APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.

Transcriptional scaffolds for heterochromatin assembly.

Heterochromatin is dynamically regulated during the cell cycle and in response to developmental signals. Recent findings from diverse systems suggest an extensive role for transcription in the assembly of heterochromatin, highlighting the emerging theme that transcription and noncoding RNAs can provide the initial scaffold for the formation of heterochromatin, which serves as a versatile recruiting platform for diverse factors involved in many cellular processes.

Multifaceted genome control by Set1 Dependent and Independent of H3K4 methylation and the Set1C/COMPASS complex.

Histone modifiers are critical regulators of chromatin-based processes in eukaryotes. The histone methyltransferase Set1, a component of the Set1C/COMPASS complex, catalyzes the methylation at lysine 4 of histone H3 (H3K4me), a hallmark of euchromatin. Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways. Set1 employs its RNA-binding RRM2 and catalytic SET domains to repress Tf2 retrotransposons and pericentromeric repeats while relying on its H3K4me function to maintain transcriptional repression at the silent mating type (mat) locus and subtelomeric regions. These repressive functions of Set1 correlate with the requirement of Set1C components to maintain repression at the mat locus and subtelomeres while dispensing Set1C in repressing Tf2s and pericentromeric repeats. We show that the contributions of several Set1C subunits to the states of H3K4me diverge considerably from those of Saccharomyces cerevisiae orthologs. Moreover, unlike S. cerevisiae, the regulation of Set1 protein level is not coupled to the status of H3K4me or histone H2B ubiquitination by the HULC complex. Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation. Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

Cell cycle control of centromeric repeat transcription and heterochromatin assembly.

Heterochromatin in eukaryotic genomes regulates diverse chromosomal processes including transcriptional silencing. However, in Schizosaccharomyces pombe RNA polymerase II (RNAPII) transcription of centromeric repeats is essential for RNA-interference-mediated heterochromatin assembly. Here we study heterochromatin dynamics during the cell cycle and its effect on RNAPII transcription. We describe a brief period during the S phase of the cell cycle in which RNAPII preferentially transcribes centromeric repeats. This period is enforced by heterochromatin, which restricts RNAPII accessibility at centromeric repeats for most of the cell cycle. RNAPII transcription during S phase is linked to loading of RNA interference and heterochromatin factors such as the Ago1 subunit of the RITS complex and the Clr4 methyltransferase complex subunit Rik1 (ref. 7). Moreover, Set2, an RNAPII-associated methyltransferase that methylates histone H3 lysine 36 at repeat loci during S phase, acts in a pathway parallel to Clr4 to promote heterochromatin assembly. We also show that phosphorylation of histone H3 serine 10 alters heterochromatin during mitosis, correlating with recruitment of condensin that affects silencing of centromeric repeats. Our analyses suggest at least two distinct modes of heterochromatin targeting to centromeric repeats, whereby RNAPII transcription of repeats and chromodomain proteins bound to methylated histone H3 lysine 9 mediate recruitment of silencing factors. Together, these processes probably facilitate heterochromatin maintenance through successive cell divisions.

Host genome surveillance for retrotransposons by transposon-derived proteins.

Transposable elements and their remnants constitute a substantial fraction of eukaryotic genomes. Host genomes have evolved defence mechanisms, including chromatin modifications and RNA interference, to regulate transposable elements. Here we describe a genome surveillance mechanism for retrotransposons by transposase-derived centromeric protein CENP-B homologues of the fission yeast Schizosaccharomyces pombe. CENP-B homologues of S. pombe localize at and recruit histone deacetylases to silence Tf2 retrotransposons. CENP-Bs also repress solo long terminal repeats (LTRs) and LTR-associated genes. Tf2 elements are clustered into 'Tf' bodies, the organization of which depends on CENP-Bs that display discrete nuclear structures. Furthermore, CENP-Bs prevent an 'extinct' Tf1 retrotransposon from re-entering the host genome by blocking its recombination with extant Tf2, and silence and immobilize a Tf1 integrant that becomes sequestered into Tf bodies. Our results reveal a probable ancient retrotransposon surveillance pathway important for host genome integrity, and highlight potential conflicts between DNA transposons and retrotransposons, major transposable elements believed to have greatly moulded the evolution of genomes.

Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.

The organization of eukaryotic genomes into distinct structural and functional domains is important for the regulation and transduction of genetic information. Here, we investigated heterochromatin and euchromatin profiles of the entire fission yeast genome and explored the role of RNA interference (RNAi) in genome organization. Histone H3 methylated at Lys4, which defines euchromatin, was not only distributed across most of the chromosomal landscape but was also present at the centromere core, the site of kinetochore assembly. In contrast, histone H3 methylated at Lys9 and its interacting protein Swi6/HP1, which define heterochromatin, coated extended domains associated with a variety of repeat elements and small islands corresponding to meiotic genes. Notably, RNAi components were distributed throughout all these heterochromatin domains, and their localization depended on Clr4/Suv39h histone methyltransferase. Sequencing of small interfering RNAs (siRNAs) associated with the RITS RNAi effector complex identified hot spots of siRNAs, which mapped to a diverse array of elements in these RNAi-heterochromatin domains. We found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs. Our analyses also uncover an important role for the RNAi machinery in maintaining genomic integrity.

A novel molecular class that recruits HDAC/MECP2 complexes to PU.1 motifs reduces neuroinflammation.

Pervasive neuroinflammation occurs in many neurodegenerative diseases, including Alzheimer's disease (AD). SPI1/PU.1 is a transcription factor located at a genome-wide significant AD-risk locus and its reduced expression is associated with delayed onset of AD. We analyzed single-cell transcriptomic datasets from microglia of human AD patients and found an enrichment of PU.1-binding motifs in the differentially expressed genes. In hippocampal tissues from transgenic mice with neurodegeneration, we found vastly increased genomic PU.1 binding. We then screened for PU.1 inhibitors using a PU.1 reporter cell line and discovered A11, a molecule with anti-inflammatory efficacy and nanomolar potency. A11 regulated genes putatively by recruiting a repressive complex containing MECP2, HDAC1, SIN3A, and DNMT3A to PU.1 motifs, thus representing a novel mechanism and class of molecules. In mouse models of AD, A11 ameliorated neuroinflammation, loss of neuronal integrity, AD pathology, and improved cognitive performance. This study uncovers a novel class of anti-inflammatory molecules with therapeutic potential for neurodegenerative disorders.

Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity.

In Parkinson's disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA-/- neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.

Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection.

Histone acetylation is important in regulating DNA accessibility. Multifunctional Sin3 proteins bind histone deacetylases (HDACs) to assemble silencing complexes that selectively target chromatin. We show that, in fission yeast, an essential HDAC, Clr6, exists in two distinct Sin3 core complexes. Complex I contains an essential Sin3 homolog, Pst1, and other factors, and predominantly targets gene promoters. Complex II contains a nonessential Sin3 homolog, Pst2, and several conserved proteins. It preferentially targets transcribed chromosomal regions and centromere cores. Defects in complex II abrogate global protective functions of chromatin, causing increased accessibility of DNA to genotoxic agents and widespread antisense transcripts that are processed by the exosome. Notably, the two Clr6 complexes differentially repress forward and reverse centromeric repeat transcripts, suggesting that these complexes regulate transcription in heterochromatin and euchromatin in similar manners, including suppression of spurious transcripts from cryptic start sites.

A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) made by Spo11 (Rec12 in fission yeast), which becomes covalently linked to the DSB ends. Like recombination events, DSBs occur at hotspots in the genome, but the genetic factors responsible for most hotspots have remained elusive. Here we describe in fission yeast the genome-wide distribution of meiosis-specific Rec12-DNA linkages, which closely parallel DSBs measured by conventional Southern blot hybridization. Prominent DSB hotspots are located approximately 65 kb apart, separated by intervals with little or no detectable breakage. Most hotspots lie within exceptionally large intergenic regions. Thus, the chromosomal architecture responsible for hotspots in fission yeast is markedly different from that of budding yeast, in which DSB hotspots are much more closely spaced and, in many regions of the genome, occur at each promoter. Our analysis in fission yeast reveals a clearly identifiable chromosomal feature that can predict the majority of recombination hotspots across a whole genome and provides a basis for searching for the chromosomal features that dictate hotspots of meiotic recombination in other organisms, including humans.

HDAC1 modulates OGG1-initiated oxidative DNA damage repair in the aging brain and Alzheimer's disease.

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired OGG1 activity, 8-oxoG accumulation at the promoters of genes critical for brain function, and transcriptional repression. Moreover, we observe elevated 8-oxoG along with reduced HDAC1 activity and downregulation of a similar gene set in the 5XFAD mouse model of Alzheimer's disease. Notably, pharmacological activation of HDAC1 alleviates the deleterious effects of 8-oxoG in aged wild-type and 5XFAD mice. Our work uncovers important roles for HDAC1 in 8-oxoG repair and highlights the therapeutic potential of HDAC1 activation to counter functional decline in brain aging and neurodegeneration.

Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes.

In Alzheimer's disease, amyloid deposits along the brain vasculature lead to a condition known as cerebral amyloid angiopathy (CAA), which impairs blood-brain barrier (BBB) function and accelerates cognitive degeneration. Apolipoprotein (APOE4) is the strongest risk factor for CAA, yet the mechanisms underlying this genetic susceptibility are unknown. Here we developed an induced pluripotent stem cell-based three-dimensional model that recapitulates anatomical and physiological properties of the human BBB in vitro. Similarly to CAA, our in vitro BBB displayed significantly more amyloid accumulation in APOE4 compared to APOE3. Combinatorial experiments revealed that dysregulation of calcineurin-nuclear factor of activated T cells (NFAT) signaling and APOE in pericyte-like mural cells induces APOE4-associated CAA pathology. In the human brain, APOE and NFAT are selectively dysregulated in pericytes of APOE4 carriers, and inhibition of calcineurin-NFAT signaling reduces APOE4-associated CAA pathology in vitro and in vivo. Our study reveals the role of pericytes in APOE4-mediated CAA and highlights calcineurin-NFAT signaling as a therapeutic target in CAA and Alzheimer's disease.

APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types.

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aß42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aß uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aß phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.

Micrococcal Nuclease Digestion of Schizosaccharomyces pombe Chromatin.

Digestion of chromatin with micrococcal nuclease (MNase) is widely used to probe nucleosome organization. Analysis of MNase digests by end-labeling techniques or overlapping quantitative polymerase chain reaction (qPCR) can be used to map locus-specific nucleosome positions. Furthermore, the application of genomic technologies can provide genome-wide views of nucleosome position and occupancy. This protocol provides a basic method for MNase digestion of Schizosaccharomyces pombe chromatin and depends on the production of permeabilized spheroplasts.

Chromatin Immunoprecipitation (ChIP) in Schizosaccharomyces pombe.

Chromatin immunoprecipitation (ChIP), the cross-linking of chromatin followed by immunoprecipitation with antibodies against a chromatin target, is a key method for measuring association of proteins with a specific genomic region(s). As a negative control, a mock ChIP experiment in which no antibody is added to the immunoprecipitation reaction is included. Enriched DNA fragments from a ChIP experiment can be analyzed in a variety of ways. For semiquantitative analysis, a region of interest can be amplified using standard polymerase chain reaction (PCR) techniques. PCR products are analyzed on agarose (or polyacrylamide) gels and band intensity calculated with a standard imaging software. ChIP enrichment is usually calculated as the ratio of ChIP to input compared with a similar ratio for a reference region not expected to be enriched for that factor. For heterochromatin analysis, housekeeping genes such as act1+ are good references. Real-time quantitative PCR (qPCR) can be used for a fully quantitative approach. If a factor is to be mapped across the genome, ChIP DNA can be amplified and labeled for microarray analysis or scrutinized on a next-generation DNA sequencing platform.

Analysis of Heterochromatin in Schizosaccharomyces pombe.

This introduction briefly describes the biology of heterochromatin in the fission yeast Schizosaccharomyces pombe We highlight some of the salient features of fission yeast that render it an excellent unicellular eukaryote for studying heterochromatin. We then discuss key aspects of heterochromatin that are of interest to those in the field, and last we introduce experimental approaches often used to investigate heterochromatin.

Reporter Gene Silencing Assays in Fission Yeast.

Reporter gene silencing assays provide a facile method for assessing the function of heterochromatin in Schizosaccharomyces pombe They use strains containing auxotrophic markers (commonly ura4+ or ade6+) located within a heterochromatic region. Transcriptional silencing of these reporters can be assessed by plating serial dilutions of cells onto minimal agar. In addition, silencing of ura4+ renders cells resistant to 5-fluoroorotic acid (5-FOA) and ade6+ silencing results in red colony color on adenine-limiting agar. Various reporters for each of the major heterochromatic domains (telomeres, centromeres, and the mating type locus) are available and, importantly, transcriptional silencing is correlated with the proper function of these regions.

Restriction of Retrotransposon Mobilization in Schizosaccharomyces pombe by Transcriptional Silencing and Higher-Order Chromatin Organization.

Uncontrolled propagation of retrotransposons is potentially detrimental to host genome integrity. Therefore, cells have evolved surveillance mechanisms to restrict the mobility of these elements. In Schizosaccharomyces pombe the Tf2 LTR retrotransposons are transcriptionally silenced and are also clustered in the nucleus into structures termed Tf bodies. Here we describe the impact of silencing and clustering on the mobility of an endogenous Tf2 element. Deletion of genes such as set1(+) (histone H3 lysine 4 methyltransferase) or abp1(+) (CENP-B homolog) that both alleviate silencing and clustering, result in a corresponding increase in mobilization. Furthermore, expression of constitutively active Sre1, a transcriptional activator of Tf2 elements, also alleviates clustering and induces mobilization. In contrast, clustering is not disrupted by loss of the HIRA histone chaperone, despite high levels of expression, and in this background, mobilization frequency is only marginally increased. Thus, mutations that compromise transcriptional silencing but not Tf bodies are insufficient to drive mobilization. Furthermore, analyses of mutant alleles that separate the transcriptional repression and clustering functions of Set1 are consistent with control of Tf2 propagation via a combination of silencing and spatial organization. Our results indicate that host surveillance mechanisms operate at multiple levels to restrict Tf2 retrotransposon mobilization.

Suppression of Meiotic Recombination by CENP-B Homologs in Schizosaccharomyces pombe.

Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of HR hot- and coldspots. Previous study reveals that the Spo11 homolog Rec12 responsible for initiation of meiotic double-strand breaks in the fission yeast Schizosaccharomyces pombe is not targeted to Tf2 retrotransposons. However, whether Tf2s are HR coldspots is not known. Here, we show that the rates of HR across Tf2s are similar to a genome average but substantially increase in mutants deficient for the CENP-B homologs. Abp1, which is the most prominent of the CENP-B family members and acts as the primary determinant of HR suppression at Tf2s, is required to prevent gene conversion and maintain proper recombination exchange of homologous alleles flanking Tf2s. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. We demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, we uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the nonhomologous end joining pathway in ensuring the suppression of HR at Tf2s. Our study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators and a DNA repair pathway to regulate meiotic recombination at interspersed repeats.