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The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2) 1 , 2 , which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies. The authors identify a molecular switch that regulates the balance between neurotoxic and neuroprotective astrocyte populations, with potential application in the treatment of glaucoma and other optic neuropathies.

A study of prostate cancer cells reveals a transcriptional activation role for long non-coding RNAs (PRNCR1 and PCGEM1) that bind to the androgen receptor, and is also observed for the truncated androgen receptor characteristic of many aggressive prostate cancers. Cancer growth influenced by long non-coding RNAs Several long non-coding RNAs (lncRNAs) are known to be overexpressed in prostate cancer. Michael Rosenfeld and colleagues have investigated the mechanistic and biological roles of two of these, known as PRNCR1 and PCGEM1. Both are found to interact with the androgen receptor (AR) dependent on specific post-translational modifications, and to enhance the looping of AR-bound enhancers to target gene promoters, leading to enhanced gene expression. They also enhance AR-mediated proliferation in prostate cancer cells and are required for tumour growth in a prostate cancer xenograft mouse model. PRNCR1 and PCGEM1 are upregulated in castration-resistant prostate cancer cell lines. The regulatory roles of lncRNAs in prostate cancer uncovered in this manuscript may open the way to new therapeutic approaches. Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis 1 , their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells 2 , and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy 3 . Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In ‘resistant’ prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo . Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours.

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles 1 , particularly the promoter antisense (PAS) transcripts 2 , 3 . Here we report the development of an assay—referred to as chromatin isolation by RNA–Cas13a complex—to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17β-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation. The authors describe a role for the non-coding antisense transcripts produced at promoters in regulating ligand-induced activation of gene transcription.

Loss of retinal ganglion cells (RGCs) in optic neuropathies results in permanent partial or complete blindness. Myocyte enhancer factor 2 (MEF2) transcription factors have been shown to play a pivotal role in neuronal systems, and in particular MEF2A knockout was shown to enhance RGC survival after optic nerve crush injury. Here we expanded these prior data to study bi-allelic, tri-allelic and heterozygous allele deletion. We observed that deletion of all MEF2A, MEF2C, and MEF2D alleles had no effect on RGC survival during development. Our extended experiments suggest that the majority of the neuroprotective effect was conferred by complete deletion of MEF2A but that MEF2D knockout, although not sufficient to increase RGC survival on its own, increased the positive effect of MEF2A knockout. Conversely, MEF2A over-expression in wildtype mice worsened RGC survival after optic nerve crush. Interestingly, MEF2 transcription factors are regulated by post-translational modification, including by calcineurin-catalyzed dephosphorylation of MEF2A Ser-408 known to increase MEF2A-dependent transactivation in neurons. However, neither phospho-mimetic nor phospho-ablative mutation of MEF2A Ser-408 affected the ability of MEF2A to promote RGC death in vivo after optic nerve injury. Together these findings demonstrate that MEF2 gene expression opposes RGC survival following axon injury in a complex hierarchy, and further support the hypothesis that loss of or interference with MEF2A expression might be beneficial for RGC neuroprotection in diseases such as glaucoma and other optic neuropathies.

Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as \"SD70,\" initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing—like approach, referred to as \"Chem-seq,\" we were next able to efficiently map the genomewide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genomewide location of small molecules, a powerful adjunt to contemporary drug development strategies.

Mammalian central nervous system (CNS) axons cannot spontaneously regenerate after injury, creating an unmet need to identify molecular regulators to promote axon regeneration and reduce the lasting impact of CNS injuries. While tubulin polymerization promoting protein family member 3 (Tppp3) is known to promote axon outgrowth in amphibians, its role in mammalian axon regeneration remains unknown. Here we investigated Tppp3 in retinal ganglion cells (RGCs) neuroprotection and axonal regeneration using an optic nerve crush (ONC) model in the rodent. Single-cell RNA sequencing identified the expression of Tppp3 in RGCs of mice, macaques, and humans. Tppp3 overexpression enhanced neurite outgrowth in mouse primary RGCs in vitro, promoted axon regeneration, and improved RGC survival after ONC. Bulk RNA sequencing indicated that Tppp3 overexpression upregulates axon regeneration genes such as Bmp4 and neuroinflammatory pathways. Our findings advance regenerative medicine by developing a new therapeutic strategy for RGC neuroprotection and axon regeneration.

Substantial evidence supports the hypothesis that enhancers are critical regulators of cell-type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of LIM domain-binding protein 1 (LDB1)/cofactor of LIM homeodomain protein 2/nuclear LIM interactor, interacting with the enhancer-binding protein achaete-scute complex homolog 1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target genes. Although LDB1-dependent activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of metastasis-associated 1 family, member 2, a component of the nucleosome remodeling deacetylase complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type. Significance The apparent importance of promoter:enhancer looping is well established; however, the molecular mechanisms of these interactions in gene activation vs. gene repression remain to be fully elucidated. Here, we report that LIM domain-binding protein 1 (LDB1) can function in transcriptional enhancer-mediated gene activation mainly at the level of transcription initiation by regulating promoter:enhancer looping, consequent to the recruitment to basic helix-loop-helix–bound enhancers in pituitary corticotrope cells. Intriguingly, LDB1 also mediates promoter:enhancer looping required for target gene repression, acting at the level of promoter pausing, by recruiting metastasis-associated 1 family, member 2 to these repressive enhancers. These findings shed light on a regulatory aspect of the molecular function of LDB1, providing a putative mechanism of enhancer-dependent transcriptional repression.

The evaluation of students' motor skills is a fundamental aspect of the physical education process, significantly contributing to the development of motor and psychomotor qualities. This study examines the impact of an experimental program utilizing badminton-specific means on the motor skills of middle school students. Hypothesis: It is hypothesized that the use of badminton-specific methods will positively influence students' motor performance. Objectives of the study were: to assess the initial level of students' motor skills, implement an experimental program based on badminton for the experimental group, and compare the motor results of the experimental group with those of the control group. Students in the experimental group demonstrated significant improvements in strength, speed, coordination, and endurance, as evidenced by enhanced performance in all four motor tests evaluated. Theimplementation of badminton-specific means proved effective not only in developing motor skills but also in increasing students' motivation towards physical activities.

It is unclear whether bidirectional non-coding RNAs transcribed from enhancer elements (eRNAs) have any functional role; here, eRNA transcription is shown to be functionally important during the activation of genes by the oestrogen receptor in human breast cancer cells. Regulatory role for eRNAs Bidirectional non-coding RNAs are transcribed from enhancer elements, but it is unclear whether these enhancer-derived RNAs (eRNAs) have a functional role or are merely a reflection of enhancer activity. Two manuscripts in this issue of Nature examine this question in the context of the positive and negative transcriptional functions of different nuclear receptors. Wenbo Li et al . provide evidence for the functional importance of eRNA transcription during the activation of genes by the oestrogen receptor in breast cancer cell lines; and Michael Lam et al . show that the repressive functions of Rev-Erb nuclear receptors in macrophages are linked to their ability to inhibit the transcription of eRNAs. Taken together these studies provide evidence for a role for eRNAs in contributing to enhancer functions. The functional importance of gene enhancers in regulated gene expression is well established 1 , 2 , 3 . In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells 4 , 5 , 6 , bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs) 7 , 8 , 9 . However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer–promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer–promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

Heart disease link to inflammatory signalling A non-coding region on chromosome 9p21 was previously shown to associate with coronary artery disease and type 2 diabetes, and the region has been implicated in regulating neighbouring genes. Here the authors identify 33 distinct enhancers within this region and show that single nucleotide polymorphisms in one of the enhancers affect STAT1 binding. They further show that in human vascular endothelium cells, the enhancer interval physically interacts with a number of specific loci, and that interferon-γ activation strongly affects the chromatin structure and transcriptional regulation of the 9p21 locus, including STAT1 binding, long-range enhancer interactions and expression of neighbouring genes. A non-coding region on chromosome 9p21 was previously shown to associate with coronary artery disease and type 2 diabetes, and the region has been implicated in regulating neighbouring genes. Here, 33 distinct enhancers within this region are identified, showing that SNPs in one of the enhancers affect STAT1 binding. Furthermore, it is shown that in human vascular endothelial cells the enhancer interval physically interacts with a number of specific loci and that IFN-γ activation strongly affects the chromatin structure and transcriptional regulation of the 9p21 locus, including STAT1 binding, long-range enhancer interactions and expression of neighbouring genes. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) 1 , 2 , 3 , 4 and type 2 diabetes 5 , 6 , 7 . Despite evidence for a role of the associated interval in neighbouring gene regulation 8 , 9 , 10 , the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome ( P  < 6.55 × 10 −33 ). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1 ) expression, which is reversed by short interfering RNA knockdown of STAT1 . Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21 . In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.