanogaster will remain a key tool for the analysis of genes required for CNS integrity. Identifying genes that are essential for maintaining neuronal integrity provides significant insight into the mechanisms underlying neurodegenerative disorders. Recessive mutants in the fly have proven invaluable for finding such genes and for highlighting key biological processes that contribute to neurodegeneration. The fruitfly Drosophila melanogaster has enabled significant advances in neurodegenerative disease research, notably in the identification of genes that are required to maintain the structural integrity of the brain, defined by recessive mutations that cause adult onset neurodegeneration. Here, we survey these genes in the fly and classify them according to five key cell biological processes. Over half of these genes have counterparts in mice or humans that are also associated with neurodegeneration. Fly genetics continues to be instrumental in the analysis of degenerative disease, with notable recent advances in our understanding of several inherited disorders, Parkinson's disease, and the central role of mitochondria in neuronal maintenance.

Hedgehog (Hh) is a member of a family of secreted proteins that direct patterning at multiple stages in both Drosophila and vertebrate development. During Drosophila embryogenesis, Hh protein is secreted by the cells of the posterior compartment of each segment. hh activates transcription of wingless (wg), gooseberry (gsb), and patched (ptc) in the cells immediately adjacent to Hh-secreting cells. Hh signaling is thought to involve the segment polarity gene cubitus interruptus (ci). ci encodes a zinc finger protein of the Gli family of sequence-specific DNA binding proteins. ci mRNA is expressed in all non-Hh expressing cells. Here we demonstrate ci activity is both necessary and sufficient to drive expression of Hh-responsive genes in the Drosophila embryos. We show that Ci is a sequence-specific DNA binding protein that drives transcription from the wg promoter in transiently transfected cells. We demonstrate that Ci binding sites in the wg promoter are necessary for this transcriptional activation. These data taken together provide strong evidence that Ci is a transcriptional effector of Hh signaling.

The fruit fly Drosophila melanogaster has brought significant advances to research in neurodegenerative disease, notably in the identification of genes that are required to maintain the structural integrity of the brain, defined by recessive mutations that cause adult-onset neurodegeneration. Here, we survey these genes in the fly and classify them according to five key cell biological processes. Over half of these genes have counterparts in mouse or human that are also associated with neurodegeneration. Fly genetics continues to be instrumental in the analysis of degenerative disease, with notable recent advances in our understanding of several inherited disorders, as well as Parkinson’s Disease and the central role of mitochondria in neuronal maintenance.

In mammals, X-chromosome inactivation (XCI) equalizes X-linked gene expression between XY males and XX females and is controlled by a specialized region known as the X-inactivation center (Xic). The Xic harbors two chromatin interaction domains, one centered around the noncoding Xist gene and the other around the antisense Tsix counterpart. Previous work demonstrated the existence of a chromatin transitional zone between the two domains. Here, we investigate the region and discover a conserved element, RS14, that presents a strong binding site for Ctcf protein. RS14 possesses an insulatory function suggestive of a boundary element and is crucial for cell differentiation and growth. Knocking out RS14 results in compromised Xist induction and aberrant XCI in female cells. These data demonstrate that a junction element between Tsix and Xist contributes to the initiation of XCI.

The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform \"identification of direct RNA interacting proteins\" (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors-including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, and modifiers-that synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. Although Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist, the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes.

Members of the Wnt gene family are present in most, if not all, metazoan animals and are required for many aspects of development. Wnt genes encode potent secreted signals which must be expressed at the correct time and place for their proper function. I address how a Wnt gene of Drosophila melanogaster, wingless (wg), is expressed precisely in one narrow stripe of cells in each segment primordium. I am principally concerned with the second of three phases of regulation of wg during embryonic development: after gastrulation, when wg depends on the Hedgehog (Hh) signal. patched (ptc) encodes a putative receptor for Hh but has the opposite effect on wg: while in a $hh\\sp-$ embryo wg expression fades, in a $ptc\\sp-$ mutant the stripes expand to about three times their original width. Two approaches were taken to study wg expression. First, wg was cloned from a related Drosophila species, D. virilis, to compare cis-acting regulatory DNA. wg is expressed in an identical pattern in both species. Upstream DNA shares substantial homology, a series of elements which on average are 87% identical. The second approach complemented this sequence analysis: functions of putative wg regulatory elements were assayed with reporter construct transgenes. Approximately 5 kb of upstream regulatory DNA is sufficient to drive the reporter gene lacZ in a pattern which duplicates the pattern of endogenous wg, depends on ptc, and responds to hh activity. Deletion of one of the conserved elements, box G, results in expansion of reporter stripes. Box G can be added back to a large deletion to restore wild type-width stripes, which in turn depend on ptc activity. Since deletion of box G causes reporter stripe expansion, the key transacting regulator which binds this element is likely to be a transcriptional repressor. Two other requirements for wg expression at this time, besides the Hh signal, are hh-independent wg autoregulation and Sloppy paired, a putative transcription factor. To determine if Slp responds to Wg, we overexpressed slp in a $wg\\sp-$ background; the results suggested that slp functions in parallel to wg (and hh as well).