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1.
Nat Commun ; 14(1): 4754, 2023 08 08.
Article in English | MEDLINE | ID: mdl-37553330

ABSTRACT

Stem cell survival versus death is a developmentally programmed process essential for morphogenesis, sizing, and quality control of genome integrity and cell fates. Cell death is pervasive during development, but its programming is little known. Here, we report that Smad nuclear interacting protein 1 (SNIP1) promotes neural progenitor cell survival and neurogenesis and is, therefore, integral to brain development. The SNIP1-depleted brain exhibits dysplasia with robust induction of caspase 9-dependent apoptosis. Mechanistically, SNIP1 regulates target genes that promote cell survival and neurogenesis, and its activities are influenced by TGFß and NFκB signaling pathways. Further, SNIP1 facilitates the genomic occupancy of Polycomb complex PRC2 and instructs H3K27me3 turnover at target genes. Depletion of PRC2 is sufficient to reduce apoptosis and brain dysplasia and to partially restore genetic programs in the SNIP1-depleted brain in vivo. These findings suggest a loci-specific regulation of PRC2 and H3K27 marks to toggle cell survival and death in the developing brain.


Subject(s)
Intracellular Signaling Peptides and Proteins , RNA-Binding Proteins , Humans , Signal Transduction/physiology , NF-kappa B , Hyperplasia , Brain
2.
bioRxiv ; 2023 May 04.
Article in English | MEDLINE | ID: mdl-37205560

ABSTRACT

53BP1 is a well-established DNA damage repair factor recently shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids. 53BP1-serine 25 phosphorylation dynamics controls 53BP1 target genes for neuronal differentiation and function, cellular response to stress, and apoptosis. Beyond 53BP1, ATM is required for phosphorylation of factors in neuronal differentiation, cytoskeleton, p53 regulation, and ATM, BNDF, and WNT signaling pathways for cortical organoid differentiation. Overall, our data suggest that 53BP1 and ATM control key genetic programs required for human cortical development.

4.
Methods Mol Biol ; 2599: 99-111, 2023.
Article in English | MEDLINE | ID: mdl-36427145

ABSTRACT

Cleavage under targets and release using nuclease (CUT & RUN) is an innovative method to profile histone modifications and chromatin-bound proteins genome-wide. CUT & RUN offers two distinct advantages of requiring much fewer cells and providing strong signal-to-noise ratios in deep-sequencing data. Here, we describe a workflow starting from dissociation and sorting of mouse embryonic brains, CUT & RUN, and DNA library preparation to deep sequencing. With our workflow, researchers can obtain high-quality sequencing data to profile histones and chromatin-associated proteins by using as few as 100,000 neural progenitor cells (NPCs).


Subject(s)
Chromatin , Neural Stem Cells , Mice , Animals , Chromatin/genetics , Endonucleases/genetics , Neural Stem Cells/metabolism , Histones/metabolism , Histone Code
5.
Proc Natl Acad Sci U S A ; 119(38): e2206147119, 2022 09 20.
Article in English | MEDLINE | ID: mdl-36095192

ABSTRACT

The neocortex, the center for higher brain function, first emerged in mammals and has become massively expanded and folded in humans, constituting almost half the volume of the human brain. Primary microcephaly, a developmental disorder in which the brain is smaller than normal at birth, results mainly from there being fewer neurons in the neocortex because of defects in neural progenitor cells (NPCs). Outer radial glia (oRGs), NPCs that are abundant in gyrencephalic species but rare in lissencephalic species, are thought to play key roles in the expansion and folding of the neocortex. However, how oRGs expand, whether they are necessary for neocortical folding, and whether defects in oRGs cause microcephaly remain important questions in the study of brain development, evolution, and disease. Here, we show that oRG expansion in mice, ferrets, and human cerebral organoids requires cyclin-dependent kinase 6 (CDK6), the mutation of which causes primary microcephaly via an unknown mechanism. In a mouse model in which increased Hedgehog signaling expands oRGs and intermediate progenitor cells and induces neocortical folding, CDK6 loss selectively decreased oRGs and abolished neocortical folding. Remarkably, this function of CDK6 in oRG expansion did not require its kinase activity, was not shared by the highly similar CDK4 and CDK2, and was disrupted by the mutation causing microcephaly. Therefore, our results indicate that CDK6 is conserved to promote oRG expansion, that oRGs are necessary for neocortical folding, and that defects in oRG expansion may cause primary microcephaly.


Subject(s)
Cyclin-Dependent Kinase 6 , Ependymoglial Cells , Microcephaly , Neocortex , Animals , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Ependymoglial Cells/cytology , Ependymoglial Cells/enzymology , Ferrets , Hedgehog Proteins/metabolism , Humans , Mice , Microcephaly/genetics , Neocortex/abnormalities , Neocortex/enzymology , Neural Stem Cells/cytology , Neural Stem Cells/enzymology , Organoids/embryology
6.
bioRxiv ; 2021 Aug 05.
Article in English | MEDLINE | ID: mdl-34075382

ABSTRACT

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans could cause coronavirus disease 2019 (COVID-19). Since its first discovery in Dec 2019, SARS-CoV-2 has become a global pandemic and caused 3.3 million direct/indirect deaths (2021 May). Amongst the scientific community's response to COVID-19, data sharing has emerged as an essential aspect of the combat against SARS-CoV-2. Despite the ever-growing studies about SARS-CoV-2 and COVID-19, to date, only a few databases were curated to enable access to gene expression data. Furthermore, these databases curated only a small set of data and do not provide easy access for investigators without computational skills to perform analyses. To fill this gap and advance open-access to the growing gene expression data on this deadly virus, we collected about 1,500 human bulk RNA-seq datasets from publicly available resources, developed a database and visualization tool, named CovidExpress (https://stjudecab.github.io/covidexpress). This open access database will allow research investigators to examine the gene expression in various tissues, cell lines, and their response to SARS-CoV-2 under different experimental conditions, accelerating the understanding of the etiology of this disease to inform the drug and vaccine development. Our integrative analysis of this big dataset highlights a set of commonly regulated genes in SARS-CoV-2 infected lung and Rhinovirus infected nasal tissues, including OASL that were under-studied in COVID-19 related reports. Our results also suggested a potential FURIN positive feedback loop that might explain the evolutional advantage of SARS-CoV-2.

7.
Epigenetics Chromatin ; 13(1): 38, 2020 09 25.
Article in English | MEDLINE | ID: mdl-32977832

ABSTRACT

BACKGROUND: UTX/KDM6A is known to interact and influence multiple different chromatin modifiers to promote an open chromatin environment to facilitate gene activation, but its molecular activities in developmental gene regulation remain unclear. RESULTS: We report that in human neural stem cells, UTX binding correlates with both promotion and suppression of gene expression. These activities enable UTX to modulate neural stem cell self-renewal, promote neurogenesis, and suppress gliogenesis. In neural stem cells, UTX has a less influence over histone H3 lysine 27 and lysine 4 methylation but more predominantly affects histone H3 lysine 27 acetylation and chromatin accessibility. Furthermore, UTX suppresses components of AP-1 and, in turn, a gliogenesis program. CONCLUSIONS: Our findings revealed that UTX coordinates dualistic gene regulation to govern neural stem cell properties and neurogenesis-gliogenesis switch.


Subject(s)
Embryonic Stem Cells/metabolism , Histone Demethylases/metabolism , Microglia/metabolism , Neural Stem Cells/metabolism , Neurogenesis , Transcription Factor AP-1/metabolism , Embryonic Stem Cells/cytology , Humans , Microglia/cytology , Neural Stem Cells/cytology , Protein Binding
8.
Nat Commun ; 11(1): 4060, 2020 08 13.
Article in English | MEDLINE | ID: mdl-32792512

ABSTRACT

Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1. In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. Our findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.


Subject(s)
Brain/embryology , Brain/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Proliferation/genetics , Cell Proliferation/physiology , Cells, Cultured , Chromatin Immunoprecipitation , Drosophila , Epigenesis, Genetic/genetics , Flow Cytometry , Fluorescent Antibody Technique , Histone-Lysine N-Methyltransferase/genetics , Immunoprecipitation , Mice , Mice, Knockout , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/physiology , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics
9.
Nat Neurosci ; 22(3): 362-373, 2019 03.
Article in English | MEDLINE | ID: mdl-30718900

ABSTRACT

UTX is a chromatin modifier required for development and neural lineage specification, but how it controls these biological processes is unclear. To determine the molecular mechanisms of UTX, we identified novel UTX protein interaction partners. Here we show that UTX and 53BP1 directly interact and co-occupy promoters in human embryonic stem cells and differentiating neural progenitor cells. Human 53BP1 contains a UTX-binding site that diverges from its mouse homolog by 41%, and disruption of the 53BP1-UTX interaction abrogated human, but not mouse, neurogenesis in vitro. The 53BP1-UTX interaction is required to upregulate key neurodevelopmental genes during the differentiation of human embryonic stem cells into neurons or into cortical organoids. 53BP1 promotes UTX chromatin binding, and in turn H3K27 modifications and gene activation, at a subset of genomic regions, including neurogenic genes. Overall, our data suggest that the 53BP1-UTX interaction supports the activation of key genes required for human neurodevelopment.


Subject(s)
Cerebral Cortex/metabolism , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Histone Demethylases/metabolism , Neural Stem Cells/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolism , Animals , Cell Differentiation , Cells, Cultured , Cerebral Cortex/growth & development , Female , Histone Code , Humans , Male , Mice, Inbred C57BL , Organoids/growth & development , Organoids/metabolism , Promoter Regions, Genetic
10.
PLoS Genet ; 12(9): e1006281, 2016 09.
Article in English | MEDLINE | ID: mdl-27622269

ABSTRACT

Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. We uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility.


Subject(s)
Argonaute Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Oogonia/metabolism , Ovary/growth & development , Proto-Oncogene Proteins c-fos/genetics , 3' Untranslated Regions , Animals , Argonaute Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Female , Oogenesis , Oogonia/cytology , Ovary/metabolism , Proto-Oncogene Proteins c-fos/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Stem Cell Niche
11.
Nat Genet ; 48(3): 283-91, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26780607

ABSTRACT

The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.


Subject(s)
Argonaute Proteins/genetics , Oogenesis/genetics , Polycomb-Group Proteins/genetics , Transcription, Genetic , Animals , Argonaute Proteins/metabolism , Chromatin/genetics , Drosophila melanogaster , Female , Gene Expression Regulation, Developmental , Germ Cells , Histones/genetics , Methylation , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 2/genetics , Polycomb-Group Proteins/biosynthesis , Stem Cells/metabolism
12.
Curr Opin Cell Biol ; 25(2): 190-4, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23465540

ABSTRACT

Piwi-interacting RNAs (piRNAs) were reported in 2006 as a novel class of small non-coding RNAs associated with Piwi proteins of the Argonaute/Piwi family. Recent studies have revealed not only the biogenesis of piRNAs and their roles in transposon silencing, but also the function of the Piwi-piRNA pathway in epigenetic and post-transcriptional regulation of gene expression. In addition, the function of this pathway in somatic cells has also been more systematically characterized. The new findings reveal the Piwi-piRNA pathway as a more general mechanism of gene regulation.


Subject(s)
Argonaute Proteins/metabolism , Drosophila Proteins/metabolism , Epigenesis, Genetic , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Animals , DNA Transposable Elements/genetics , Gene Silencing , Histones/chemistry , Histones/metabolism
13.
PLoS Genet ; 5(3): e1000435, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19325889

ABSTRACT

Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.


Subject(s)
Genomic Instability , Heterochromatin/genetics , Histones/metabolism , Animals , DNA Damage , DNA Repair , Drosophila/genetics , Methylation , Methyltransferases/deficiency , Mitosis , Mutation , RNA Interference
14.
Cell ; 139(7): 1290-302, 2009 Dec 24.
Article in English | MEDLINE | ID: mdl-20064375

ABSTRACT

Polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in embryonic stem (ES) cells and during development. Here we show that Jarid2/Jumonji, a protein enriched in pluripotent cells and a founding member of the Jumonji C (JmjC) domain protein family, is a PRC2 subunit in ES cells. Genome-wide ChIP-seq analyses of Jarid2, Ezh2, and Suz12 binding reveal that Jarid2 and PRC2 occupy the same genomic regions. We further show that Jarid2 promotes PRC2 recruitment to the target genes while inhibiting PRC2 histone methyltransferase activity, suggesting that it acts as a "molecular rheostat" that finely calibrates PRC2 functions at developmental genes. Using Xenopus laevis as a model we demonstrate that Jarid2 knockdown impairs the induction of gastrulation genes in blastula embryos and results in failure of differentiation. Our findings illuminate a mechanism of histone methylation regulation in pluripotent cells and during early cell-fate transitions.


Subject(s)
Nerve Tissue Proteins/metabolism , Repressor Proteins/metabolism , Animals , Embryonic Stem Cells/metabolism , Gene Knockdown Techniques , Humans , Mice , Mitochondria/metabolism , Polycomb Repressive Complex 2 , Polycomb-Group Proteins , RNA/metabolism , Retinoblastoma-Binding Protein 2/metabolism
15.
Curr Opin Genet Dev ; 18(2): 204-11, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18372168

ABSTRACT

In this review we summarize recent studies that demonstrate the importance of epigenetic mechanisms for maintaining genome integrity, specifically with respect to repeated DNAs within heterochromatin. Potential problems that arise during replication, recombination, and repair of repeated sequences are counteracted by post-translational histone modifications and associated proteins, including the cohesins. These factors appear to ensure repeat stability by multiple mechanisms: suppressing homologous recombination, controlling the three-dimensional organization of damaged repeats to reduce the probability of aberrant recombination, and promoting the use of less problematic repair pathways. The presence of such systems may facilitate repeat and chromosome evolution, and their failure can lead to genome instability, chromosome rearrangements, and the onset of pathogenesis.


Subject(s)
DNA/genetics , Epigenesis, Genetic/genetics , Heterochromatin/genetics , Animals , Biological Evolution , Cell Nucleus/genetics , Humans , Tandem Repeat Sequences
16.
Nat Cell Biol ; 9(1): 25-35, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17159999

ABSTRACT

Investigations aimed at identifying regulators of nuclear architecture in Drosophila demonstrated that cells lacking H3K9 methylation and RNA interference (RNAi) pathway components displayed disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared with wild type. We also observed a substantial increase in extrachromosomal circular (ecc) repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 and ecc DNA formation is not induced by removal of cohesin. We conclude that the structural integrity and organization of repeated DNAs and nucleoli are regulated by the H3K9 methylation and RNAi pathways, and other regulators of heterochromatin-mediated silencing. In addition, repeated DNA stability involves suppression of non-homologous end joining (NHEJ) or other recombination pathways. These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.


Subject(s)
Cell Nucleolus/genetics , DNA Methylation , Drosophila/genetics , Histones/metabolism , RNA Interference , Animals , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA, Ribosomal/metabolism , DNA, Satellite/metabolism , Drosophila Proteins/genetics , Heterochromatin/genetics , Heterochromatin/physiology , Models, Genetic , Nuclear Proteins/genetics , Protein Inhibitors of Activated STAT/genetics , Repressor Proteins/genetics , Cohesins
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