Biology subjects

Hatten, M. E.

Publications and source records attributed to Hatten, M. E..

13 recordsLinked to original sources

bioRxiv2026

Long-term maintenance of H3K27me3 in postmitotic neurons is dispensable for gene expression regulation

Neuronal maturation is associated with extensive changes in gene expression and chromatin organization. However, the molecular mechanisms that control the epigenetic landscape in terminally differentiated neurons remain poorly understood. Here, we show that maturing cerebellar granule cells undergo a striking and specific increase in the levels of the repressive histone modification H3K27me3 across different genomic regions, including individual genes, broad intergenic regions, and gene clusters. The accumulation of H3K27me3 coincides with a developmental switch from EZH2 to EZH1 and colocalizes with H3K36me2 and DNA non-CpG methylation. Using mice with a conditional deletion in the catalytic domain of EZH1, we demonstrate that the maintenance of H3K27me3 in mature neurons depends on EZH1. Unexpectedly, an almost complete loss of H3K27me3 in postmitotic GCs induces minimal changes in gene expression and chromatin accessibility at 7 months of age. Using single-nucleus RNA sequencing (snRNAseq) from the mouse neocortex, we show that, similarly to GCs, the loss of EZH1-mediated H3K27me3 also has a minimal impact on cortical neuron gene expression. The amino acid composition of EZH1 suggests reduced sensitivity to H3K36 methylation, providing a potential basis for its activity in chromatin contexts that are not permissive for EZH2. Together, our results show that a postmitotic switch from EZH2 to EZH1 establishes novel chromatin domains in neurons with a minimal role in transcriptional maintenance.

molecular biology↗

bioRxiv2024

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2{Delta}ex3-4;Trp53-/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

cancer biology↗

bioRxiv2024

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. We recently reported a family with a paternally inherited intragenic ASTN2 duplication with a range of neurodevelopmental disorders, including autism spectrum disorder (ASD), learning difficulties, and speech and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity and repetitive behaviors, altered social behaviors, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors were also prominent in Astn2 cKO animals. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrates a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

neuroscience↗

bioRxiv2023

Altered chromatin occupancy of patient-associated H4 mutants misregulate neuronal differentiation

Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.

molecular biology↗

bioRxiv2023

SEL1L-HRD1 ER-associated degradation is a new ataxia gene

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD); however, definitive evidence for the importance of SEL1L in HRD1 ERAD is lacking. Here we report that attenuation of the interaction between SEL1L and HRD1 impairs HRD1 ERAD function and has pathological consequences in mice. Our data show that SEL1L variant p.Ser658Pro (SEL1LS658P) previously identified in Finnish Hound suffering cerebellar ataxia is a recessive hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Mechanistically, SEL1LS658P variant attenuates the SEL1L-HRD1 interaction and causes HRD1 dysfunction by generating electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes revealed that the SEL1L-HRD1 interaction is prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L recruits not only the lectins OS9 and ERLEC1, but the E2 UBE2J1 and retrotranslocon DERLIN, to HRD1. These data underscore the pathophysiological importance and disease relevance of the SEL1L-HRD1 complex, and identify a key step in organizing the HRD1 ERAD complex.

cell biology↗

bioRxiv2023

Histone bivalency regulates the timing of cerebellar granule cell development

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

neuroscience↗

bioRxiv2021

Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, the cerebellum is now recognized to contribute to human cognitive evolution having a broad range of non-motor cognitive functions in addition to motor control. Emerging studies highlight the complexity of human cerebellar histogenesis, compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. Here we report a rapid and simple protocol for the directed derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC), and strategies to decrease culture variability; a common limitation in hPSC studies. Upon transplantation into juvenile mice, early postmitotic hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification (TRAP)-seq, the ATOH1 lineage most closely resembled human cerebellar tissue in the second trimester. Unexpectedly, TRAP-seq identified a heterochronic shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, within granule cell progenitors (GCPs) in the human external granule layer. This molecular divergence may provide the mechanism by which the GCP pool persists into year two post birth in humans, but only lasts for two weeks in mice. Our approach provides a scalable in vitro model of the human ATOH1 lineage that yields cerebellar granule cells within 48 days as well as a strategy for identifying uniquely human cellular and molecular characteristics.

developmental biology↗

bioRxiv2020

Novel genetic features of human and mouse Purkinje cell differentiation defined by comparative transcriptomics.

Comparative transcriptomics between differentiating human pluripotent stem cells (hPSC) and developing mouse neurons offers a powerful approach to compare genetic and epigenetic pathways in human and mouse neurons. To analyze human Purkinje cell (PC) differentiation, we optimized a protocol to generate hPSC-PCs that formed synapses when cultured with mouse cerebellar glia and granule cells and fired large calcium currents, measured with the genetically encoded calcium indicator jRGECO1a. To directly compare global gene expression of hPSC-PCs with developing mouse PCs, we used translating ribosomal affinity purification (TRAP). As a first step, we used Tg(Pcp2-L10a-Egfp) TRAP mice to profile actively transcribed genes in developing postnatal mouse PCs, and used metagene projection to identify the most salient patterns of PC gene expression over time. We then created a transgenic Pcp2-L10a-Egfp TRAP hESC line to profile gene expression in differentiating hPSC-PCs, finding that the key gene expression pathways of differentiated hPSC-PCs most closely matched those of late juvenile, mouse PCs (P21). Comparative bioinformatics identified classical PC gene signatures as well as novel mitochondrial and autophagy gene pathways during the differentiation of both mouse and human PCs. In addition, we identified genes expressed in hPSC-PCs but not mouse PCs and confirmed protein expression of a novel human PC gene, CD40LG, expressed in both hPSC-PCs and native human cerebellar tissue. This study therefore provides the first direct comparison of hPSC-PC and mouse PC gene expression and a robust method for generating differentiated hPSC-PCs with human-specific gene expression for modeling developmental and degenerative cerebellar disorders. Significance StatementTo compare global gene expression features of differentiating human pluripotent stem cell-derived Purkinje cells (hPSC-PC) and developing mouse Purkinje cells (PC) we derived hPSC-PCs and compared gene expression datasets from human and mouse PCs. We optimized a differentiation protocol that generated hPSC-PCs most similar in gene expression to mouse P21 PCs. Metagene projection analysis of mouse PC gene expression over postnatal development identified both classical PC marker genes as well as novel mitochondrial and autophagy gene pathways. These key gene expression patterns were conserved in differentiating hPSC-PCs. We further identified differences in timing and expression of key gene sets between mouse and hPSC-PCs and confirmed expression of a novel human PC marker, CD40LG, in human cerebellar tissue.

developmental biology↗

bioRxiv2019

The proteasome regulator PI31 is required for protein homeostasis, synapse maintenance and neuronal survival in mice

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 26S assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse CNS suggests a key role for PI31 in human developmental neurological disorders.\n\nStatement of SignificanceThe conserved proteasome-binding protein PI31 serves as an adapter to couple proteasomes with cellular motors to mediate their transport to distal tips of neurons where protein breakdown occurs. We generated global and conditional PI31 knockout mouse strains and show that this protein is required for protein homeostasis, and that its conditional inactivation in neurons disrupts synaptic structures and long-term survival. This work establishes a critical role for PI31 and local protein degradation in the maintenance of neuronal architecture, circuitry and function. Because mutations that impair PI31 function cause neurodegenerative diseases in humans, reduced PI31 activity may contribute to age-related neurodegenerative diseases.

neuroscience↗

bioRxiv2018

Astrotactins 1 and 2 have similar membrane topology and mature via endoproteolytic cleavage catalyzed by signal peptidase

Astrotactins 1 and 2 (Astn1, Astn2) are membrane proteins that function in glial-guided migration, receptor trafficking and synaptic plasticity in brain, as well as in planar polarity pathways in skin. Here, we have mapped the topologies of mouse Astn1 and Astn2 in rough microsomal membranes (RMs) using glycosylation mapping and protease-protection assays, and find that Astn2 has a cleaved N-terminal signal peptide (SP), an N-terminal domain located in the lumen of the RMs (topologically equivalent to the extracellular surface in cells), two transmembrane helices (TMHs), and a large C-terminal lumenal domain. We find that Astn1 has the same topology as Astn2 but see no evidence of SP cleavage. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second TMH; importantly, we identify the endoprotease responsible for the maturation of Astn1 and Astn2 as signal peptidase. Differences in the degree of the maturation of Astn1 and Astn2 possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on the CNS neuronal membranes and to the different functions of Astn1 and Astn2.

neuroscience↗

bioRxiv2018

N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

Prior studies demonstrate that Astrotactin (ASTN1) provides a neuronal receptor for glial-guided CNS migration. Here we report that ASTN1 binds N-cadherin (CDH2) and that the ASTN1:CDH2 interaction supports cell-cell adhesion. To test the function of ASTN1:CDH2 binding in glial-guided neuronal migration, we generated a conditional loss of Cdh2 in cerebellar granule cells and in glia. Granule cell migration was slowed in cerebellar slice cultures after a conditional loss of neuronal Cdh2, and more severe migration defects occurred after a conditional loss of glial Cdh2. Expression of a mutant form of ASTN1 that does not bind CDH2, in granule cells, also slowed migration. Moreover, in vitro chimeras of granule cells and glia showed impaired neuron-glia attachment in the absence of glial, but not neuronal, Cdh2. Thus, cis and trans bindings of ASTN1 to neuronal and glial CDH2 form an asymmetric neuron-glial bridge complex that promotes glial-guided neuronal migration.

developmental biology↗

bioRxiv2018

ASTN2 modulates synaptic strength by trafficking and degradation of surface proteins

Surface protein dynamics dictate synaptic connectivity and function in neuronal circuits. ASTN2, a gene disrupted by copy number variations (CNVs) in neurodevelopmental disorders, including autism spectrum, was previously shown to regulate the surface expression of ASTN1 in glial-guided neuronal migration. Here, we demonstrate that ASTN2 binds to and regulates the surface expression of multiple synaptic proteins in post-migratory neurons by endocytosis, resulting in modulation of synaptic activity. In cerebellar Purkinje cells (PCs), by immuno-gold electron microscopy, ASTN2 localizes primarily to endocytic and autophagocytic vesicles in the cell soma and in subsets of dendritic spines. Overexpression of ASTN2 in PCs, but not of ASTN2 lacking the FNIII-domain commonly disrupted by CNVs in patients including in a family presented here, increases inhibitory and excitatory postsynaptic activity and reduces levels of ASTN2 binding partners. Our data suggest a fundamental role for ASTN2 in dynamic regulation of surface proteins by endocytic trafficking and protein degradation.

neuroscience↗

bioRxiv2018

Differential 3’ Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.

neuroscience↗