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Coronavirus disease 2019 (COVID-19) patients have manifested a variety of neurological complications, and there is still much to reveal regarding the neurotropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human stem cell-derived brain organoids offer a valuable in vitro approach to study the cellular effects of SARS-CoV-2 on the brain. Here we used human embryonic stem cell-derived cortical organoids to investigate whether SARS-CoV-2 could infect brain tissue in vitro and found that cortical organoids could be infected at low viral titers and within 6 h. Importantly, we show that glial cells and cells of the choroid plexus were preferentially targeted in our model, but not neurons. Interestingly, we also found expression of angiotensin-converting enzyme 2 in SARS-CoV-2 infected cells; however, viral replication and cell death involving DNA fragmentation does not occur. We believe that our model is a tractable platform to study the cellular effects of SARS-CoV-2 infection in brain tissue., Graphical abstract, In this article, Hsieh and colleagues investigated the neurotropism of the virus responsible for COVID-19, SARS-CoV-2. Using human ESC-derived brain organoids, this study found that SARS-CoV-2 can infect these organoids at low viral loads, but does not replicate within 4 days post infection. Specifically, SARS-CoV-2 preferentially targeted glial cells and cells of the choroid plexus in this model.

In the mammalian hippocampus, adult-born granule cells (abGCs) contribute to the function of the dentate gyrus (DG). Disruption of the DG circuitry causes spontaneous recurrent seizures (SRS), which can lead to epilepsy. Although abGCs contribute to local inhibitory feedback circuitry, whether they are involved in epileptogenesis remains elusive. Here, we identify a critical window of activity associated with the aberrant maturation of abGCs characterized by abnormal dendrite morphology, ectopic migration, and SRS. Importantly, in a mouse model of temporal lobe epilepsy, silencing aberrant abGCs during this critical period reduces abnormal dendrite morphology, cell migration, and SRS. Using mono-synaptic tracers, we show silencing aberrant abGCs decreases recurrent CA3 back-projections and restores proper cortical connections to the hippocampus. Furthermore, we show that GABA-mediated amplification of intracellular calcium regulates the early critical period of activity. Our results demonstrate that aberrant neurogenesis rewires hippocampal circuitry aggravating epilepsy in mice., Adult-born granule cells integrate in hippocampal circuitry and contribute to hippocampal function. Here, the authors show that a critical period of neuronal activity regulates aberrant neurogenesis to rewire hippocampal circuitry and drive seizures in a mouse model of epilepsy.

Pregnant women are at greater risk of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), because of their altered immunity and strained cardiovascular system. Emerging studies of placenta, embryos, and cerebral organoids suggest that fetal organs including brain could also be vulnerable to coronavirus disease 2019 (COVID-19). Additionally, a case study from Paris has reported transient neurological complications in neonates born to pregnant mothers. However, it remains poorly understood whether the fetal brain expresses cellular components that interact with Spike protein (S) of coronaviruses, which facilitates fusion of virus and host cell membrane and is the primary protein in viral entry. To address this question, we analyzed the expression of known (ACE2, TMPRSS2, and FURIN) and novel (ZDHHC5, GOLGA7, and ATP1A1) S protein interactors in publicly available fetal brain bulk and single cell RNA sequencing datasets. Bulk RNA sequencing analysis across multiple regions of fetal brain spanning 8 weeks post conception (wpc)−37wpc indicates that two of the known S protein interactors are expressed at low levels with median normalized gene expression values ranging from 0.08 to 0.06 (ACE2) and 0.01–0.02 (TMPRSS2). However, the third known S protein interactor FURIN is highly expressed (11.1–44.09) in fetal brain. Interestingly, all three novel S protein interactors are abundantly expressed throughout fetal brain development with median normalized gene expression values ranging from 20.38–21.60 (ZDHHC5), 92.47–68.35 (GOLGA7), and 65.45–194.5 (ATP1A1). Moreover, the peaks of expression of novel interactors is around 12–26wpc. Using publicly available single cell RNA sequencing datasets, we further show that novel S protein interactors show higher co-expression with neurons than with neural progenitors and astrocytes. These results suggest that even though two of the known S protein interactors are present at low levels in fetal brain, novel S protein interactors are abundantly present and could play a direct or indirect role in SARS-CoV-2 fetal brain pathogenesis, especially during the 2nd and 3rd trimesters of pregnancy.

SummaryMounting evidence suggests that cognitive deficits associated with various neurological disorders may arise in part from a small population of dysregulated adult-born neurons in the dentate gyrus (DG). How these dysregulated adult-born neurons contribute to brain-wide network maladaptation and subsequent cognitive deficits remains unknown. Using an established mouse model with a small number of time-stamped dysregulated adult-born immature neurons and spatial memory deficits, we performed resting state functional magnetic resonance imaging and found that approximately 500 deficient immature neurons (in vivo fiber photometry recording, we demonstrated that dysregulated adult-born neurons induce aberrant activity and synchrony in local hippocampal CA3 and CA1 regions, as well as distal medial-dorsal thalamus and IC regions during a spatial memory process. These results suggest that a few hundred dysregulated adult-born immature neurons can impact brain-wide network dynamics across several anatomically discrete regions and collectively contribute to impaired cognitive functions.

Summary: In the adult brain, new dentate granule cells integrate into neural circuits and participate in hippocampal functioning. However, when and how they initiate this integration remain poorly understood. Using retroviral and live-imaging methods, we find that new neurons undergo neurite remodeling for competitive horizontal-to-radial repositioning in the dentate gyrus prior to circuit integration. Gene expression profiling, lipidomics analysis, and molecular interrogation of new neurons during this period reveal a rapid activation of sphingolipid signaling mediated by sphingosine-1-phosphate receptor 1. Genetic manipulation of this G protein-coupled receptor reveals its requirement for successful repositioning of new neurons. This receptor is also activated by hippocampus-engaged behaviors, which enhances repositioning efficiency. These findings reveal that activity-dependent sphingolipid signaling regulates cellular repositioning of new dentate granule cells. The competitive horizontal-to-radial repositioning of new neurons may provide a gating strategy in the adult brain to limit the integration of new neurons into pre-existing circuits. : Yang et al. show that prior to circuit integration, adult-born dentate granule cells undergo horizontal-to-radial transitioning, which is regulated by sphingolipid signaling via sphingosine-1-phosphate receptor 1. Keywords: G protein-coupled receptor, electrophysiological maturation, dentate granule cell, environmental enrichment, lipidomics, adult hippocampal neurogenesis, circuit integration initiation, sphingosine-1-phosphate receptor 1, hippocampus behavior-engaged activation

Epilepsy is a disorder of the central nervous system characterized by spontaneous recurrent seizures. Although current therapies exist to control the number and severity of clinical seizures, there are no pharmacological cures or disease-modifying treatments available. Use of transgenic mouse models has allowed an understanding of neural stem cells in their relation to epileptogenesis in mesial temporal lobe epilepsy. Further, with the significant discovery of factors necessary to reprogram adult somatic cell types into pluripotent stem cells, it has become possible to study monogenic epilepsy-in-a-dish using patient-derived neurons. This discovery along with some of the newest technological advances in recapitulating brain development in a dish has brought us closer than ever to a platform in which to study and understand the mechanisms of this disease. These technologies will be critical in understanding the mechanism of epileptogenesis and ultimately lead to improved therapies and precision medicine for patients with epilepsy.

Summary Currently, all methods for converting non-neuronal cells into neurons involve injury to the brain; however, whether neuronal transdifferentiation can occur long after the period of insult remains largely unknown. Here, we use the transcription factor NEUROD1, previously shown to convert reactive glial cells to neurons in the cortex, to determine whether astrocyte-to-neuron transdifferentiation can occur under physiological conditions. We utilized adeno-associated virus 9 (AAV9), which crosses the blood-brain barrier without injury, to deliver NEUROD1 to astrocytes through an intravascular route. Interestingly, we found that a small, but significant number of non-reactive astrocytes converted to neurons in the striatum, but not the cortex. Moreover, astrocytes cultured to minimize their proliferative potential also exhibited limited neuronal transdifferentiation with NEUROD1 expression. Our results show that a single transcription factor can induce astrocyte-to-neuron conversion under physiological conditions, potentially facilitating future clinical approaches long after the acute injury phase., Highlights • Intravascular AAV9 efficiently targets astrocytes in the cortex and striatum • NEUROD1 can convert a small, but significant number of astrocytes to neurons in vivo • NEUROD1 induces limited neuronal transdifferentiation in vitro • Injury-related factors may be important in neuronal conversion strategies, In this article, Hsieh and colleagues use AAV9 viral infection to selectively target and overexpress NEUROD1 in astrocytes in the absence of injury to the CNS. The authors show that NEUROD1 is capable of converting a small, but significant number of astrocytes to neurons in vivo in the absence of reactive gliosis.

Three-dimensional (3D) organoid models derived from human pluripotent stem cells provide a platform for studying human development and understanding disease mechanisms. Most studies that examine biallelic inactivation of the cell cycle regulator Retinoblastoma 1 (RB1) and the link to retinoblastoma is in mice, however, less is known regarding the pathophysiological role of RB1 during human retinal development. To study the role of RB1 in early human retinal development and tumor formation, we generated retinal organoids from CRISPR/Cas9-derived RB1-null human embryonic stem cells (hESCs). We showed that RB is abundantly expressed in retinal progenitor cells in retinal organoids and loss of RB1 promotes S-phase entry. Furthermore, loss of RB1 resulted in widespread apoptosis and reduced the number of photoreceptor, ganglion, and bipolar cells. Interestingly, RB1 mutation in retinal organoids did not result in retinoblastoma formation in vitro or in the vitreous body of NOD/SCID immunodeficient mice. Together, our work identifies a crucial function for RB1 in human retinal development and suggests that RB1 deletion alone is not sufficient for tumor development, at least in human retinal organoids.

Considerable evidence suggests loss of function mutations in the chromatin remodeler, CHD2, contribute to a broad spectrum of human neurodevelopmental disorders. However, it is unknown how CHD2 mutations lead to impaired brain function. Here we report mice with heterozygous mutations in Chd2 exhibit deficits in neuron proliferation and a shift in neuronal excitability that included divergent changes in excitatory and inhibitory synaptic function. Further in vivo experiments show Chd2(+/−) mice displayed aberrant cortical rhythmogenesis and severe deficits in long-term memory, consistent with phenotypes observed in humans. We identified broad, age-dependent transcriptional changes in Chd2(+/−) mice, including alterations in neurogenesis, synaptic transmission and disease-related genes. Deficits in interneuron density and memory caused by Chd2(+/−) were reproduced by Chd2 mutation restricted to a subset of inhibitory neurons and corrected by interneuron transplantation. Our results provide initial insight into how Chd2 haploinsufficiency leads to aberrant cortical network function and impaired memory.

In March 2016, meeting organizers Sebastian Jessberger and Hongjun Song brought together over 100 scientists from around the world to Cancun, Mexico to present the latest research on neurogenesis. The meeting covered diverse aspects of embryonic and adult neurogenesis with a focus on novel technologies, including chemogenetics and optogenetics, live cell two-photon imaging, cell fate reprogramming and human pluripotent stem cell models. This Meeting Review describes the exciting work that was presented and some of the emerging themes from the meeting.

Background Antimicrobial resistance (AMR) to carbapenems in Enterobacteriaceae such as Klebsiella pneumoniae (KPN) is a major global public health concern. Infections caused by these pathogens are associated with high morbidity and mortality and perpetuated by limited safe alternative treatment options. This study aims to describe the antimicrobial susceptibility patterns amongst KPN to the carbapenems Latin America. Methods Surveillance laboratory data from 2000 to 2014 were obtained through the ReLAVRA network from 19 countries in Latin America. Longitudinal trends of mean percentage non-susceptibility for the region were conducted and evaluated with a significance level of P < 0.05. Results A total of 209,972 and 181,128 KPN isolates were reported from 2000 to 2014 for antibiotic susceptibility to imipenem and meropenem, respectively. From 2000 to 2014 an increasing trend was observed in the reported % KPN NS to imipenem (P < 0.0001) from 0.6% to 11.6% with an average annual percentage increase (AAPI) of 36.3% [95% CI: 39.8%–33%]. (Figure 1). Similarly, the % KPN NS to meropenem increased (P < 0.0001) from 0% in 2000 to 12.3% in 2014 with an AAPI of 49.5% [95% CI: 54%–44.6%] (Figure 2). For both antibiotics, the last 5 years of the timeframe (2010 to 2014) showed the highest rate of increase in NS. NS to carbapenems varied significantly between reporting countries, with the highest % KPN NS to imipenem and meropenem reported by Brazil, Guatemala, Nicaragua, and Peru. Conclusion The increase in KPN NS to carbapenems observed in Latin America threatens effective treatment of infections caused by this pathogen. The extremely limited treatment options could lead to further increases in morbidity and mortality. Strengthening health systems and core country capacity to identify and deal with these emerging high-risk pathogens and resistance mechanisms, through surveillance is vital to inform public health actions, control measures, mitigate outbreaks and support further development of Public health actions against AMR at country and regional level. Disclosures All authors: No reported disclosures.

Adult neurogenesis, the generation of new neurons in the adult brain, occurs in the hippocampal dentate gyrus (DG) and the olfactory bulb (OB) of all mammals, but the functions of these new neurons are not entirely clear. Originally, adult-born neurons were considered to have excitatory effects on the DG network, but recent studies suggest a net inhibitory effect. Therefore, we hypothesized that selective removal of newborn neurons would lead to increased susceptibility to the effects of a convulsant. This hypothesis was tested by evaluating the response to the chemoconvulsant kainic acid (KA) in mice with reduced adult neurogenesis, produced either by focal X-irradiation of the DG, or by pharmacogenetic deletion of dividing radial glial precursors. In the first 4 hrs after KA administration, when mice have the most robust seizures, mice with reduced adult neurogenesis had more severe convulsive seizures, exhibited either as a decreased latency to the first convulsive seizure, greater number of convulsive seizures, or longer convulsive seizures. Nonconvulsive seizures did not appear to change or they decreased. Four-21 hrs after KA injection, mice with reduced adult neurogenesis showed more interictal spikes (IIS) and delayed seizures than controls. Effects were greater when the anticonvulsant ethosuximide was injected 30 min prior to KA administration; ethosuximide allows forebrain seizure activity to be more easily examined in mice by suppressing seizures dominated by the brainstem. These data support the hypothesis that reduction of adult-born neurons increases the susceptibility of the brain to effects of KA.

Transcription factors bind to specific DNA sequences and control the transcription rate of nearby genes in the genome. This activation or repression of gene expression is further potentiated by epigenetic modifications of histones with active and silent marks, respectively. Resident adult stem cells in the hematopoietic system, skin, and brain exist in a non-proliferative quiescent resting state. When quiescent stem cells become activated and transition to dividing progenitors and distinct cell types, they can replenish and repair tissue. Thus, determination of the position of transcription factor binding and histone epigenetic modification on the chromatin is an essential step toward understanding the gene regulation of quiescent and proliferative adult stem cells for potential applications in regenerative medicine. Genome-wide transcription factor occupancy and histone modifications on the genome can be obtained by assessing DNA-protein interaction through next-generation chromatin immunoprecipitation sequencing technology (ChIP-seq). This chapter outlines the protocol to perform, analyze, and validate ChIP-seq experiments that can be used to identify protein-DNA interactions and histone marks on the chromatin. The methods described here are applicable to quiescent and proliferative neural stem cells, and a wide range of other cellular systems.

Adult neurogenesis is significantly increased in the hippocampus of rodent models of temporal lobe epilepsy (TLE). These adult-generated neurons have recently been shown to play a contributing role in the development of spontaneous recurrent seizures (SRS). In order to eventually target pro-epileptic adult neurogenesis in the clinical setting, it will be important to identify molecular players involved in the control of aberrant neurogenesis after seizures. Here, we focused on NeuroD1 (ND1), a member of the bHLH family of transcription factors previously shown to play an essential role in the differentiation and maturation of adult-generated neurons in the hippocampus. Wild-type mice treated with pilocarpine to induce status epilepticus (SE) showed a significant up-regulation of NeuroD1+ immature neuroblasts located in both the granule cell layer (GCL), and ectopically localized to the hilar region of the hippocampus. As expected, conditional knockout (cKO) of NeuroD1 in Nestin-expressing stem/progenitors and their progeny led to a reduction in the number of NeuroD1+ adult-generated neurons after pilocarpine treatment compared to WT littermates. Surprisingly, there was no change in SRS in NeuroD1 cKO mice, suggesting that NeuroD1 cKO fails to reduce aberrant neurogenesis below the threshold needed to impact SRS. Consistent with this conclusion, the total number of adult-generated neurons in the pilocarpine model, especially the total number of Prox1+ hilar ectopic granule cells were unchanged after NeuroD1 cKO, suggesting strategies to reduce SRS will need to achieve a greater removal of aberrant adult-generated neurons.

The tumor suppressor retinoblastoma protein (RB) regulates S-phase cell cycle entry via E2F transcription factors. Knockout (KO) mice have shown that RB plays roles in cell migration, differentiation and apoptosis, in developing and adult brain. In addition, the RB family is required for self-renewal and survival of human embryonic stem cells (hESCs). Since little is known about the role of RB in human brain development, we investigated its function in cerebral organoids differentiated from gene-edited hESCs lacking RB. We show that RB is abundantly expressed in neural stem and progenitor cells in organoids at 15 and 28 days of culture. RB loss promoted S-phase entry in DCX+ cells and increased apoptosis in Sox2+ neural stem and progenitor cells, and in DCX+ and Tuj1+ neurons. Associated with these cell cycle and pro-apoptotic effects, we observed increased CCNA2 and BAX gene expression, respectively. Moreover, we observed aberrant Tuj1+ neuronal migration in RB-KO organoids and upregulation of the gene encoding VLDLR, a receptor important in reelin signaling. Corroborating the results in RB-KO organoids in vitro, we observed ectopically localized Tuj1+ cells in RB-KO teratomas grown in vivo Taken together, these results identify crucial functions for RB in the cerebral organoid model of human brain development.

The maintenance of the resident adult neural stem/progenitor cell (NSPC) pool depends on the precise balance of proliferation, differentiation, and maintenance of the undifferentiated state. Identifying the mechanisms that regulate this balance in adult hippocampal NSPCs can provide insight into basic stem cell self-renewal principles important for tissue homeostasis and preventing tumor formation. Pharmacological inhibition of histone deacetylases (HDACs), a class of histone-modifying enzymes, have promising effects in cancer cells, yet the specific roles of individual HDACs in stem cell proliferation is unclear. Here using conditional KO (cKO) mice and in vitro cell culture, we show that histone deacetylase 3 (HDAC3) is required for the proliferation of adult NSPCs. Detailed cell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defect in cell cycle progression through the gap 2/mitosis (G2/M) but not the S phase. Moreover, HDAC3 controls G2/M phase progression mainly through posttranslational stabilization of the G2/M cyclin-dependent kinase 1 (CDK1). These results demonstrate that HDAC3 plays a critical role in NSPC proliferation and suggest that strategies aimed at pharmacological modulation of HDAC3 may be beneficial for tissue regeneration and controlling tumor cell growth.

BackgroundShigellosis is the second leading cause of diarrheal death globally. The global burden has been complicated by the emergence of Shigella strains resistant to first line antibiotic treatments such as ciprofloxacin. This study aims to describe the epidemiologic distribution of the most common Shigella species, and their antimicrobial susceptibility patterns to ciprofloxacin and nalidixic acid (NA) in Latin America.MethodsLaboratory data from 19 countries were obtained through the Latin American Network for Antimicrobial Resistance Surveillance (ReLAVRA) from 2000-2015. The Clinical Laboratory Standards Institute reduced susceptibility breakpoints for Enterobacteriaceae was used to interpret the disc diffusion tests for Shigella susceptibility to ciprofloxacin and NA. Negative binominal regression was used to analyze longitudinal trends of Shigella isolates antimicrobial susceptibility.Results79,548 Shigella isolates were tested and reported between 2000-2015. The most common isolated species were S. flexneri (49%), and S. sonnei (28%). There was a steady increase in the proportion of S. sonnei isolates within the region(pConclusionThere is an increasing trend in Shigella nonsusceptibility to ciprofloxacin and NA, including among the most common shigella species, in Latin America. This rise of nonsusceptibility among Shigella species to commonly used treatments such as ciprofloxacin is alarming and threatens the control and management of this currently treatable infection. Improved data quality, collection and reporting is needed in Latin America to respond effectively to the rising trends observed. This includes the need for quality isolate level epidemiological data; molecular data, and data on antibiotic consumption and use.

Seizures can give rise to enduring changes that reflect alterations in gene-expression patterns, intracellular and intercellular signaling, and ultimately network alterations that are a hallmark of epilepsy. A growing body of literature suggests that long-term changes in gene transcription associated with epilepsy are mediated via modulation of chromatin structure. One transcription factor in particular, repressor element 1-silencing transcription factor (REST), has received a lot of attention due to the possibility that it may control fundamental transcription patterns that drive circuit excitability, seizures, and epilepsy. REST represses a suite of genes in the nervous system by utilizing nuclear protein complexes that were originally identified as mediators of epigenetic inheritance. Epigenetics has traditionally referred to mechanisms that allow a heritable change in gene expression in the absence of DNA mutation. However a more contemporaneous definition acknowledges that many of the mechanisms used to perpetuate epigenetic traits in dividing cells are utilized by neurons to control activity-dependent gene expression. This review surveys what is currently understood about the role of epigenetic mechanisms in epilepsy. We discuss how REST controls gene expression to affect circuit excitability and neurogenesis in epilepsy. We also discuss how the repressor methyl-CpG-binding protein 2 (MeCP2) and activator cyclic AMP response element binding protein (CREB) regulate neuronal activity and are themselves controlled by activity. Finally we highlight possible future directions in the field of epigenetics and epilepsy.

Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state., The transcription factor REST plays a crucial role in maintaining the adult neural stem cell pool. To better understand how REST maintains quiescence in neural progenitors, the authors use ChIP-seq and RNA-seq and find that REST regulates represses ribosome biogenesis, cell cycle and neuronal genes.

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation.

Adult neurogenesis occurs throughout life in the mammalian hippocampus and is essential for memory and mood control. There is significant interest in identifying ways to promote neurogenesis and ensure maintenance of these hippocampal functions. Previous work with a synthetic small molecule, isoxazole 9 (Isx-9), highlighted its neuronal-differentiating properties in vitro. However, the ability of Isx-9 to drive neurogenesis in vivo or improve hippocampal function was unknown. Here we show that Isx-9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult-generated dentate gyrus neurons. Isx-9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx-9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization. Molecular exploration of Isx-9-induced regulation of neurogenesis (via FACS and microarray of SGZ stem and progenitor cells) suggested the involvement of the myocyte-enhancer family of proteins (Mef2). Indeed, transgenic-mediated inducible knockout of all brain-enriched Mef2 isoforms (Mef2a/c/d) specifically from neural stem cells and their progeny confirmed Mef2's requirement for Isx-9-induced increase in hippocampal neurogenesis. Thus, Isx-9 enhances hippocampal neurogenesis and memory in vivo, and its effects are reliant on Mef2, revealing a novel cell-intrinsic molecular pathway regulating adult neurogenesis.—Petrik, D., Jiang, Y., Birnbaum, S. G., Powell, C. M., Kim, M.-S., Hsieh, J., Eisch, A. J. Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule.

The adult brain contains a reservoir of neural stem cells (NSCs) that generates functional neurons in a process called adult neurogenesis. Integration of new neurons into mature neural circuits maintains brain tissue homeostasis essential for learning, olfaction and behavior. Even subtle disruptions in NSC self-renewal/differentiation can result in substantial changes in neuronal production rates, contributing to neuropsychiatric symptoms, cognitive dysfunction and epilepsy. Recent studies have revealed pivotal roles for epigenetic regulators of gene expression. Epigenetic and genetic regulation allows a rich array of possibilities to fine-tune neuronal gene expression and offers potential therapeutic opportunities to modulate brain function related to adult neurogenesis. Here we discuss the role of epigenetic mechanisms underlying NSC fate and translational strategies for the future.

Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila Norp A phospholipase C which mediates rhabdomeric phototransduction. The Plcb4^(−/−) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

In the central nervous system (CNS), neural stem cells (NSCs) differentiate into neurons, astrocytes, and oligodendrocytes--these cell lineages are considered unidirectional and irreversible under normal conditions. The introduction of a defined set of transcription factors has been shown to directly convert terminally differentiated cells into pluripotent stem cells, reinforcing the notion that preserving cellular identity is an active process. Indeed, recent studies highlight that tumor suppressor genes (TSGs) such as Ink4a/Arf and p53, control the barrier to efficient reprogramming, leaving open the question whether the same TSGs function to maintain the differentiated state. During malignancy or following brain injury, mature astrocytes have been reported to re-express neuronal genes and re-gain neurogenic potential to a certain degree, yet few studies have addressed the underlying mechanisms due to a limited number of cellular models or tools to probe this process. Here, we use a synthetic small-molecule (isoxazole) to demonstrate that highly malignant EGFRvIII-expressing Ink4a/Arf(-/-); Pten(-/-) astrocytes downregulated their astrocyte character, re-entered the cell cycle, and upregulated neuronal gene expression. As a collateral discovery, isoxazole small-molecules blocked tumor cell proliferation in vitro, a phenotype likely coupled to activation of neuronal gene expression. Similarly, histone deacetylase inhibitors induced neuronal gene expression and morphologic changes associated with the neuronal phenotype, suggesting the involvement of epigenetic-mediated gene activation. Our study assesses the contribution of specific genetic pathways underlying the de-differentiation potential of astrocytes and uncovers a novel pharmacological tool to explore astrocyte plasticity, which may bring insight to reprogramming and anti-tumor strategies.

MicroRNAs are a class of small RNA regulators that are involved in numerous cellular processes, including development, proliferation, differentiation, and plasticity. The emerging concept is that microRNAs play a central role in controlling the balance between stem cell self-renewal and fate determination by regulating the expression of stem cell regulators. This review will highlight recent advances in the regulation of neural stem cell self-renewal and neurogenesis by microRNAs. It will cover microRNA functions during the entire process of neurogenesis, from neural stem cell self-renewal and fate determination to neuronal maturation, synaptic formation, and plasticity. The interplay between microRNAs and both cell-intrinsic and -extrinsic stem cell players, including transcription factors, epigenetic regulators, and extrinsic signaling molecules will be discussed. This is a summary of the topics covered in the mini-symposium on microRNA regulation of neural stem cells and neurogenesis in SFN 2010 and is not meant to be a comprehensive review of the subject.

In mature, differentiated neurons in the central nervous system (CNS), epigenetic mechanisms – including DNA methylation, histone modification, and regulatory noncoding RNAs – play critical roles in encoding experience and environmental stimuli into stable, behaviorally-meaningful changes in gene expression. For example, epigenetic changes in mature hippocampal neurons have been implicated in learning and memory and in a variety of neuropsychiatric disorders, including depression. With all the recent (and warranted) attention given to epigenetic modifications in mature neurons, it is easy to forget that epigenetic mechanisms were initially described for their ability to promote differentiation and drive cell fate in embryonic and early postnatal development, including neurogenesis. Given the discovery of ongoing neurogenesis in the adult brain and the intriguing links among adult hippocampal neurogenesis, hippocampal function, and neuropsychiatric disorders, it is timely to complement the ongoing discussions on the role of epigenetics in mature neurons with a review on what is currently known about the role of epigenetics in adult hippocampal neurogenesis. The process of adult hippocampal neurogenesis is complex, with neural stem cells (NSCs) giving rise to fate-restricted progenitors and eventually mature dentate gyrus granule cells. Notably, neurogenesis occurs within an increasingly well-defined “neurogenic niche”, where mature cellular elements like vasculature, astrocytes, and neurons release signals that can dynamically regulate neurogenesis. Here we review the evidence that key stages and aspects of adult neurogenesis are driven by epigenetic mechanisms. We discuss the intrinsic changes occurring within NSCs and their progeny that are critical for neurogenesis. We also discuss how extrinsic changes occurring in cellular components in the niche can result in altered neurogenesis. Finally we describe the potential relevance of epigenetics for understanding the relationship between hippocampal neurogenesis in neuropsychiatric disorders. We propose that a more thorough understanding of the molecular and genetic mechanisms that control the complex process of neurogenesis, including the proliferation and differentiation of NSCs, will lead to novel therapeutics for the treatment of neuropsychiatric disorders.

The molecular mechanism by which neural progenitor cells commit to a specified lineage of the central nervous system remains unknown. We show that HDAC1 and HDAC2 redundantly control neuronal development and are required for neuronal specification. Mice lacking HDAC1 or HDAC2 in neuronal precursors show no overt histoarchitectural phenotypes, whereas deletion of both HDAC1 and HDAC2 in developing neurons results in severe hippocampal abnormalities, absence of cerebellar foliation, disorganization of cortical neurons, and lethality by postnatal day 7. These abnormalities in brain formation can be attributed to a failure of neuronal precursors to differentiate into mature neurons and to excessive cell death. These results reveal redundant and essential roles for HDAC1 and HDAC2 in the progression of neuronal precursors to mature neurons in vivo.

Oligodendrocyte development is regulated by the interaction of repressors and activators in a complex transcriptional network. We found that two histone-modifying enzymes, HDAC1 and HDAC2, were required for oligodendrocyte formation. Genetic deletion of both Hdac1 and Hdac2 in oligodendrocyte lineage cells resulted in stabilization and nuclear translocation of beta-catenin, which negatively regulates oligodendrocyte development by repressing Olig2 expression. We further identified the oligodendrocyte-restricted transcription factor TCF7L2/TCF4 as a bipartite co-effector of beta-catenin for regulating oligodendrocyte differentiation. Targeted disruption of Tcf7l2 in mice led to severe defects in oligodendrocyte maturation, whereas expression of its dominant-repressive form promoted precocious oligodendrocyte specification in developing chick neural tube. Transcriptional co-repressors HDAC1 and HDAC2 compete with beta-catenin for TCF7L2 interaction to regulate downstream genes involved in oligodendrocyte differentiation. Thus, crosstalk between HDAC1/2 and the canonical Wnt signaling pathway mediated by TCF7L2 serves as a regulatory mechanism for oligodendrocyte differentiation.

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5 , using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs.

Myocyte enhancer factor (Mef)-2 transcription factors are implicated in activity-dependent neuronal processes during development, but the role of MEF2 in neural stem/progenitor cells (NSPCs) in the adult brain is unknown. We used a transgenic mouse in which Mef2a, -c, and -d were inducibly deleted in adult nestin-expressing NSPCs and their progeny. Recombined cells in the hippocampal granule cell layer were visualized and quantified by yellow fluorescent protein (YFP) expression. In control mice, postmitotic neurons expressed Mef2a, -c, and -d, whereas type 1 stem cells and proliferating progenitors did not. Based on this expression, we hypothesized that Mef2a, -c, and -d deletion in adult nestin-expressing NSPCs and their progeny would result in fewer mature neurons. Control mice revealed an increase in YFP+ neurons and dendrite formation over time. Contrary to our hypothesis, inducible Mef2 KO mice also displayed an increase in YFP+ neurons over time—but with significantly stunted dendrites—suggesting an uncoupling of neuron survival and dendritogenesis. We also found non–cell-autonomous effects after Mef2a, -c, and -d deletion. These in vivo findings indicate a surprising functional role for Mef2a, -c, and -d in cell- and non–cell-autonomous control of adult hippocampal neurogenesis that is distinct from its role during development.—Latchney, S. E., Jiang, Y., Petrik, D. P., Eisch, A. J., Hsieh, J. Inducible knockout of Mef2a, -c, and -d from nestin-expressing stem/progenitor cells and their progeny unexpectedly uncouples neurogenesis and dendritogenesis in vivo.

Unraveling the mechanisms by which neural stem cells generate distinct cell types remains a central challenge in central nervous system (CNS) biology. Recent studies have shown that epigenetic gene regulation plays an important role in the control of cell growth and differentiation. These epigenetic controls cover a wide spectrum, including the interaction of chromatin remodeling enzymes with neurogenic transcription factors, the maintenance of genome stability in neuronal cells and the involvement of noncoding RNAs in neural fate specification. Extracellular signaling systems that control the growth and differentiation of neural stem cells act, at least in part, by interfacing with these diverse epigenetic mechanisms.

Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded (ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. The NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with NRSF/REST transcriptional machinery, resulting in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST into cells with neuronal identity that can express neuronal genes. The mechanism of action appears to be mediated through a dsRNA/protein interaction, rather than through siRNA or miRNA. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of noncoding RNAs as key regulators of cell behavior at both transcriptional and posttranscriptional levels.

Diverse environmental stimuli regulate continuous production of new neurons in restricted adult brain regions, such as the mammalian hippocampus. Certain forms of “physiological” stimulation (e.g., exercise, experience, and normal aging) promote neurogenesis, presumably for cellular replacement and function. In contrast, “pathological” stimulation arising from brain injury (e.g., traumatic brain injury and stroke) also promotes neurogenesis, presumably for repair or replacement of dying/degenerating neurons. In most of these cases, the direct cause-and-effect relationship between adult hippocampal neurogenesis and the functional role of adult-generated neurons—whether it is for normal tissue homeostasis or repair of damaged tissue—remains unclear. A major neurological disorder that warrants investigation is epilepsy, where a single seizure episode is sufficient to massively increase neural stem/progenitor cell proliferation and adult hippocampal neurogenesis. Based on morphological and functional changes of adult-generated granule neurons after seizures, it has been postulated that seizure-induced neurogenesis may lead to ectopic dentate granule cell production and neuronal circuit formation (“aberrant neurogenesis”) and spontaneous recurrent seizures (“epileptogenesis”). Moreover, studies show that blocking aberrant neurogenesis leads to a reduction in epileptogenesis, suggesting a causative role of seizure-induced neurogenesis in epilepsy development. Thus, a better understanding of the cellular and molecular mechanisms governing aberrant neurogenesis might prevent epilepsy development. On a molecular level, seizure activity has been shown to mediate epigenetic changes, including DNA methylation and histone modification. Since epigenetic alterations are typically heritable and associated with long-lasting changes in gene expression, epigenetic dysregulation of gene expression may control behavior of hippocampal neural stem cells and their progeny after seizures and contribute to epilepsy. In this chapter, we describe the seizure-induced cellular alterations within the adult hippocampal dentate gyrus that are associated with epilepsy development. We also discuss several recent examples of the molecular control of hippocampal neurogenesis after seizures, focusing on epigenetic mechanisms.

Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

The cellular basis of adult neurogenesis is neural stem cells residing in restricted areas of the adult brain. These cells self-renew and are multipotent. The maintenance of "stemness" and commitment to differentiation are tightly controlled by intricate molecular networks. Epigenetic mechanisms, including chromatin remodeling, DNA methylation, and noncoding RNAs (ncRNAs), have profound regulatory roles in mammalian gene expression. Significant advances have been made regarding the dynamic roles of epigenetic modulation and function. It has become evident that epigenetic regulators are key players in neural-stem-cell self-renewal, fate specification, and final maturation of new neurons, therefore, adult neurogenesis. Altered epigenetic regulation can result in a number of neurological and neurodevelopmental disorders. Here, we review recent discoveries that advance our knowledge in epigenetic regulation of mammalian neural stem cells and neurogenesis. Insights from studies of epigenetic gene regulation in neurogenesis may lead to new therapies for the treatment of neurodevelopmental disorders.

Stem cells have captured our imagination and generated hope, representing a source of replacement cells to treat a host of medical conditions. Tucked away in specialized niches, stem cells maintain tissue function and rejuvenate organs. Balancing the equation between cellular supply and demand is especially important in the adult brain, as neural stem cells (NSCs) in two discrete regions, the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ) next to the lateral ventricles, continuously self-renew and differentiate into neurons in a process called adult neurogenesis. Through the interplay of intrinsic and extrinsic factors, adult neurogenic niches ensure neuronal turnover throughout life, contributing to plasticity and homeostatic processes in the brain. This review summarizes recent progress on the molecular control of adult neurogenesis in the SGZ and SVZ, focusing on the role of specific transcription factors that mediate the progression from NSCs to lineage-committed progenitors and, ultimately, the generation of mature neurons and glia.

Multipotent neural stem cells (NSCs) possess the ability to self-renew and differentiate into both neurons and glia. However, the detailed mechanisms underlying NSC fate decisions are not well understood. Recent work suggests that the interaction between cell type specific transcription factors and microRNAs (miRNAs) is important as resident neural stem/progenitor cells give rise to functionally mature neurons. Recently, we demonstrated that the transcriptional repressor REST (RE1-silencing transcription factor) is essential to prevent precocious neuronal differentiation and maintain NSC self-renewal in the adult hippocampus. Here we show that REST is required for orchestrating the expression of distinct subsets of miRNAs in primary mouse NSC cultures, a physiologically relevant cell type. Using miRNA array profiling, we identified known REST-regulated miRNA genes, as well as previously uncharacterized REST-dependent miRNAs. Interestingly, in response to proliferation and differentiation stimuli, REST-regulated miRNAs formed distinct clusters and displayed variable expression dynamics. These results suggest that REST functions in a context-dependent manner through its target miRNAs for mediating neuronal production.

In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.

The cellular and molecular mechanisms that control neural stem cell lineage decisions during development and throughout adulthood remain a central challenge. Epigenetic mechanisms and chromatin remodeling have emerged to play fundamental roles in gene regulation. Studies have implicated chromatin structure as an active mediator underlying brain function, neural cell growth and differentiation, and plasticity. These epigenetic mechanisms include cell type-specific transcriptional regulators, histone modifications and chromatin remodeling enzymes, and small regulatory noncoding RNAs. Environmental signals that control growth and function in the brain act, at least in part, by interfacing with these diverse epigenetic mechanisms.

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

Neural stem cells generate distinct cell types for tissue formation and cell replacement during development and throughout adulthood. Neural development and plasticity are determined by both extrinsic and intrinsic factors that interface to regulate gene programs for controlling neuronal cell fate and function. Recent reports have shown that chromatin remodeling and epigenetic gene regulation play an important role in such diverse areas as neural cell fate specification and synaptic development and function. These epigenetic mechanisms include cell-type-specific transcriptional regulators, histone modifications and chromatin remodeling enzymes, and the activity of retrotransposons.

DNA hydroxylation catalyzed by Tet dioxygenases occurs abundantly in embryonic stem cells and neurons in mammals. However, its biological function in vivo is largely unknown. Here we demonstrate that Tet1 plays an important role in regulating neural progenitor cell proliferation in adult mouse brain. Mice lacking Tet1 exhibit impaired hippocampal neurogenesis accompanied by poor learning and memory. In adult neural progenitor cells deficient in Tet1, a cohort of genes involved in progenitor proliferation were hypermethylated and down-regulated. Our results indicate that Tet1 is positively involved in the epigenetic regulation of neural progenitor cell proliferation in the adult brain.

Although the brain is generally considered a terminally differentiated organ, new nerve cells are made every day through a process called “adult neurogenesis,” which occurs in specialized regions like the hippocampal dentate gyrus ( 1 ). Stem cells in the brain sample electrical signals (activity) from neighboring neurons, deciding which genes to express and which signaling pathways to launch toward developing their own neuronal identity. Why would stem cells be able to respond to exogenous neuronal electrical activity, which can be considered a highly specialized function? Indeed, it seems counterintuitive insofar as one of the defining functions of all stem cells is to actively maintain the undifferentiated state.

Neurogenesis occurs throughout adult life in the dentate gyrus of mammalian hippocampus and has been suggested to play an important role in cognitive function. Multiple trophic factors including IGF-I have been demonstrated to regulate hippocampal neurogenesis. Ames dwarf mice live considerably longer than normal animals and maintain physiological function at youthful levels, including cognitive function, despite a deficiency of circulating GH and IGF-I. Here we show an increase in numbers of newly generated cells [bromodeoxyuridine (BrdU) positive] and newborn neurons (neuronal nuclear antigen and BrdU positive) in the dentate gyrus of adult dwarf mice compared with normal mice using BrdU labeling. Despite the profound suppression of hippocampal GH expression, hippocampal IGF-I protein levels are up-regulated and the corresponding mRNAs are as high in Ames dwarf as in normal mice. Our results suggest that local/hippocampal IGF-I expression may have induced the increase in hippocampal neurogenesis, and increased neurogenesis might contribute to the maintenance of youthful levels of cognitive function during aging in these long-lived animals.

It has become apparent that chromatin modification plays a critical role in the regulation of cell-type-specific gene expression. Here, we show that an inhibitor of histone deacetylase, valproic acid (VPA), induced neuronal differentiation of adult hippocampal neural progenitors. In addition, VPA inhibited astrocyte and oligodendrocyte differentiation, even in conditions that favored lineage-specific differentiation. Among the VPA-up-regulated, neuron-specific genes, a neurogenic basic helix–loop–helix transcription factor, NeuroD , was identified. Overexpression of NeuroD resulted in the induction and suppression of neuronal and glial differentiation, respectively. These results suggest that VPA promotes neuronal fate and inhibits glial fate simultaneously through the induction of neurogenic transcription factors including NeuroD.

▪ Abstract Mechanisms for repetition of DNA pose both opportunities and challenges to a functional genome: opportunities for increasing gene expression by amplification of useful sequences, and challenges of controlling amplification by unwanted sequences such as transposons and viruses. Experiments in numerous organisms have suggested the likely existence of a general mechanism for recognition of repeated character in DNA. This review focuses (a) on the nature of these recognition mechanisms, and (b) on types of chromatin modification and gene silencing that are used to control repeated DNA.