The role of Niemann-Pick type C2 in zebrafish embryonic development.

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

Cerebrospinal Fluid Protein Biomarker Discovery in CLN3.

Syndromic CLN3-Batten is a fatal, pediatric, neurodegenerative disease caused by variants in CLN3, which encodes the endolysosomal transmembrane CLN3 protein. No approved treatment for CLN3 is currently available. The protracted and asynchronous disease presentation complicates the evaluation of potential therapies using clinical disease progression parameters. Biomarkers as surrogates to measure the progression and effect of potential therapeutics are needed. We performed proteomic discovery studies using cerebrospinal fluid (CSF) samples from 28 CLN3-affected and 32 age-similar non-CLN3 individuals. Proximal extension assay (PEA) of 1467 proteins and untargeted data-dependent mass spectrometry [MS; MassIVE FTP server (ftp://MSV000090147@massive.ucsd.edu)] were used to generate orthogonal lists of protein marker candidates. At an adjusted p-value of <0.1 and threshold CLN3/non-CLN3 fold-change ratio of 1.5, PEA identified 54 and MS identified 233 candidate biomarkers. Some of these (NEFL, CHIT1) have been previously linked with other neurologic conditions. Others (CLPS, FAM217B, QRICH2, KRT16, ZNF333) appear to be novel. Both methods identified 25 candidate biomarkers, including CHIT1, NELL1, and ISLR2 which had absolute fold-change ratios >2. NELL1 and ISLR2 regulate axonal development in neurons and are intriguing new candidates for further investigation in CLN3. In addition to identifying candidate proteins for CLN3 research, this study provides a comparison of two large-scale proteomic discovery methods in CSF.

Transcriptome of HPßCD-treated Niemann-Pick disease type C1 cells highlights GPNMB as a biomarker for therapeutics.

The rare, fatal neurodegenerative disorder Niemann-Pick disease type C1 (NPC1) arises from lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. The timing and severity of NPC1 clinical presentation is extremely heterogeneous. This study analyzed RNA-Seq data from 42 NPC1 patient-derived, primary fibroblast cell lines to determine transcriptional changes induced by treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a compound currently under investigation in clinical trials. A total of 485 HPßCD-responsive genes were identified. Pathway enrichment analysis of these genes showed significant involvement in cholesterol and lipid biosynthesis. Furthermore, immunohistochemistry of the cerebellum as well as measurements of plasma from Npc1m1N null mice treated with HPßCD and adeno-associated virus gene therapy suggests that one of the identified genes, GPNMB, may serve as a useful biomarker of treatment response in NPC1 disease. Overall, this large NPC1 patient-derived dataset provides a comprehensive foundation for understanding the genomic response to HPßCD treatment.

Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin organization.

The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human ß-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the ß-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

Role of LDB1 in the transition from chromatin looping to transcription activation.

Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1) protein is recruited to the ß-globin locus via LMO2 and is required for looping of the ß-globin locus control region (LCR) to the active ß-globin promoter. We show that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2, sufficient to completely restore LCR-promoter looping and transcription in LDB1-depleted cells. The looping function of the DD is unique and irreplaceable by heterologous DDs. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the coregulators FOG1 and the nucleosome remodeling and deacetylating (NuRD) complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior, as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1 in these processes.

The zinc-finger protein CLAMP promotes gypsy chromatin insulator function in Drosophila.

Chromatin insulators are DNA-protein complexes that establish independent higher-order DNA domains to influence transcription. Insulators are functionally defined by two properties: they can block communication between an enhancer and a promoter, and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is knocked down, gypsy-dependent enhancer-blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap, particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, whereas depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.

Shep regulates Drosophila neuronal remodeling by controlling transcription of its chromatin targets.

Neuronal remodeling is crucial for formation of the mature nervous system and disruption of this process can lead to neuropsychiatric diseases. Global gene expression changes in neurons during remodeling as well as the factors that regulate these changes remain poorly defined. To elucidate this process, we performed RNA-seq on isolated Drosophila larval and pupal neurons and found upregulated synaptic signaling and downregulated gene expression regulators as a result of normal neuronal metamorphosis. We further tested the role of alan shepard (shep), which encodes an evolutionarily conserved RNA-binding protein required for proper neuronal remodeling. Depletion of shep in neurons prevents the execution of metamorphic gene expression patterns, and shep-regulated genes correspond to Shep chromatin and/or RNA-binding targets. Reduced expression of a Shep-inhibited target gene that we identified, brat, is sufficient to rescue neuronal remodeling defects of shep knockdown flies. Our results reveal direct regulation of transcriptional programs by Shep to regulate neuronal remodeling during metamorphosis.

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology.

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.

A compendium of RNA-binding motifs for decoding gene regulation.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function.

A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.

Identification of Novel Pathways Associated with Patterned Cerebellar Purkinje Neuron Degeneration in Niemann-Pick Disease, Type C1.

: Niemann-Pick disease, type C1 (NPC1) is a lysosomal disease characterized by progressive cerebellar ataxia. In NPC1, a defect in cholesterol transport leads to endolysosomal storage of cholesterol and decreased cholesterol bioavailability. Purkinje neurons are sensitive to the loss of NPC1 function. However, degeneration of Purkinje neurons is not uniform. They are typically lost in an anterior-to-posterior gradient with neurons in lobule X being resistant to neurodegeneration. To gain mechanistic insight into factors that protect or potentiate Purkinje neuron loss, we compared RNA expression in cerebellar lobules III, VI, and X from control and mutant mice. An unexpected finding was that the gene expression differences between lobules III/VI and X were more pronounced than those observed between mutant and control mice. Functional analysis of genes with anterior to posterior gene expression differences revealed an enrichment of genes related to neuronal cell survival within the posterior cerebellum. This finding is consistent with the observation, in multiple diseases, that posterior Purkinje neurons are, in general, resistant to neurodegeneration. To our knowledge, this is the first study to evaluate anterior to posterior transcriptome-wide changes in gene expression in the cerebellum. Our data can be used to not only explore potential pathological mechanisms in NPC1, but also to further understand cerebellar biology.

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner.

Chromatin insulators are DNA-protein complexes that are situated throughout the genome that are proposed to contribute to higher-order organization and demarcation into distinct transcriptional domains. Mounting evidence in different species implicates RNA and RNA-binding proteins as regulators of chromatin insulator activities. Here, we identify the Drosophila hnRNP M homolog Rumpelstiltskin (Rump) as an antagonist of gypsy chromatin insulator enhancer-blocking and barrier activities. Despite ubiquitous expression of Rump, decreasing Rump levels leads to improvement of barrier activity only in tissues outside of the central nervous system (CNS). Furthermore, rump mutants restore insulator body localization in an insulator mutant background only in non-CNS tissues. Rump associates physically with core gypsy insulator proteins, and chromatin immunoprecipitation and sequencing analysis of Rump demonstrates extensive colocalization with a subset of insulator sites across the genome. The genome-wide binding profile and tissue specificity of Rump contrast with that of Shep, a recently identified RNA-binding protein that antagonizes gypsy insulator activity primarily in the CNS. Our findings indicate parallel roles for RNA-binding proteins in mediating tissue-specific regulation of chromatin insulator activity.

metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA.

Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA-protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP- and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep associates with chromatin cotranscriptionally, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity.

Messenger RNA is a functional component of a chromatin insulator complex.

Chromatin insulators are DNA protein complexes situated throughout the genome capable of demarcating independent transcriptional domains. Previous studies point to an important role for RNA in gypsy chromatin insulator function in Drosophila; however, the identity of these putative insulator-associated RNAs is not currently known. Here we utilize RNA-immunoprecipitation and high throughput sequencing (RIP-seq) to isolate RNAs stably associated with gypsy insulator complexes. Strikingly, these RNAs correspond to specific sense-strand, spliced and polyadenylated mRNAs, including two insulator protein transcripts. In order to assess the functional significance of these associated mRNAs independent of their coding function, we expressed untranslatable versions of these transcripts in developing flies and observed both alteration of insulator complex nuclear localization as well as improvement of enhancer-blocking activity. Together, these data suggest a novel, noncoding mechanism by which certain mRNAs contribute to chromatin insulator function.

Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila.

Chromatin insulators are functionally conserved DNA-protein complexes situated throughout the genome that organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the precise mechanisms are not yet understood. Here we identify the exosome, the highly conserved major cellular 3' to 5' RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. Genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32 and CTCF insulator proteins. Colocalization occurs mainly at promoters but also boundary elements such as Mcp, Fab-8, scs and scs', which overlaps with a promoter. Surprisingly, exosome associates primarily with promoters but not gene bodies of active genes, arguing against simple cotranscriptional recruitment to RNA substrates. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is required specifically for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel positive relationship between exosome and chromatin insulators throughout the genome.

Tissue-specific regulation of chromatin insulator function.

Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type-specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP-seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.

CTCF-mediated transcriptional regulation through cell type-specific chromosome organization in the ß-globin locus.

The principles underlying the architectural landscape of chromatin beyond the nucleosome level in living cells remains largely unknown despite its potential to play a role in mammalian gene regulation. We investigated the three-dimensional folding of a 1 Mbp region of human chromosome 11 containing the ß-globin genes by integrating looping interactions of the CCCTC-binding insulator protein CTCF determined comprehensively by chromosome conformation capture (3C) into a polymer model of chromatin. We find that CTCF-mediated cell type-specific interactions in erythroid cells are organized to favor contacts known to occur in vivo between the ß-globin locus control region (LCR) and genes. In these cells, the modeled ß-globin domain folds into a globule with the LCR and the active globin genes on the periphery. In contrast, in non-erythroid cells, the globule is less compact with few but dominant CTCF interactions driving the genes away from the LCR. This leads to a decrease in contact frequencies that can exceed 1000-fold depending on the stiffness of the chromatin and the exact position of the genes. Our findings show that an ensemble of CTCF contacts functionally affects spatial distances between control elements and target genes contributing to chromosomal organization required for transcription.

Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles.

Introduction: Enhancer of zeste homolog 2 (Ezh2) is responsible for trimethylation of histone 3 at lysine 27 (H3K27me3), resulting in repression of gene expression. Here, we explore the role of Ezh2 in forebrain GABAergic interneuron development. Methods: We removed Ezh2 in the MGE by generating Nkx2-1Cre;Ezh2 conditional knockout mice. We then characterized changes in MGE-derived interneuron fate and electrophysiological properties in juvenile mice, as well as alterations in gene expression, chromatin accessibility and histone modifications in the MGE. Results: Loss of Ezh2 increases somatostatin-expressing (SST+) and decreases parvalbumin-expressing (PV+) interneurons in the forebrain. We observe fewer MGE-derived interneurons in the first postnatal week, indicating reduced interneuron production. Intrinsic electrophysiological properties in SST+ and PV+ interneurons are normal, but PV+ interneurons display increased axonal complexity in Ezh2 mutant mice. Single nuclei multiome analysis revealed differential gene expression patterns in the embryonic MGE that are predictive of these cell fate changes. Lastly, CUT&Tag analysis revealed that some genomic loci are particularly resistant or susceptible to shifts in H3K27me3 levels in the absence of Ezh2, indicating differential selectivity to epigenetic perturbation. Discussion: Thus, loss of Ezh2 in the MGE alters interneuron fate, morphology, and gene expression and regulation. These findings have important implications for both normal development and potentially in disease etiologies.

Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration.

In neurons, RNA granules are transported along the axon for local translation away from the soma. Recent studies indicate that some of this transport involves hitchhiking of RNA granules on lysosome-related vesicles. In the present study, we leveraged the ability to prevent transport of these vesicles into the axon by knockout of the lysosome-kinesin adaptor BLOC-one-related complex (BORC) to identify a subset of axonal mRNAs that depend on lysosome-related vesicles for transport. We found that BORC knockout causes depletion of a large group of axonal mRNAs mainly encoding ribosomal and mitochondrial/oxidative phosphorylation proteins. This depletion results in mitochondrial defects and eventually leads to axonal degeneration in human induced pluripotent stem cell (iPSC)-derived and mouse neurons. Pathway analyses of the depleted mRNAs revealed a mechanistic connection of BORC deficiency with common neurodegenerative disorders. These results demonstrate that mRNA transport on lysosome-related vesicles is critical for the maintenance of axonal homeostasis and that its failure causes axonal degeneration.