RESUMO
The hypothalamus regulates innate social behaviors, including mating and aggression. These behaviors can be evoked by optogenetic stimulation of specific neuronal subpopulations within MPOA and VMHvl, respectively. Here, we perform dynamical systems modeling of population neuronal activity in these nuclei during social behaviors. In VMHvl, unsupervised analysis identified a dominant dimension of neural activity with a large time constant (>50 s), generating an approximate line attractor in neural state space. Progression of the neural trajectory along this attractor was correlated with an escalation of agonistic behavior, suggesting that it may encode a scalable state of aggressiveness. Consistent with this, individual differences in the magnitude of the integration dimension time constant were strongly correlated with differences in aggressiveness. In contrast, approximate line attractors were not observed in MPOA during mating; instead, neurons with fast dynamics were tuned to specific actions. Thus, different hypothalamic nuclei employ distinct neural population codes to represent similar social behaviors.
Assuntos
Comportamento Sexual Animal , Núcleo Hipotalâmico Ventromedial , Animais , Comportamento Sexual Animal/fisiologia , Núcleo Hipotalâmico Ventromedial/fisiologia , Hipotálamo/fisiologia , Agressão/fisiologia , Comportamento SocialRESUMO
Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
Assuntos
Alelos , Proteína 9 Associada à CRISPR/metabolismo , Cromossomos Humanos/genética , Embrião de Mamíferos/metabolismo , Animais , Sequência de Bases , Blastocisto/metabolismo , Ciclo Celular/genética , Linhagem Celular , Deleção Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Implantação do Embrião/genética , Proteínas do Olho/genética , Fertilização , Edição de Genes , Rearranjo Gênico/genética , Loci Gênicos , Genoma Humano , Genótipo , Heterozigoto , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mutação INDEL/genética , Camundongos , Mitose , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3'-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.
Assuntos
Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Linhagem Celular , Endonucleases/genética , Endonucleases/metabolismo , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Genômica , Células HeLa , Humanos , Mutagênese Insercional/genéticaRESUMO
Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.
Assuntos
Gammaretrovirus/genética , Phascolarctidae/genética , RNA Interferente Pequeno/genética , Animais , Elementos de DNA Transponíveis , Gammaretrovirus/metabolismo , Gammaretrovirus/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Genoma , Células Germinativas/metabolismo , Células Germinativas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Phascolarctidae/virologia , Splicing de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.
Assuntos
Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Imunoprecipitação da Cromatina , Retrovirus Endógenos/genética , Feminino , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oligorribonucleotídeos Antissenso/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Interferência de RNA , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteína 28 com Motivo Tripartido/antagonistas & inibidores , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Regulação para Cima , NucleolinaRESUMO
Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/química , Elementos Nucleotídeos Longos e Dispersos , Proteínas Associadas à Matriz Nuclear/química , Poliadenilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteínas de Ligação a RNA/química , RNA/química , Processamento Alternativo , Animais , Sítios de Ligação , Éxons , Células HeLa , Humanos , Íntrons , Camundongos , Mutação , Motivos de Nucleotídeos , Filogenia , Ligação Proteica , Mapeamento de Interação de Proteínas , Splicing de RNARESUMO
LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Uridina/metabolismo , Animais , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , RNA Nucleotidiltransferases/antagonistas & inibidores , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Retroelementos/genéticaRESUMO
Deafness or hearing deficits are debilitating conditions. They are often caused by loss of sensory hair cells or defects in their function. In contrast to mammals, nonmammalian vertebrates robustly regenerate hair cells after injury. Studying the molecular and cellular basis of nonmammalian vertebrate hair cell regeneration provides valuable insights into developing cures for human deafness. In this review, we discuss the current literature on hair cell regeneration in the context of other models for sensory cell regeneration, such as the retina and the olfactory epithelium. This comparison reveals commonalities with, as well as differences between, the different regenerating systems, which begin to define a cellular and molecular blueprint of regeneration. In addition, we propose how new technical advances can address outstanding questions in the field.
Assuntos
Células-Tronco Adultas/metabolismo , Orelha Interna/metabolismo , Células Ciliadas Auditivas/fisiologia , Mucosa Olfatória/metabolismo , Regeneração/fisiologia , Retina/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Citocinas/metabolismo , Orelha Interna/citologia , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Regeneração/genética , Retina/citologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ferimentos e Lesões/genética , Ferimentos e Lesões/metabolismoRESUMO
The zebrafish visual system has become a paradigmatic preparation for behavioral and systems neuroscience. Around 40 types of retinal ganglion cells (RGCs) serve as matched filters for stimulus features, including light, optic flow, prey, and objects on a collision course. RGCs distribute their signals via axon collaterals to 12 retinorecipient areas in forebrain and midbrain. The major visuomotor hub, the optic tectum, harbors nine RGC input layers that combine information on multiple features. The retinotopic map in the tectum is locally adapted to visual scene statistics and visual subfield-specific behavioral demands. Tectal projections to premotor centers are topographically organized according to behavioral commands. The known connectivity in more than 20 processing streams allows us to dissect the cellular basis of elementary perceptual and cognitive functions. Visually evoked responses, such as prey capture or loom avoidance, are controlled by dedicated multistation pathways that-at least in the larva-resemble labeled lines. This architecture serves the neuronal code's purpose of driving adaptive behavior.
Assuntos
Células Ganglionares da Retina , Colículos Superiores , Vias Visuais , Peixe-Zebra , Animais , Vias Visuais/fisiologia , Peixe-Zebra/fisiologia , Células Ganglionares da Retina/fisiologia , Colículos Superiores/fisiologia , Percepção Visual/fisiologiaRESUMO
RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Íntrons , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , RNA de Cadeia Dupla , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Interferons/metabolismo , Interferons/genética , Animais , Células HEK293 , Camundongos , Transcriptoma , Éxons , Sítios de Splice de RNA , Elementos Alu/genéticaRESUMO
The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.
Assuntos
Inativação Gênica , Humanos , Células HEK293 , Histonas/metabolismo , Histonas/genética , Retroelementos/genética , Epigênese Genética , Elementos Nucleotídeos Longos e Dispersos/genética , Transdução de Sinais , Interferons/metabolismo , Interferons/imunologia , Interferons/genética , Células HeLaRESUMO
Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Proteínas de Ligação à Região de Interação com a Matriz , RNA Polimerase II , Receptores de Estrogênio , Transcrição Gênica , Humanos , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Regulação da Expressão Gênica , Ligação Proteica , Células HEK293 , Genoma HumanoRESUMO
In mammals, dosage compensation involves two parallel processes: (1) X inactivation, which equalizes X chromosome dosage between males and females, and (2) X hyperactivation, which upregulates the active X for X-autosome balance. The field currently favors models whereby dosage compensation initiates "de novo" during mouse development. Here, we develop "So-Smart-seq" to revisit the question and interrogate a comprehensive transcriptome including noncoding genes and repeats in mice. Intriguingly, de novo silencing pertains only to a subset of Xp genes. Evolutionarily older genes and repetitive elements demonstrate constitutive Xp silencing, adopt distinct signatures, and do not require Xist to initiate silencing. We trace Xp silencing backward in developmental time to meiotic sex chromosome inactivation in the male germ line and observe that Xm hyperactivation is timed to Xp silencing on a gene-by-gene basis. Thus, during the gamete-to-embryo transition, older Xp genes are transmitted in a "pre-inactivated" state. These findings have implications for the evolution of imprinting.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Feminino , Camundongos , Masculino , Animais , Inativação do Cromossomo X/genética , Impressão Genômica , Células Germinativas , Epigênese Genética , Embrião de Mamíferos , RNA Longo não Codificante/genética , Cromossomo X/genética , Mamíferos/genéticaRESUMO
Long interspersed element 1 (LINE-1) is the only protein-coding transposon that is active in humans. LINE-1 propagates in the genome using RNA intermediates via retrotransposition. This activity has resulted in LINE-1 sequences occupying approximately one-fifth of our genome. Although most copies of LINE-1 are immobile, â¼100 copies are retrotransposition-competent. Retrotransposition is normally limited via epigenetic silencing, DNA repair, and other host defense mechanisms. In contrast, LINE-1 overexpression and retrotransposition are hallmarks of cancers. Here, we review mechanisms of LINE-1 regulation and how LINE-1 may promote genetic heterogeneity in tumors. Finally, we discuss therapeutic strategies to exploit LINE-1 biology in cancers.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Neoplasias , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Neoplasias/genética , Neoplasias/terapia , RNA , Proteínas/genética , Epigênese GenéticaRESUMO
Transposable elements have created the majority of the sequence in many genomes. In mammals, LINE-1 retrotransposons have been expanding for more than 100 million years as distinct, consecutive lineages; however, the drivers of this recurrent lineage emergence and disappearance are unknown. Most human genome assemblies provide a record of this ancient evolution, but fail to resolve ongoing LINE-1 retrotranspositions. Utilizing the human CHM1 long-read-based haploid assembly, we identified and cloned all full-length, intact LINE-1s, and found 29 LINE-1s with measurable in vitro retrotransposition activity. Among individuals, these LINE-1s varied in their presence, their allelic sequences, and their activity. We found that recently retrotransposed LINE-1s tend to be active in vitro and polymorphic in the population relative to more ancient LINE-1s. However, some rare allelic forms of old LINE-1s retain activity, suggesting older lineages can persist longer than expected. Finally, in LINE-1s with in vitro activity and in vivo fitness, we identified mutations that may have increased replication in ancient genomes and may prove promising candidates for mechanistic investigations of the drivers of LINE-1 evolution and which LINE-1 sequences contribute to human disease.
Assuntos
Genoma Humano , Elementos Nucleotídeos Longos e Dispersos , Animais , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos , Mamíferos/genética , Mutação , Evolução MolecularRESUMO
Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.
Assuntos
Metilação de DNA , Elementos de DNA Transponíveis , DNA de Neoplasias , Epigênese Genética , Epigenoma , Regulação Neoplásica da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Sequenciamento por Nanoporos , Neoplasias , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de ÓrgãosRESUMO
p53 is a potent tumor suppressor and commonly mutated in human cancers. Recently, we demonstrated that p53 genes act to restrict retrotransposons in germline tissues of flies and fish but whether this activity is conserved in somatic human cells is not known. Here we show that p53 constitutively restrains human LINE1s by cooperatively engaging sites in the 5'UTR and stimulating local deposition of repressive histone marks at these transposons. Consistent with this, the elimination of p53 or the removal of corresponding binding sites in LINE1s, prompted these retroelements to become hyperactive. Concurrently, p53 loss instigated chromosomal rearrangements linked to LINE sequences and also provoked inflammatory programs that were dependent on reverse transcriptase produced from LINE1s. Taken together, our observations establish that p53 continuously operates at the LINE1 promoter to restrict autonomous copies of these mobile elements in human cells. Our results further suggest that constitutive restriction of these retroelements may help to explain tumor suppression encoded by p53, since erupting LINE1s produced acute oncogenic threats when p53 was absent.
Assuntos
Regulação da Expressão Gênica/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Linhagem Celular , Deleção de Genes , Rearranjo Gênico/genética , Código das Histonas/genética , Humanos , Imunidade/genética , Elementos Nucleotídeos Longos e Dispersos/imunologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína Supressora de Tumor p53/genéticaRESUMO
Despite being the predominant genetic elements in mammalian genomes, retrotransposons were often dismissed as genomic parasites with ambiguous biological significance. However, recent studies reveal their functional involvement in early embryogenesis, encompassing crucial processes such as zygotic genome activation (ZGA) and cell fate decision. This review underscores the paradigm shift in our understanding of retrotransposon roles during early preimplantation development, as well as their rich functional reservoir that is exploited by the host to provide cis-regulatory elements, noncoding RNAs, and functional proteins. The rapid advancement in long-read sequencing, low input multiomics profiling, advanced in vitro systems, and precise gene editing techniques encourages further dissection of retrotransposon functions that were once obscured by the intricacies of their genomic footprints.
Assuntos
Genoma , Retroelementos , Animais , Retroelementos/genética , Zigoto , Desenvolvimento Embrionário/genética , Mamíferos/genéticaRESUMO
Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.
Assuntos
Sistema da Linha Lateral , Peixe-Zebra , Animais , Peixe-Zebra/genética , Forma Celular , Animais Geneticamente Modificados , Células Ciliadas Auditivas/fisiologiaRESUMO
Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of â¼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.