LINE-1 specific nuclear organization in mice olfactory sensory neurons☆
Graphical abstract
LINE-1 copies are aggregated around central constitutive heterochromatin block and frequently occupy the same position as facultative heterochromatin mark in the nuclei of mice olfactory epithelium neurons. Even though LINE-1 copies are transcribed in olfactory epithelium, they are enriched for heterochromatin marks at olfactory receptor gene loci.
Introduction
Long Interspersed Elements 1 (LINE-1) is the major class of interspersed repeats in mammalian nuclei, comprising almost 20% of the mouse genome (Waterston et al., 2002). LINE-1 copies lack long terminal repeats and are classified as autonomous retrotransposons since they require an RNA intermediate to duplicate and encode the proteins necessary for their retrotransposition (Kazazian, 2004). An intact LINE-1 copy has 6 Kb in length, consisting basically of a 5′ internal promoter, two open reading frames (ORFs), and a 3′ termini polyadenylate sequence (Kazazian, 2004). ORF1 encodes a nucleic acid-binding protein and ORF2 an endonuclease with reverse transcriptase activity (Dombroski et al., 1994; Feng et al., 1996; Hohjoh and Singer, 1996; Mathias et al., 1991). A full-length LINE-1 RNA may be translated in the cytoplasm and assembled with its encoded proteins, moving back to the nucleus where ORF2 protein reinserts a new LINE-1 copy in the genome. LINE-1 activity has been implicated in the transcriptional regulation of nearby genes (Faulkner et al., 2009) and facultative heterochromatin deposition during X chromosome silencing (Chow et al., 2010). But LINE-1 new insertions in the genome may cause rupture of coding and regulatory sequences, therefore these elements are silenced by DNA methylation and heterochromatin (Rangasamy, 2013; Smith et al., 2012; Zamudio et al., 2015). Interestingly, there are allelic variants at the promoter region of some human LINE-1 copies which affect a conserved Yin Yang 1 (YY1) transcription factor binding site and impact DNA methylation and silencing of these elements in pluripotent and differentiated cells (Sanchez-Luque et al., 2019). These LINE-1 variants may be involved in retrotransposition during hippocampal neuron differentiation, contributing to somatic mosaicism (Sanchez-Luque et al., 2019).
Even though many LINE-1 copies are capable of transcription, the majority of them are transpositionally incompetent due to mutations and truncations in their sequences (Sanchez-Luque et al., 2019; Hardies et al., 1986). Successive waves of LINE-1 expansion and decline have shaped mammalian genomes, therefore these elements are classified in different subfamilies (Sookdeo et al., 2013). Subfamilies L1Md_A, L1Md_T, L1Md_Gf, and L1Md_F have recently evolved in the murine genome and their copies are currently active in mice (Chow et al., 2010; Goodier et al., 2001; Naas et al., 1998).
In the last decades, retrotransposons were upgraded from junk DNA to functional genome elements, and much progress has been made to understand their profound impact in mammalian genome (Faulkner and Billon, 2018; Fadloun et al., 2013; Faulkner et al., 2009; Goodier and Kazazian, 2008; Hurst and Werren, 2001). Recently, their activity has been associated with neuronal diversity in the central nervous system (Sanchez-Luque et al., 2019; Bedrosian et al., 2018; Bundo et al., 2014; Erwin et al., 2014; Gage and Muotri, 2012; Muotri et al., 2005).
Younger and longer LINE-1 copies are significantly enriched in the olfactory receptor (OR) gene loci in humans and mice (Allen et al., 2003; Kambere and Lane, 2009). It was suggested that this LINE-1 enrichment could be involved in the transcriptional regulation of these receptors (Allen et al., 2003; Kambere and Lane, 2009).
OR genes are members of one of the largest gene family in mammals, with thousands of alleles distributed in almost all mouse chromosomes (Godfrey et al., 2004). But each mature olfactory sensory neuron (OSN) must express only a single allele from a single OR gene in order to achieve the correct axon coalescence with central nervous system, leading to successful odorant recognition (Chess et al., 1994; Feinstein and Mombaerts, 2004; Hanchate et al., 2015). Recently, it was shown that OR genes monogenic and monoallelic expression depend not only on epigenetic silencing (Magklara et al., 2011) but also on the OSN heterochromatin organization (Clowney et al., 2012). OSNs present a peculiar nuclear architecture where OR genes concentrate around a large central constitutive heterochromatin block, enriched for H3K9me3, surrounded by a domain of facultative heterochromatin, enriched for H3K27me3 (Armelin-Correa et al., 2014a; Clowney et al., 2012; Tan et al., 2019). Therefore, OSNs have an ‘inside-out’ chromatin configuration where heterochromatin concentrates in a central block, in contrast to the conventional nuclear organization, where heterochromatin is localized in the nuclear periphery (Le Gros et al., 2016; Tan et al., 2019).
OSNs are the majority of cells in the adult mouse olfactory epithelium (OE), a pseudostratified neuroepithelium that requires continuous neurogenesis due to its direct contact with the external environment (Leung et al., 2007; Schwob, 2002). Horizontal basal cells (HBCs), which are localized in contact with the epithelial basal lamina, are capable of repopulating the entire epithelium in the case of profound lesions (Leung et al., 2007). The globose basal cells (GBCs) reside just above HBCs and are self-renewing progenitor cells (GBCprog) (Calof et al., 2002; Leung et al., 2007). HBCs and GBCs compose the basal cell layer in OE. GBCprogs give rise to neuronal precursors cells (GBCprec) which migrate radially in OE and differentiate in immature neurons (OSNim). OSNim express the growth-associated protein 43 (Gap43) (Verhaagen et al., 1989). As OSNim differentiation advances, Gap43 is downregulated and the expression of the transduction molecules downstream the odorant receptors gradually begins (Hanchate et al., 2015; Rodriguez-Gil et al., 2015). At the end of the process, the mature olfactory sensory neuron (OSNm) has acquired the full repertoire of molecules required for OR signaling transduction and express the olfactory marker protein (Omp) (Farbman and Margolis, 1980; Hanchate et al., 2015). GBC can also differentiate in glial-like sustentacular cells (SUS), with cell bodies populating the OE apical layer and cell processes extending from the surface to the basal lamina of the epithelium (Carr et al., 1991; Leung et al., 2007).
Despite significant advances in understanding the role of retrotransposons activity during neuronal differentiation (Bedrosian et al., 2018; Bundo et al., 2014; Coufal et al., 2011; Coufal et al., 2009; Gage and Muotri, 2012; Muotri et al., 2005), little is known about LINE-1 nuclear organization in neurons (Solovei et al., 2009). Olfactory neurons have a striking inverted nuclear architecture leading to OR genes silencing and the mechanisms involved in this organization are not fully understood (Alexander and Lomvardas, 2014; Armelin-Correa et al., 2014a; Armelin-Correa et al., 2014b; Clowney et al., 2012; Leung et al., 2007). Herein we demonstrate, using Immuno DNA FISH, that LINE-1 copies are organized and concentrated in a large cluster around the central constitutive heterochromatin block only in OSN nuclei. Also, these LINE-1 clusters partially colocalize with the facultative heterochromatin in OSNs, and this peculiar organization cannot be detected in basal or sustentacular cells. Analysis of ChIP-seq data using antibodies against H3K27me3 and H3K9me3 revealed that LINE-1 copies located at different OR gene loci present different patterns of heterochromatin marks enrichment in the olfactory epithelium. Finally, we show that LINE-1 retrotransposons are transcribed not only in OE but also in the vomeronasal epithelium (VNE), another neuroepithelium member of the olfactory system. Our results suggest that LINE-1 copies are transcribed in olfactory epithelium and that these retrotransposons may participate in the organization of OSN specific nuclear architecture which is critical for the appropriate OR gene expression in these neurons.
Section snippets
Animals
All the experiments were performed with biological material from male C57BL/6 mice with 21 days of birth, provided by the Center for Development of Experimental Models for Medicine and Biology (CEDEME) at the Federal University of Sao Paulo (UNIFESP). We have used only male mice in this study because it was described that in the nuclei of female mice cells LINE-1 transcription participates in X chromosome inactivation (Chow et al., 2010). The animals were kept in plastic cages under a 12/12 h
LINE-1 are concentrate around the constitutive heterochromatin block in olfactory neurons nuclei
Due to the enrichment of LINE-1 copies in OR gene loci (Allen et al., 2003; Kambere and Lane, 2009), we decided to describe the distribution of these retrotransposons in the nuclei of OSNs using DNA FISH probes previously employed in mouse retina (Solovei et al., 2009). OSNs present an inverted chromatin distribution, showing a large central constitutive heterochromatin block in the nucleus (Armelin-Correa et al., 2014a, Armelin-Correa et al., 2014b). This central nuclear constitutive
Discussion
This study is the first to examine LINE-1 nuclear organization in mouse olfactory epithelium cells. LINE-1 is the most abundant interspersed repeat in the mammalian genome, comprising approximately 20% of its DNA content (Ostertag and Kazazian, 2001). There are around 600.000 LINE-1 copies in the mouse genome, but just 5% of these copies are capable of retrotransposition (DeBerardinis et al., 1998; Goodier et al., 2001; Naas et al., 1998). Even though most of the copies have lost their capacity
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
CRediT authorship contribution statement
Leonardo Fontoura Ormundo:Investigation, Formal analysis.Cleiton Fagundes Machado:Investigation, Formal analysis, Writing - original draft, Writing - review & editing.Erika Demasceno Sakamoto:Investigation.Viviane Simões da Silva:Investigation.Lucia Armelin-Correa:Funding acquisition, Project administration, Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing.
Declaration of competing interest
None.
References (85)
- et al.
Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells
Mol. Cell
(2014) - et al.
Increased l1 retrotransposition in the neuronal genome in schizophrenia
Neuron
(2014) - et al.
Identification of a new non-neuronal cell type in rat olfactory epithelium
Neuroscience
(1991) - et al.
Allelic inactivation regulates olfactory receptor gene expression
Cell
(1994) - et al.
LINE-1 activity in facultative heterochromatin formation during X chromosome inactivation
Cell
(2010) - et al.
Nuclear aggregation of olfactory receptor genes governs their monogenic expression
Cell
(2012) - et al.
Dnmt3a regulates global gene expression in olfactory sensory neurons and enables odorant-induced transcription
Neuron
(2014) - et al.
Olfactory marker protein during ontogeny: immunohistochemical localization
Dev. Biol.
(1980) - et al.
A contextual model for axonal sorting into glomeruli in the mouse olfactory system
Cell
(2004) - et al.
Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition
Cell
(1996)
Local and cis effects of the H element on expression of odorant receptor genes in mouse
Cell
Retrotransposons revisited: the restraint and rehabilitation of parasites
Cell
Soft X-ray tomography reveals gradual chromatin compaction and reorganization during neurogenesis in vivo
Cell Rep.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method
Methods
Heterochromatin-mediated gene silencing facilitates the diversification of olfactory neurons
Cell Rep.
An epigenetic signature for monoallelic olfactory receptor expression
Cell
Enhancer interaction networks as a means for singular olfactory receptor expression
Cell
A multigene family encoding a diverse array of putative pheromone receptors in mammals
Cell
How retrotransposons shape genome regulation
Curr. Opin. Gen. Dev.
Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution
Cell
Nuclear architecture as an epigenetic regulator of neural development and function
Neuroscience
High concentrations of long interspersed nuclear element sequence distinguish monoallelically expressed genes
Proc. Natl. Acad. Sci. U. S. A.
Nuclear compartmentalization of odorant receptor genes
Proc. Natl. Acad. Sci. U. S. A.
Nuclear architecture and gene silencing in olfactory sensory neurons
Bioarchitecture
Early life experience drives structural variation of neural genomes in mice
Science
Somatic expression of LINE-1 elements in human tissues
Nucleic Acids Res.
The evolution of LINE-1 in vertebrates
Genome Biol. Evol.
Developmental and cell type specificity of LINE-1 expression in mouse testis: implications for transposition
Mol. Cell. Biol.
Progenitor cells of the olfactory receptor neuron lineage
Microsc. Res. Tech.
L1 retrotransposition in human neural progenitor cells
Nature
Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells
Proc. Natl. Acad. Sci. U. S. A.
Rapid amplification of a retrotransposon subfamily is evolving the mouse genome
Nat. Genet.
An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae
Mol. Cell. Biol.
Mobile DNA elements in the generation of diversity and complexity in the brain
Nat. Rev. Neurosci.
L1-associated genomic regions are deleted in somatic cells of the healthy human brain
Nat. Neurosci.
Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA
Nat. Struct. Mol. Biol.
Heterochromatin drives compartmentalization of inverted and conventional nuclei
Nature
L1 retrotransposition in the soma: a field jumping ahead
Mob. DNA
The regulated retrotransposon transcriptome of mammalian cells
Nat. Genet.
What makes each brain unique
Sci. Am.
The mouse olfactory receptor gene family
Proc. Natl. Acad. Sci. U. S. A.
A novel active L1 retrotransposon subfamily in the mouse
Genome Res.
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Acknowledgments: We thank Dr. Taiza Stumpp and Dr. Bettina Malnic for helpful comments and suggestions; and all members of the Molecular Neuroscience Laboratory (IQ-USP) and Developmental Biology Laboratory (EPM-UNIFESP) for suggestions and support with reagents and laboratory equipment. We also thank Dr. Alexandre Bruni for support with the microscope. We are in debt with Victor Pereira de Sa Xavier for skillful computer and data assistance.
Grant information: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) and by grant #2016/07782-2, São Paulo Research Foundation (FAPESP) Brazil.
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These authors had equal contribution to this work.