Highly conserved shifts in ubiquitin-proteasome system (UPS) activity drive mitochondrial remodeling during quiescence.

Defects in cellular proteostasis and mitochondrial function drive many aspects of infertility, cancer, and other age-related diseases. All of these conditions rely on quiescent cells, such as oocytes and adult stem cells, that reduce their activity and remain dormant as part of their roles in tissue homeostasis, reproduction, and even cancer recurrence. Using a multi-organism approach, we show that dynamic shifts in the ubiquitin proteasome system drive mitochondrial remodeling during cellular quiescence. In contrast to the commonly held view that the ubiquitin-proteasome system (UPS) is primarily regulated by substrate ubiquitination, we find that increasing proteasome number and their recruitment to mitochondria support mitochondrial respiratory quiescence (MRQ). GSK3 triggers proteasome recruitment to the mitochondria by phosphorylating outer membrane proteins, such as VDAC, and suppressing mitochondrial fatty acid oxidation. This work defines a process that couples dynamic regulation of UPS activity to coordinated shifts in mitochondrial metabolism in fungi, Drosophila, and mammals during quiescence.

Heritable shifts in redox metabolites during mitochondrial quiescence reprogramme progeny metabolism.

Changes in maternal diet and metabolic defects in mothers can profoundly affect health and disease in their progeny. However, the biochemical mechanisms that induce the initial reprogramming events at the cellular level have remained largely unknown owing to limitations in obtaining pure populations of quiescent oocytes. Here, we show that the precocious onset of mitochondrial respiratory quiescence causes a reprogramming of progeny metabolic state. The premature onset of mitochondrial respiratory quiescence drives the lowering of Drosophila oocyte NAD+ levels. NAD+ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine in embryos and lower levels of histone H3 lysine 27 trimethylation, resulting in enhanced intestinal lipid metabolism in progeny. In addition, we show that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells.

aFARP-ChIP-seq, a convenient and reliable method for genome profiling in as few as 100 cells with a capability for multiplexing ChIP-seq.

Much effort has been devoted to understand how chromatin modification regulates development and disease. Despite recent progress, however, it remains difficult to obtain high-quality epigenomic maps using chromatin-immunoprecipitation-coupled deep sequencing (ChIP-seq) in samples with low-cell numbers. Here, we present an Atlantis dsDNase-based technology, aFARP-ChIP-seq, that provides accurate profiling of genome-wide histone modifications in as few as 100 cells. By mapping histone lysine trimethylation (H3K4me3) and acetylation (H3K27Ac) in group I innate lymphoid cells (ILC1) sorted from different tissues in parallel, aFARP-ChIP-seq uncovers putative active promoter and enhancer landscapes of several tissue-specific Natural Killer cells (NK) and ILC1. aFARP-ChIP-seq is also highly effective in mapping transcription factor binding sites in small number of cells. Thus, aFARP-ChIP-seq offers multiplexing mapping of both epigenome and transcription factor binding sites using a small number of cells.

Cell-type-specific role of lamin-B1 in thymus development and its inflammation-driven reduction in thymus aging.

Cellular architectural proteins often participate in organ development and maintenance. Although functional decay of some of these proteins during aging is known, the cell-type-specific developmental role and the cause and consequence of their subsequent decay remain to be established especially in mammals. By studying lamins, the nuclear structural proteins, we demonstrate that lamin-B1 functions specifically in the thymic epithelial cells (TECs) for proper thymus organogenesis. An up-regulation of proinflammatory cytokines in the intra-thymic myeloid immune cells during aging accompanies a gradual reduction of lamin-B1 in adult TECs. We show that these cytokines can cause senescence and lamin-B1 reduction of the young adult TECs. Lamin-B1 supports the expression of TEC genes that can help maintain the adult TEC subtypes we identified by single-cell RNA-sequencing, thymic architecture, and function. Thus, structural proteins involved in organ building and maintenance can undergo inflammation-driven decay which can in turn contribute to age-associated organ degeneration.

Muscle stem cell renewal suppressed by Gas1 can be reversed by GDNF in mice.

Muscle undergoes progressive weakening and regenerative dysfunction with age due in part to the functional decline of skeletal muscle stem cells (MuSCs). MuSCs are heterogeneous but whether their gene expression changes with age and the implication of such changes are unclear. Here we show that in mice, Growth arrest-specific gene 1 (Gas1) is expressed in a small subset of young MuSCs with its expression progressively increasing in larger fractions of MuSCs later in life. Over-expression of Gas1 in young MuSCs and inactivation of Gas1 in aged MuSCs support that Gas1 reduces the quiescence and self-renewal capacity of MuSCs. Gas1 reduces Ret signaling, which is required for MuSC quiescence and self-renewal. Indeed, we show that the Ret ligand, Glial Cell-Derived Neurotrophic Factor (GDNF), can counteract Gas1 by stimulating Ret signaling and enhancing MuSC self-renewal and regeneration, thus improving muscle function. We propose that strategies aimed to target this pathway can be exploited to improve the regenerative decline of muscle stem cells.

Lamins Organize the Global Three-Dimensional Genome from the Nuclear Periphery.

Lamins are structural components of the nuclear lamina (NL) that regulate genome organization and gene expression, but the mechanism remains unclear. Using Hi-C, we show that lamins maintain proper interactions among the topologically associated chromatin domains (TADs) but not their overall architecture. Combining Hi-C with fluorescence in situ hybridization (FISH) and analyses of lamina-associated domains (LADs), we reveal that lamin loss causes expansion or detachment of specific LADs in mouse ESCs. The detached LADs disrupt 3D interactions of both LADs and interior chromatin. 4C and epigenome analyses further demonstrate that lamins maintain the active and repressive chromatin domains among different TADs. By combining these studies with transcriptome analyses, we found a significant correlation between transcription changes and the interaction changes of active and inactive chromatin domains These findings provide a foundation to further study how the nuclear periphery impacts genome organization and transcription in development and NL-associated diseases.

Lamin B1 is required for mature neuron-specific gene expression during olfactory sensory neuron differentiation.

B-type lamins are major constituents of the nuclear lamina in all metazoan cells, yet have specific roles in the development of certain cell types. Although they are speculated to regulate gene expression in developmental contexts, a direct link between B-type lamins and developmental gene expression in an in vivo system is currently lacking. Here, we identify lamin B1 as a key regulator of gene expression required for the formation of functional olfactory sensory neurons. By using targeted knockout in olfactory epithelial stem cells in adult mice, we show that lamin B1 deficient neurons exhibit attenuated response to odour stimulation. This deficit can be explained by decreased expression of genes involved in mature neuron function, along with increased expression of genes atypical of the olfactory lineage. These results support that the broadly expressed lamin B1 regulates expression of a subset of genes involved in the differentiation of a specific cell type.

Low-Cell-Number Epigenome Profiling Aids the Study of Lens Aging and Hematopoiesis.

Understanding how chromatin modification regulates development and disease can be limited by available material. Despite recent progress, balancing high-quality and reliable mapping using chromatin-immunoprecipitation-based deep sequencing (ChIP-seq) remains a challenge. We report two techniques, recovery via protection (RP)-ChIP-seq and favored amplification RP-ChIP-seq (FARP-ChIP-seq), that provide reproducible mapping in as few as 500 cells. RP-ChIP-seq allows detection of age-associated epigenetic changes in a single mouse lens, whereas FARP-ChIP-seq accurately maps histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), and multi-potent progenitors (MPPs) from one mouse. These datasets not only highlight genes that may be involved in lens aging but also indicate a lack of H3K4me3/H3K27me3 bivalency on hematopoietic genes in HSCs.

Neuroprotection by astrocytes in brain ischemia: importance of microRNAs.

Astrocytes have been shown to protect neurons and increase their survival in many pathological settings. Manipulating astrocyte functions is thus an important strategy to enhance neuronal survival and improve outcome following cerebral ischemia. Increasing evidence supports the involvement of microRNAs (miRNA), some of them being astrocyte-enriched, in the regulation of cerebral ischemia. This mini review will focus on several recently reported astrocyte-enriched miRNAs (miR-181 and miR-29 families and miR-146a), their validated targets, regional expression and effects on outcome after cerebral ischemia.

Probenecid protects against transient focal cerebral ischemic injury by inhibiting HMGB1 release and attenuating AQP4 expression in mice.

Stroke results in inflammation, brain edema, and neuronal death. However, effective neuroprotectants are not available. Recent studies have shown that high mobility group box-1 (HMGB1), a proinflammatory cytokine, contributes to ischemic brain injury. Aquaporin 4 (AQP4), a water channel protein, is considered to play a pivotal role in ischemia-induced brain edema. More recently, studies have shown that pannexin 1 channels are involved in cerebral ischemic injury and the cellular inflammatory response. Here, we examined whether the pannexin 1 channel inhibitor probenecid could reduce focal ischemic brain injury by inhibiting cerebral inflammation and edema. Transient focal ischemia was induced in C57BL/6J mice by middle cerebral artery occlusion (MCAO) for 1 h. Infarct volume, neurological score and cerebral water content were evaluated 48 h after MCAO. Immunostaining, western blot analysis and ELISA were used to assess the effects of probenecid on the cellular inflammatory response, HMGB1 release and AQP4 expression. Administration of probenecid reduced infarct size, decreased cerebral water content, inhibited neuronal death, and reduced inflammation in the brain 48 h after stroke. In addition, HMGB1 release from neurons was significantly diminished and serum HMGB1 levels were substantially reduced following probenecid treatment. Moreover, AQP4 protein expression was downregulated in the cortical penumbra following post-stroke treatment with probenecid. These results suggest that probenecid, a powerful pannexin 1 channel inhibitor, protects against ischemic brain injury by inhibiting cerebral inflammation and edema.

Identification of loop nucleotide polymorphisms affecting microRNA processing and function.

MicroRNAs are short 21-22 nucleotide single strand RNAs that are involved in post-transcriptional regulation of gene expression. Most microRNAs are first transcribed as long primary microRNAs and then undergo a two step-wise sequential processing to yield single-stranded mature microRNAs. It has been suggested that the loop region of primary microRNAs plays an important role in regulating microRNA biogenesis and target recognition. However, despite the fact that several single nucleotide polymorphisms have been identified in mature microRNA sequences and are related to human diseases, it remains unclear whether and how the single nucleotide polymorphisms in the loop regions of primary microRNAs would affect the biogenesis and function of microRNAs. Herein, we provide evidence that primary microRNAs loop nucleotides control the accuracy and efficiency of microRNA processing. Accordingly, we identified 32 single nucleotide polymorphisms in the loop regions of human primary microRNAs using bioinformatics, and further validated three loss-of-function and one gain-of-function single nucleotide polymorphisms using dual-luciferase assays. Thus, these results reveal a critical regulatory role encoded in the loop nucleotides of primary microRNAs for microRNA processing and function.

Astrocyte-enriched miR-29a targets PUMA and reduces neuronal vulnerability to forebrain ischemia.

Following transient forebrain ischemia, astrocytes play a key role in determining whether or not neurons in the hippocampal CA1 sector go on to die in a delayed fashion. MicroRNAs (miRNAs) are a novel class of RNAs that control gene expression at the post-transcriptional level and the miR-29 family is highly expressed in astrocytes. In this study we assessed levels of miR-29 in hippocampus following forebrain ischemia and found that after transient forebrain ischemia and short periods of reperfusion, miR-29a significantly increased in the resistant dentate gyrus, but decreased in the vulnerable CA1 region of the hippocampus. We demonstrate that miR-29a targets BH3-only proapoptotic BCL2 family member PUMA by luciferase reporter assay and by Western blot. Comparing primary neuron and astrocyte cultures, and postnatal brain, we verified the strongly astrocytic expression of miR-29a. We further found that miR-29a mimic protects and miR-29a inhibitor aggravates cell injury and mitochondrial function after ischemia-like stresses in vitro. Lastly, by overexpressing and reducing miR-29a we demonstrate the protective effect of miR-29a on CA1 delayed neuronal death after forebrain ischemia. Our data suggest that by targeting a pro-apoptotic BCL2 family member, increasing levels of miR-29a might emerge as a strategy for protection against ischemia-reperfusion injury.

Modulating the strength and threshold of NOTCH oncogenic signals by mir-181a-1/b-1.

Oncogenes, which are essential for tumor initiation, development, and maintenance, are valuable targets for cancer therapy. However, it remains a challenge to effectively inhibit oncogene activity by targeting their downstream pathways without causing significant toxicity to normal tissues. Here we show that deletion of mir-181a-1/b-1 expression inhibits the development of Notch1 oncogene-induced T cell acute lymphoblastic leukemia (T-ALL). mir-181a-1/b-1 controls the strength and threshold of Notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of NOTCH and pre-T cell receptor (TCR) signaling pathways. Importantly, although Notch oncogenes utilize normal thymic progenitor cell genetic programs for tumor transformation, comparative analyses of mir-181a-1/b-1 function in normal thymocyte and tumor development demonstrate that mir-181a-1/b-1 can be specifically targeted to inhibit tumor development with little toxicity to normal development. Finally, we demonstrate that mir-181a-1/b-1, but not mir-181a-2b-2 and mir-181-c/d, controls the development of normal thymic T cells and leukemia cells. Together, these results illustrate that NOTCH oncogene activity in tumor development can be selectively inhibited by targeting the molecular networks controlled by mir-181a-1/b-1.

miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo.

MicroRNAs (miRNA) are short (~22nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke.

miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes.

Mitochondria are central to the execution of apoptosis, and the Bcl-2 protein family of pro- and anti-apoptotic proteins interacts with mitochondria to regulate apoptosis. Using bioinformatics we predicted that miR-181, a microRNA expressed in brain, could target the 3'UTRs of Bcl-2 family members Bcl-2-L11/Bim, Mcl-1, and Bcl-2. Using the luciferase reporter assay we confirmed these targets. We used mimic and inhibitor to alter miR-181a levels in primary astrocyte cultures and found miR-181a reduction was associated with increased Bcl-2 and Mcl-1 protein levels. Decreased miR-181a levels reduced glucose deprivation induced apoptosis, mitochondrial dysfunction, and loss of mitochondrial membrane potential in astrocytes.

Loop nucleotides control primary and mature miRNA function in target recognition and repression.

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5' end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation.

MicroRNA programs in normal and aberrant stem and progenitor cells.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

The potential functions of primary microRNAs in target recognition and repression.

Major RNA products of a microRNA (miRNA) gene--the long primary transcript (pri-miRNA), the ∼70-nucleotide (nt) precursor miRNA (pre-miRNA), and the ∼21-nt mature miRNA--all contain the same sequence required for target gene recognition. Thus, it is intrinsically difficult to discern the contribution of individual RNA species or to rule out a function of miRNA precursor species in target repression. Here, we describe a novel approach to dissect the functional contribution of pri-miRNA without compromising important cellular pathways. We show that pri-let-7 has a direct function in target repression in the absence of properly processed mature let-7. Moreover, we show that loop nucleotides provide regulatory controls of the activity of pri-let-7 by modulating interactions between pri-let-7 and target RNAs in vitro and in vivo. Finally, we show that human let-7a-3 pri-miRNA can directly interact with target mRNAs. These findings illustrate that the regulatory information encoded in structured pri-miRNAs may be translated into function through direct interactions with target mRNAs.

Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development.

BACKGROUND: Mature miRNAs can often be classified into large families, consisting of members with identical seeds (nucleotides 2 through 7 of the mature miRNAs) and highly homologous approximately 21-nucleotide (nt) mature miRNA sequences. However, it is unclear whether members of a miRNA gene family, which encode identical or nearly identical mature miRNAs, are functionally interchangeable in vivo. METHODS AND FINDINGS: We show that mir-181a-1, but not mir-181c, can promote CD4 and CD8 double-positive (DP) T cell development when ectopically expressed in thymic progenitor cells. The distinct activities of mir-181a-1 and mir-181c are largely determined by their unique pre-miRNA loop nucleotides-not by the one-nucleotide difference in their mature miRNA sequences. Moreover, the activity of mir-181a-1 on DP cell development can be quantitatively influenced by nucleotide changes in its pre-miRNA loop region. We find that both the strength and the functional specificity of miRNA genes can be controlled by the pre-miRNA loop nucleotides. Intriguingly, we note that mutations in the pre-miRNA loop regions affect pre-miRNA and mature miRNA processing, but find no consistent correlation between the effects of pre-miRNA loop mutations on the levels of mature miRNAs and the activities of the mir-181a-1/c genes. CONCLUSIONS: These results demonstrate that pre-miRNA loop nucleotides play a critical role in controlling the activity of miRNA genes and that members of the same miRNA gene families could have evolved to achieve different activities via alterations in their pre-miRNA loop sequences, while maintaining identical or nearly identical mature miRNA sequences.