Caenorhabditis elegans establishes germline versus soma by balancing inherited histone methylation.

Formation of a zygote is coupled with extensive epigenetic reprogramming to enable appropriate inheritance of histone methylation and prevent developmental delays. In Caenorhabditis elegans, this reprogramming is mediated by the H3K4me2 demethylase SPR-5 and the H3K9 methyltransferase, MET-2. In contrast, the H3K36 methyltransferase MES-4 maintains H3K36me2/3 at germline genes between generations to facilitate re-establishment of the germline. To determine whether the MES-4 germline inheritance pathway antagonizes spr-5; met-2 reprogramming, we examined the interaction between these two pathways. We found that the developmental delay of spr-5; met-2 mutant progeny is associated with ectopic H3K36me3 and the ectopic expression of MES-4-targeted germline genes in somatic tissues. Furthermore, the developmental delay is dependent upon MES-4 and the H3K4 methyltransferase, SET-2. We propose that MES-4 prevents crucial germline genes from being repressed by antagonizing maternal spr-5; met-2 reprogramming. Thus, the balance of inherited histone modifications is necessary to distinguish germline versus soma and prevent developmental delay.This article has an associated 'The people behind the papers' interview.

A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory.

Epigenetic information undergoes extensive reprogramming in the germline between generations. This reprogramming may be essential to establish a developmental ground state in the zygote. We show that mutants in spr-5, the Caenorhabditis elegans ortholog of the H3K4me2 demethylase LSD1/KDM1, exhibit progressive sterility over many generations. This sterility correlates with the misregulation of spermatogenesis-expressed genes and transgenerational accumulation of the histone modification dimethylation of histone H3 on lysine 4 (H3K4me2). This suggests that H3K4me2 can serve as a stable epigenetic memory, and that erasure of H3K4me2 by LSD/KDM1 in the germline prevents the inappropriate transmission of this epigenetic memory from one generation to the next. Thus, our results provide direct mechanistic insights into the processes that are required for epigenetic reprogramming between generations.

Unique molecular characteristics and microglial origin of Kv1.3 channel-positive brain myeloid cells in Alzheimer's disease.

Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer's disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aß) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11b+CD45+ CNS-MPs acutely isolated from an Aß mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11b+Kv1.3+ CNS-MPs, CD11b+CD45int Kv1.3neg microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers (Tmem119, P2ry12) and are distinct from peripheral Ly6chigh/Ly6clow monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of CD11c, and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3+ CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aß burden, increased CD11c+ CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes (IL1b). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype.

Hansel, Gretel, and the Consequences of Failing to Remove Histone Methylation Breadcrumbs.

Like breadcrumbs in the forest, cotranscriptionally acquired histone methylation acts as a memory of prior transcription. Because it can be retained through cell divisions, transcriptional memory allows cells to coordinate complex transcriptional programs during development. However, if not reprogrammed properly during cell fate transitions, it can also disrupt cellular identity. In this review, we discuss the consequences of failure to reprogram histone methylation during three crucial epigenetic reprogramming windows: maternal reprogramming at fertilization, embryonic stem cell (ESC) differentiation, and the continuous maintenance of cell identity in differentiated cells. In addition, we discuss how following the wrong breadcrumb trail of transcriptional memory provides a framework for understanding how heterozygous loss-of-function mutations in histone-modifying enzymes may cause severe neurodevelopmental disorders.

The inhibition of LSD1 via sequestration contributes to tau-mediated neurodegeneration.

Tauopathies are a class of neurodegenerative diseases associated with pathological tau. Despite many advances in our understanding of these diseases, the direct mechanism through which tau contributes to neurodegeneration remains poorly understood. Previously, our laboratory implicated the histone demethylase LSD1 in tau-induced neurodegeneration by showing that LSD1 localizes to pathological tau aggregates in Alzheimer's disease cases, and that it is continuously required for the survival of hippocampal and cortical neurons in mice. Here, we utilize the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. In addition, we show that reducing LSD1 in these mice is sufficient to highly exacerbate tau-mediated neurodegeneration and tau-induced gene expression changes. Finally, we find that overexpressing LSD1 in the hippocampus of tauopathy mice, even after pathology has formed, is sufficient to significantly delay neurodegeneration and counteract tau-induced expression changes. These results suggest that inhibiting LSD1 via sequestration contributes to tau-mediated neurodegeneration. Thus, LSD1 is a promising therapeutic target for tauopathies such as Alzheimer's disease.

SPR-5 and MET-2 function cooperatively to reestablish an epigenetic ground state during passage through the germ line.

The Caenorhabditis elegans LSD1 H3K4me2 demethylase SPR-5 reprograms epigenetic transcriptional memory during passage through the germ line. Here we show that mutants in the H3K9me2 methyltransferase, met-2, result in transgenerational epigenetic effects that parallel spr-5 mutants. In addition, we find that spr-5;met-2 double mutants have a synergistic effect on sterility, H3K4me2, and spermatogenesis expression. These results implicate MET-2 as a second histone-modifying enzyme in germ-line reprogramming and suggest a model in which SPR-5 and MET-2 function cooperatively to reestablish an epigenetic ground state required for the continued immortality of the C. elegans germ line. Without SPR-5 and MET-2, we find that the ability to express spermatogenesis genes is transgenerationally passed on to the somatic cells of the subsequent generation. This indicates that H3K4me2 may act in the maintenance of cell fate. Finally, we demonstrate that reducing H3K4me2 causes a large increase in H3K9me2 added by the SPR-5;MET-2 reprogramming mechanism. This finding suggests a novel histone code interaction in which the input chromatin environment dictates the output chromatin state. Taken together, our results provide evidence for a broader reprogramming mechanism in which multiple enzymes coordinately regulate histone information during passage through the germ line.

Epigenetic patterns maintained in early Caenorhabditis elegans embryos can be established by gene activity in the parental germ cells.

Epigenetic information, such as parental imprints, can be transmitted with genetic information from parent to offspring through the germ line. Recent reports show that histone modifications can be transmitted through sperm as a component of this information transfer. How the information that is transferred is established in the parent and maintained in the offspring is poorly understood. We previously described a form of imprinted X inactivation in Caenorhabditis elegans where dimethylation on histone 3 at lysine 4 (H3K4me2), a mark of active chromatin, is excluded from the paternal X chromosome (Xp) during spermatogenesis and persists through early cell divisions in the embryo. Based on the observation that the Xp (unlike the maternal X or any autosome) is largely transcriptionally inactive in the paternal germ line, we hypothesized that transcriptional activity in the parent germ line may influence epigenetic information inherited by and maintained in the embryo. We report that chromatin modifications and histone variant patterns assembled in the germ line can be retained in mature gametes. Furthermore, despite extensive chromatin remodeling events at fertilization, the modification patterns arriving with the gametes are largely retained in the early embryo. Using transgenes, we observe that expression in the parental germline correlates with differential chromatin assembly that is replicated and maintained in the early embryo. Expression in the adult germ cells also correlates with more robust expression in the somatic lineages of the offspring. These results suggest that differential expression in the parental germ lines may provide a potential mechanism for the establishment of parent-of-origin epigenomic content. This content can be maintained and may heritably affect gene expression in the offspring.

Lineage Tracing and Single-Cell RNA-seq in C. elegans to Analyze Transgenerational Epigenetic Phenotypes Inherited from Germ Cells.

The last several years have seen an increasing number of examples of transgenerational epigenetic inheritance, in which phenotypes are inherited for three or more generations without changes to the underlying DNA sequence. One model system that has been particularly useful for studying transgenerational epigenetic inheritance is C. elegans. Their short generation time and hermaphroditic reproduction have allowed multiple transgenerational phenotypes to be identified, including aging, fertility, and behavior. However, it is still not clear how transgenerational epigenetic inheritance from the germline affects embryogenesis. Fortunately, the C. elegans embryo has a unique property that makes it ideal for addressing this question: they develop via an invariant lineage, with each cell undergoing stereotypical cell divisions to adopt the same cell fate in every individual embryo. Because of this invariant cell lineage, automated lineage tracing and single-cell RNA-seq can be employed to determine how transgenerational epigenetic inheritance from the germline affects developmental timing and cell fate. Unfortunately, difficulties with these techniques have severely limited their adoption in the community. Here, we provide a practical guide to automated lineage tracing coupled with single-cell RNA-seq to facilitate their use in studying transgenerational epigenetic inheritance in C. elegans embryos.

SPR-1/CoREST facilitates the maternal epigenetic reprogramming of the histone demethylase SPR-5/LSD1.

Maternal reprogramming of histone methylation is critical for reestablishing totipotency in the zygote, but how histone-modifying enzymes are regulated during maternal reprogramming is not well characterized. To address this gap, we asked whether maternal reprogramming by the H3K4me1/2 demethylase SPR-5/LSD1/KDM1A, is regulated by the chromatin co-repressor protein, SPR-1/CoREST, in Caenorhabditis elegans and mice. In C. elegans, SPR-5 functions as part of a reprogramming switch together with the H3K9 methyltransferase MET-2. By examining germline development, fertility, and gene expression in double mutants between spr-1 and met-2, as well as fertility in double mutants between spr-1 and spr-5, we find that loss of SPR-1 results in a partial loss of SPR-5 maternal reprogramming function. In mice, we generated a separation of function Lsd1 M448V point mutation that compromises CoREST binding, but only slightly affects LSD1 demethylase activity. When maternal LSD1 in the oocyte is derived exclusively from this allele, the progeny phenocopy the increased perinatal lethality that we previously observed when LSD1 was reduced maternally. Together, these data are consistent with CoREST having a conserved function in facilitating maternal LSD1 epigenetic reprogramming.

Ectopic transcription due to inappropriately inherited histone methylation may interfere with the ongoing function of terminally differentiated cells.

Many human neurodevelopmental disorders are caused by de novo mutations in histone modifying enzymes. These patients have craniofacial defects, developmental delay, intellectual disability and behavioral abnormalities, but it remains unclear how the mutations lead to such developmental defects. Here we take advantage of the invariant C. elegans lineage along with a unique double mutant in the H3K4me1/2 demethylase SPR-5/LSD1/KDM1A and the H3K9 methyltransferase MET-2/SETDB1 to address this question. We demonstrate that spr-5; met-2 double mutant worms have a severe chemotaxis defect that is dependent upon the ectopic expression of germline genes in somatic tissues. In addition, by performing single-cell RNAseq, we find that germline genes begin to be ectopically expression widely in spr-5; met-2 embryos. However, surprisingly we found that spr-5; met-2 mutants have no somatic lineage defects prior to the 200-cell stage of embryogenesis. This suggests that the altered chemotaxis behavior may be due to ongoing defect in terminally differentiated cells rather than a defect in development. To test this directly, we used RNAi to shut off the ectopic expression of germline genes in L2 spr-5; met-2 larvae, which have a fully formed nervous system. Remarkably, we find that shutting off the ectopic germline expression rescues normal chemotaxis behavior in the same adult worms that previously had a chemotaxis defect at the L2 stage. This suggests that ongoing ectopic transcription can block normal behavior in a fully intact nervous system. These data raise the possibility that intellectual disability and altered behavior in neurodevelopmental syndromes, caused by mutations in histone modifying enzymes, could be due to ongoing ectopic transcription and may be reversible.

The Pipeline CURE: An Iterative Approach to Introduce All Students to Research Throughout a Biology Curriculum.

Participation in research provides personal and professional benefits for undergraduates. However, some students face institutional barriers that prevent their entry into research, particularly those from underrepresented groups who may stand to gain the most from research experiences. Course-based undergraduate research experiences (CUREs) effectively scale research availability, but many only last for a single semester, which is rarely enough time for a novice to develop proficiency. To address these challenges, we present the Pipeline CURE, a framework that integrates a single research question throughout a biology curriculum. Students are introduced to the research system - in this implementation, C. elegans epigenetics research - with their first course in the major. After revisiting the research system in several subsequent courses, students can choose to participate in an upper-level research experience. In the Pipeline, students build resilience via repeated exposure to the same research system. Its iterative, curriculum-embedded approach is flexible enough to be implemented at a range of institutions using a variety of research questions. By uniting evidence-based teaching methods with ongoing scientific research, the Pipeline CURE provides a new model for overcoming barriers to participation in undergraduate research.

Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region.

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg(2+), implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

A Resource for the Allele-Specific Analysis of DNA Methylation at Multiple Genomically Imprinted Loci in Mice.

Genomically imprinted loci are expressed mono-allelically, dependent upon the parent of origin. Their regulation not only illuminates how chromatin regulates gene expression but also how chromatin can be reprogrammed every generation. Because of their distinct parent-of-origin regulation, analysis of imprinted loci can be difficult. Single nucleotide polymorphisms (SNPs) are required to accurately assess these elements allele specifically. However, publicly available SNP databases lack robust verification, making analysis of imprinting difficult. In addition, the allele-specific imprinting assays that have been developed employ different mouse strains, making it difficult to systemically analyze these loci. Here, we have generated a resource that will allow the allele-specific analysis of many significant imprinted loci in a single hybrid strain of Mus musculus This resource includes verification of SNPs present within 10 of the most widely used imprinting control regions and allele-specific DNA methylation assays for each gene in a C57BL/6J and CAST/EiJ hybrid strain background.

Neuroepigenetic mechanisms in disease.

Epigenetics allows for the inheritance of information in cellular lineages during differentiation, independent of changes to the underlying genetic sequence. This raises the question of whether epigenetic mechanisms also function in post-mitotic neurons. During the long life of the neuron, fluctuations in gene expression allow the cell to pass through stages of differentiation, modulate synaptic activity in response to environmental cues, and fortify the cell through age-related neuroprotective pathways. Emerging evidence suggests that epigenetic mechanisms such as DNA methylation and histone modification permit these dynamic changes in gene expression throughout the life of a neuron. Accordingly, recent studies have revealed the vital importance of epigenetic players in the central nervous system and during neurodegeneration. Here, we provide a review of several of these recent findings, highlighting novel functions for epigenetics in the fields of Rett syndrome, Fragile X syndrome, and Alzheimer's disease research. Together, these discoveries underscore the vital importance of epigenetics in human neurological disorders.

A Model for Epigenetic Inhibition via Transvection in the Mouse.

Transvection is broadly defined as the ability of one locus to affect its homologous locus in trans Although it was first discovered in the 1950s, there are only two known cases in mammals. Here, we report another instance of mammalian transvection induced by the Cre/LoxP system, which is widely used for conditional gene targeting in the mouse. We attempted to use the germline-expressed Vasa-Cre transgene to engineer a mouse mutation, but observe a dramatic reduction of LoxP recombination in mice that inherit an already deleted LoxP allele in trans A similar phenomenon has previously been observed with another Cre that is expressed during meiosis: Sycp-1-Cre This second example of LoxP inhibition in trans reinforces the conclusion that certain meiotically expressed Cre alleles can initiate transvection in mammals. However, unlike the previous example, we find that the inhibition of LoxP recombination is not due to DNA methylation. In addition, we demonstrate that LoxP inhibition is easily alleviated by adding an extra generation to our crossing scheme. This finding confirms that the LoxP sites are inhibited via an epigenetic mechanism, and provides a method for the use of other Cre transgenes associated with a similar LoxP inhibition event. Furthermore, the abrogation of LoxP inhibition by the simple addition of an extra generation in our crosses establishes a unique mouse system for future studies to uncover the mechanism of transvection in mammals.

LSD1 protects against hippocampal and cortical neurodegeneration.

To investigate the mechanisms that maintain differentiated cells, here we inducibly delete the histone demethylase LSD1/KDM1A in adult mice. Loss of LSD1 leads to paralysis, along with widespread hippocampus and cortex neurodegeneration, and learning and memory defects. We focus on the hippocampus neuronal cell death, as well as the potential link between LSD1 and human neurodegenerative disease and find that loss of LSD1 induces transcription changes in common neurodegeneration pathways, along with the re-activation of stem cell genes, in the degenerating hippocampus. These data implicate LSD1 in the prevention of neurodegeneration via the inhibition of inappropriate transcription. Surprisingly, we also find that transcriptional changes in the hippocampus are similar to Alzheimer's disease (AD) and frontotemporal dementia (FTD) cases, and LSD1 is specifically mislocalized to pathological protein aggregates in these cases. These data raise the possibility that pathological aggregation could compromise the function of LSD1 in AD and FTD."LSD1 is a histone demethylase that plays many roles during development. Here, the authors provide evidence that loss of LSD1 in adult mice leads to paralysis and neurodegeneration in the hippocampus and cortex and suggest a potential link between LSD1 and human neurodegenerative disease.

KDM1A/LSD1 regulates the differentiation and maintenance of spermatogonia in mice.

The proper regulation of spermatogenesis is crucial to ensure the continued production of sperm and fertility. Here, we investigated the function of the H3K4me2 demethylase KDM1A/LSD1 during spermatogenesis in developing and adult mice. Conditional deletion of Kdm1a in the testis just prior to birth leads to fewer spermatogonia and germ cell loss before 3 weeks of age. These results demonstrate that KDM1A is required for spermatogonial differentiation, as well as germ cell survival, in the developing testis. In addition, inducible deletion of Kdm1a in the adult testis results in the abnormal accumulation of meiotic spermatocytes, as well as apoptosis and progressive germ cell loss. These results demonstrate that KDM1A is also required during adult spermatogenesis. Furthermore, without KDM1A, the stem cell factor OCT4 is ectopically maintained in differentiating germ cells. This requirement for KDM1A is similar to what has been observed in other stem cell populations, suggesting a common function. Taken together, we propose that KDM1A is a key regulator of spermatogenesis and germ cell maintenance in the mouse.

Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally.

Somatic cell nuclear transfer has established that the oocyte contains maternal factors with epigenetic reprogramming capacity. Yet the identity and function of these maternal factors during the gamete to embryo transition remains poorly understood. In C. elegans, LSD1/KDM1A enables this transition by removing H3K4me2 and preventing the transgenerational inheritance of transcription patterns. Here we show that loss of maternal LSD1/KDM1A in mice results in embryonic arrest at the 1-2 cell stage, with arrested embryos failing to undergo the maternal-to-zygotic transition. This suggests that LSD1/KDM1A maternal reprogramming is conserved. Moreover, partial loss of maternal LSD1/KDM1A results in striking phenotypes weeks after fertilization; including perinatal lethality and abnormal behavior in surviving adults. These maternal effect hypomorphic phenotypes are associated with alterations in DNA methylation and expression at imprinted genes. These results establish a novel mammalian paradigm where defects in early epigenetic reprogramming can lead to defects that manifest later in development.

Restoring totipotency through epigenetic reprogramming.

Epigenetic modifications are implicated in the maintenance and regulation of transcriptional memory by marking genes that were previously transcribed to facilitate transmission of these expression patterns through cell division. During germline specification and maintenance, extensive epigenetic modifications are acquired. Yet somehow at fertilization, the fusion of the highly differentiated sperm and egg results in formation of the totipotent zygote. This massive change in cell fate implies that the selective erasure and maintenance of epigenetic modifications at fertilization may be critical for the re-establishment of totipotency. In this review, we discuss recent studies that provide insight into the extensive epigenetic reprogramming that occurs around fertilization and the mechanisms that may be involved in the re-establishment of totipotency in the embryo.