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1.
Cell ; 187(12): 3072-3089.e20, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38781967

RESUMO

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.


Assuntos
Matriz Extracelular , Mucosa Intestinal , Animais , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Matriz Extracelular/metabolismo , Miosina Tipo II/metabolismo , Mesoderma/metabolismo , Mesoderma/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Morfogênese , Metaloproteinases da Matriz/metabolismo
2.
Nat Cell Biol ; 26(2): 250-262, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38321203

RESUMO

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.


Assuntos
Duodeno , Intestino Delgado , Humanos , Camundongos , Animais , Intestino Delgado/metabolismo , Duodeno/metabolismo , Intestinos , Jejuno/metabolismo , Íleo/metabolismo , Mamíferos
3.
bioRxiv ; 2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37790430

RESUMO

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar-ẟ and Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.

4.
J Clin Invest ; 133(20)2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37643009

RESUMO

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Mucinas , Humanos , Animais , Camundongos , Mucinas/genética , Mucinas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Inibidores do Fator de Necrose Tumoral , Células Epiteliais/metabolismo , Diferenciação Celular , Fatores de Necrose Tumoral , Homeostase
5.
bioRxiv ; 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37425793

RESUMO

Tissue folding generates structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, the numerous finger-like protrusions that are essential for nutrient absorption. However, the molecular and mechanical mechanisms driving the initiation and morphogenesis of villi remain a matter of debate. Here, we identify an active mechanical mechanism that simultaneously patterns and folds intestinal villi. We find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. At the cell-level, this occurs through a process dependent upon matrix metalloproteinase-mediated tissue fluidization and altered cell-ECM adhesion. By combining computational models with in vivo experiments, we reveal these cellular features manifest at the tissue-level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active de-wetting of a thin liquid film.

6.
Cell Stem Cell ; 30(4): 433-449.e8, 2023 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-37028407

RESUMO

Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers; that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.


Assuntos
Mucosa Intestinal , Nicho de Células-Tronco , Animais , Camundongos , Mucosa Intestinal/metabolismo , Intestinos , Transdução de Sinais , Diferenciação Celular , Proliferação de Células
7.
Cell Stem Cell ; 29(8): 1262-1272.e5, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35931034

RESUMO

The intestinal epithelium undergoes continuous renewal and has an exceptional capacity to regenerate after injury. Maintenance and proliferation of intestinal stem cells (ISCs) are regulated by their surrounding niche, largely through Wnt signaling. However, it remains unclear which niche cells produce signals during different injury states, and the role of endothelial cells (ECs) as a component of the ISC niche during homeostasis and after injury has been underappreciated. Here, we show that lymphatic endothelial cells (LECs) reside in proximity to crypt epithelial cells and secrete molecules that support epithelial renewal and repair. LECs are an essential source of Wnt signaling in the small intestine, as loss of LEC-derived Rspo3 leads to a lower number of stem and progenitor cells and hinders recovery after cytotoxic injury. Together, our findings identify LECs as an essential niche component for optimal intestinal recovery after cytotoxic injury.


Assuntos
Células Endoteliais , Intestinos , Proliferação de Células , Células Epiteliais , Mucosa Intestinal , Células-Tronco
8.
iScience ; 25(6): 104374, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35633935

RESUMO

Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the ß-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction. Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one ß-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected ß-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage. Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.

10.
NPJ Genom Med ; 6(1): 77, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34556655

RESUMO

Current genetic testenhancer and narrows the diagnostic intervals for rare diseases provide a diagnosis in only a modest proportion of cases. The Full-Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGA's utility for diagnosis of rare diseases in a clinical setting. FGA identified structural variants and small variants with an overall diagnostic yield of 40% (20 of 50 cases) and 35% in exome-negative cases (8 of 23 cases), 4 of these were structural variants. FGA detected and mapped structural variants that are missed by short reads, including non-coding duplication, and phased variants across long distances of more than 180 kb. With the prioritization algorithm, longer DNA technologies could replace multiple tests for monogenic disorders and expand the range of variants detected. Our study suggests that genomes produced from technologies like FGA can improve variant detection and provide higher resolution genome maps for future application.

11.
Nat Commun ; 11(1): 5482, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127893

RESUMO

The current human reference genome is predominantly derived from a single individual and it does not adequately reflect human genetic diversity. Here, we analyze 338 high-quality human assemblies of genetically divergent human populations to identify missing sequences in the human reference genome with breakpoint resolution. We identify 127,727 recurrent non-reference unique insertions spanning 18,048,877 bp, some of which disrupt exons and known regulatory elements. To improve genome annotations, we linearly integrate these sequences into the chromosomal assemblies and construct a Human Diversity Reference. Leveraging this reference, an average of 402,573 previously unmapped reads can be recovered for a given genome sequenced to ~40X coverage. Transcriptomic diversity among these non-reference sequences can also be directly assessed. We successfully map tens of thousands of previously discarded RNA-Seq reads to this reference and identify transcription evidence in 4781 gene loci, underlining the importance of these non-reference sequences in functional genomics. Our extensive datasets are important advances toward a comprehensive reference representation of global human genetic diversity.


Assuntos
Variação Genética , Genoma Humano , População/genética , Mapeamento Cromossômico , Biologia Computacional , Expressão Gênica , Genômica , Técnicas de Genotipagem , Humanos , Anotação de Sequência Molecular , RNA-Seq , Análise de Sequência de DNA , Transcriptoma , Sequenciamento Completo do Genoma
12.
Genetics ; 214(1): 179-191, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31754017

RESUMO

Sequences encoding Olduvai protein domains (formerly DUF1220) show the greatest human lineage-specific increase in copy number of any coding region in the genome and have been associated, in a dosage-dependent manner, with brain size, cognitive aptitude, autism, and schizophrenia. Tandem intragenic duplications of a three-domain block, termed the Olduvai triplet, in four NBPF genes in the chromosomal 1q21.1-0.2 region, are primarily responsible for the striking human-specific copy number increase. Interestingly, most of the Olduvai triplets are adjacent to, and transcriptionally coregulated with, three human-specific NOTCH2NL genes that have been shown to promote cortical neurogenesis. Until now, the underlying genomic events that drove the Olduvai hyperamplification in humans have remained unexplained. Here, we show that the presence or absence of an alternative first exon of the Olduvai triplet perfectly discriminates between amplified (58/58) and unamplified (0/12) triplets. We provide sequence and breakpoint analyses that suggest the alternative exon was produced by an nonallelic homologous recombination-based mechanism involving the duplicative transposition of an existing Olduvai exon found in the CON3 domain, which typically occurs at the C-terminal end of NBPF genes. We also provide suggestive in vitro evidence that the alternative exon may promote instability through a putative G-quadraplex (pG4)-based mechanism. Lastly, we use single-molecule optical mapping to characterize the intragenic structural variation observed in NBPF genes in 154 unrelated individuals and 52 related individuals from 16 families and show that the presence of pG4-containing Olduvai triplets is strongly correlated with high levels of Olduvai copy number variation. These results suggest that the same driver of genomic instability that allowed the evolutionarily recent, rapid, and extreme human-specific Olduvai expansion remains highly active in the human genome.


Assuntos
Proteínas de Transporte/genética , Genoma Humano , Expansão das Repetições de Trinucleotídeos , Animais , Sequência de Bases , Variações do Número de Cópias de DNA , Evolução Molecular , Quadruplex G , Amplificação de Genes , Dosagem de Genes , Instabilidade Genômica , Recombinação Homóloga , Humanos , Primatas , Domínios Proteicos , Homologia de Sequência
13.
Front Microbiol ; 10: 577, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30949159

RESUMO

Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34+ hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of resistant and permissive cell types, and the cellular features characterizing the latter, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of ∼7000 individual cells at day 1 post-infection using the 10× genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit - Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells permissive to CMV replication, and provide a possible rationale for their preferential infection.

14.
PLoS One ; 14(1): e0208237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30645582

RESUMO

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , DNA Intergênico/genética , Edição de Genes , Inativação Gênica , Genótipo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fenótipo , Deleção de Sequência/genética , Regulação para Cima/genética , gama-Globinas/genética
15.
Sci Transl Med ; 8(360): 360ra134, 2016 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-27733558

RESUMO

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the ß-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.


Assuntos
Células-Tronco Adultas/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/terapia , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Hemoglobina Falciforme/genética , Adulto , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mutação , Polimorfismo de Nucleotídeo Único , Pesquisa Translacional Biomédica
16.
BMC Genomics ; 16: 882, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26519295

RESUMO

BACKGROUND: To examine the contributions of sequence and function conservation in the evolution of enhancers, we systematically identified enhancers whose sequences are not conserved among distant groups of vertebrate species, but have homologous function and are likely to be derived from a common ancestral sequence. Our approach combined comparative genomics and epigenomics to identify potential enhancer sequences in the genomes of three groups of distantly related vertebrate species. RESULTS: We searched for sequences that were conserved within groups of closely related species but not between groups of more distant species, and were associated with an epigenetic mark of enhancer activity. To facilitate inferring orthology between non-conserved sequences, we limited our search to introns whose orthology could be unambiguously established by mapping the bracketing exons. We show that a subset of these non-conserved but syntenic sequences from the mouse and zebrafish genomes have homologous functions in a zebrafish transgenic enhancer assay. The conserved expression patterns driven by these enhancers are probably associated with short transcription factor-binding motifs present in the divergent sequences. CONCLUSIONS: We have identified numerous potential enhancers with divergent sequences but a conserved function. These results indicate that selection on function, rather than sequence, may be a common mode of enhancer evolution; evidence for selection at the sequence level is not a necessary criterion to define a gene regulatory element.


Assuntos
Sequência Conservada , Elementos Facilitadores Genéticos , Variação Genética , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Biologia Computacional/métodos , Evolução Molecular , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
17.
BMC Genomics ; 16: 462, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26076733

RESUMO

BACKGROUND: Piwi-interacting RNAs (piRNAs) are a class of small RNAs; distinct types of piRNAs are expressed in the mammalian testis at different stages of development. The function of piRNAs expressed in the adult testis is not well established. We conducted a detailed characterization of piRNAs aligning at or near the 3' UTRs of protein-coding genes in a deep dataset of small RNAs from adult mouse testis. RESULTS: We identified 2710 piRNA clusters associated with 3' UTRs, including 1600 that overlapped genes not previously associated with piRNAs. 35% of the clusters extend beyond the annotated transcript; we find that these clusters correspond to, and are likely derived from, novel polyadenylated mRNA isoforms that contain previously unannotated extended 3'UTRs. Extended 3' UTRs, and small RNAs derived from them, are also present in somatic tissues; a subset of these somatic 3'UTR small RNA clusters are absent in mice lacking MIWI2, indicating a role for MIWI2 in the metabolism of somatic small RNAs. CONCLUSIONS: The finding that piRNAs are processed from extended 3' UTRs suggests a role for piRNAs in the remodeling of 3' UTRs. The presence of both clusters and extended 3'UTRs in somatic cells, with evidence for involvement of MIWI2, indicates that this pathway is more broadly distributed than currently appreciated.


Assuntos
Regiões 3' não Traduzidas/genética , RNA Interferente Pequeno/genética , Animais , Proteínas Argonautas/genética , Masculino , Camundongos , RNA Mensageiro/genética , Testículo/metabolismo
18.
Biomark Cancer ; 6: 37-47, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25520563

RESUMO

Small noncoding RNAs circulating in the blood may serve as signaling molecules because of their ability to carry out a variety of cellular functions. We have previously described tRNA- and YRNA-derived small RNAs circulating as components of larger complexes in the blood of humans and mice; the characteristics of these small RNAs imply specific processing, secretion, and physiological regulation. In this study, we have asked if changes in the serum abundance of these tRNA and YRNA fragments are associated with a diagnosis of cancer. We used deep sequencing and informatics analysis to catalog small RNAs in the sera of breast cancer cases and normal controls. 5' tRNA halves and YRNA fragments are abundant in both groups, but we found that a breast cancer diagnosis is associated with changes in levels of specific subtypes. This prompted us to look at existing sequence datasets of serum small RNAs from 42 breast cancer cases, taken at the time of diagnosis. We find significant changes in the levels of specific 5' tRNA halves and YRNA fragments associated with clinicopathologic characteristics of the cancer. Although these findings do not establish causality, they suggest that circulating 5' tRNA halves and YRNA fragments with known cellular functions may participate in breast cancer syndromes and have potential as circulating biomarkers. Larger studies with multiple types of cancer are needed to adequately evaluate their potential use for the development of noninvasive cancer screening.

19.
Bioessays ; 36(12): 1138-44, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25220261

RESUMO

Drosophila melanogaster is often considered to lack genomic 5-methylcytosine (m(5) C), an opinion reinforced by two whole genome bisulfite-sequencing studies that failed to find m(5) C. New evidence, however, indicates that genomic methylation is indeed present in the fly, albeit in small quantities and in unusual patterns. At embryonic stage 5, m(5) C occurs in short strand-specific regions that cover ∼1% of the genome, at tissue levels suggesting a distribution restricted to a subset of nuclei. Its function is not obvious, but methylation in subsets of nuclei would obscure functional associations since transcript levels and epigenetic modifications are assayed in whole embryos. Surprisingly, Mt2, the fly's only candidate DNA methyltransferase, is not necessary for the observed methylation. Full evaluation of the functions of genome methylation in Drosophila must await discovery and experimental inactivation of the DNA methyltransferase, as well as a better understanding of the pattern and developmental regulation of genomic m(5) C.


Assuntos
5-Metilcitosina/metabolismo , Núcleo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epigênese Genética , Genoma , Animais , Núcleo Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Motivos de Nucleotídeos
20.
Genome Res ; 24(5): 821-30, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24558263

RESUMO

Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: Its location and function have not been established. We have used a novel and highly sensitive genomewide cytosine methylation assay to detect and map genome methylation in stage 5 Drosophila embryos. The methylation we observe with this method is highly localized and strand asymmetrical, limited to regions covering ∼1% of the genome, dynamic in early embryogenesis, and concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. Gene body methylation is associated with lower expression, and many genes containing methylated regions have developmental or transcriptional functions. The only known DNA methyltransferase in Drosophila is the DNMT2 homolog MT2, but lines deficient for MT2 retain genomic methylation, implying the presence of a novel methyltransferase. The association of methylation with a lower expression of specific developmental genes at stage 5 raises the possibility that it participates in controlling gene expression during the maternal-zygotic transition.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genoma de Inseto , Motivos de Nucleotídeos , Animais , Composição de Bases , Ilhas de CpG , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento
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