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1.
Nat Commun ; 15(1): 1761, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409161

RESUMO

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.


Assuntos
Reprogramação Celular , Cromatina , Animais , Camundongos , Diferenciação Celular/genética , Reprogramação Celular/genética , Metaplasia , Fatores de Transcrição/metabolismo
2.
Stem Cell Reports ; 18(7): 1436-1450, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37352852

RESUMO

Over the last several years, a method has emerged that endows adult hepatocytes with in vitro proliferative capacity, producing chemically induced liver progenitors (CLiPs). However, there is a growing controversy regarding the origin of these cells. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro using rat and mouse models. Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary epithelial cell-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. This study advances our understanding of the origin of CLiPs and motivates the application of this technique in liver regenerative medicine.


Assuntos
Hepatócitos , Células-Tronco , Camundongos , Ratos , Animais , Células-Tronco/metabolismo , Fígado , Células Epiteliais/metabolismo , Diferenciação Celular , Proliferação de Células
3.
bioRxiv ; 2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36824858

RESUMO

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

4.
Elife ; 102021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33620315

RESUMO

Cancer patients often harbor occult metastases, a potential source of relapse that is targetable only through systemic therapy. Studies of this occult fraction have been limited by a lack of tools with which to isolate discrete cells on spatial grounds. We developed PIC-IT, a photoconversion-based isolation technique allowing efficient recovery of cell clusters of any size - including single-metastatic cells - which are largely inaccessible otherwise. In a murine pancreatic cancer model, transcriptional profiling of spontaneously arising microcolonies revealed phenotypic heterogeneity, functionally reduced propensity to proliferate and enrichment for an inflammatory-response phenotype associated with NF-κB/AP-1 signaling. Pharmacological inhibition of NF-κB depleted microcolonies but had no effect on macrometastases, suggesting microcolonies are particularly dependent on this pathway. PIC-IT thus enables systematic investigation of metastatic heterogeneity. Moreover, the technique can be applied to other biological systems in which isolation and characterization of spatially distinct cell populations is not currently feasible.


Assuntos
Perfilação da Expressão Gênica , Oncologia/métodos , Metástase Neoplásica/diagnóstico , Neoplasias Pancreáticas/fisiopatologia , Fenótipo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processos Fotoquímicos
5.
Hepatology ; 74(1): 444-457, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33423324

RESUMO

BACKGROUND AND AIMS: Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. APPROACH AND RESULTS: We applied the techniques of bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, and assay for transposase-accessible chromatin with high-throughput sequencing to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGF-ß signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGF-ß signaling component mothers against decapentaplegic homolog 4 (Smad4). Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. Although some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single-cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. CONCLUSION: Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGF-ß signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGF-ß can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of BECs and bile ducts in the adult liver.


Assuntos
Plasticidade Celular/genética , Regeneração Hepática/genética , Fígado/fisiologia , Animais , Ductos Biliares/citologia , Diferenciação Celular/genética , Epigênese Genética , Células Epiteliais/fisiologia , Hepatócitos/fisiologia , Hepatócitos/transplante , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Transgênicos , RNA-Seq , Análise de Célula Única , Proteína Smad4/genética
6.
Cancer Discov ; 11(3): 736-753, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33158848

RESUMO

Although immunotherapy has revolutionized cancer care, patients with pancreatic ductal adenocarcinoma (PDA) rarely respond to these treatments, a failure that is attributed to poor infiltration and activation of T cells in the tumor microenvironment (TME). We performed an in vivo CRISPR screen and identified lysine demethylase 3A (KDM3A) as a potent epigenetic regulator of immunotherapy response in PDA. Mechanistically, KDM3A acts through Krueppel-like factor 5 (KLF5) and SMAD family member 4 (SMAD4) to regulate the expression of the epidermal growth factor receptor (EGFR). Ablation of KDM3A, KLF5, SMAD4, or EGFR in tumor cells altered the immune TME and sensitized tumors to combination immunotherapy, whereas treatment of established tumors with an EGFR inhibitor, erlotinib, prompted a dose-dependent increase in intratumoral T cells. This study defines an epigenetic-transcriptional mechanism by which tumor cells modulate their immune microenvironment and highlights the potential of EGFR inhibitors as immunotherapy sensitizers in PDA. SIGNIFICANCE: PDA remains refractory to immunotherapies. Here, we performed an in vivo CRISPR screen and identified an epigenetic-transcriptional network that regulates antitumor immunity by converging on EGFR. Pharmacologic inhibition of EGFR is sufficient to rewire the immune microenvironment. These results offer a readily accessible immunotherapy-sensitizing strategy for PDA.This article is highlighted in the In This Issue feature, p. 521.


Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/genética , Animais , Biomarcadores Tumorais/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Terapia Combinada , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genômica/métodos , Humanos , Imunidade/genética , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Mutação , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Transcriptoma , Resultado do Tratamento
7.
Cancer Discov ; 10(6): 854-871, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32188706

RESUMO

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. SIGNIFICANCE: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer.This article is highlighted in the In This Issue feature, p. 747.


Assuntos
Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Neoplasias/genética , Transição Epitelial-Mesenquimal , Humanos
8.
Dev Cell ; 48(4): 425-426, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30782410

RESUMO

To maintain proper organ size, nature has devised trans-organ communication systems-involving both paracrine and circulating regulatory factors-to safeguard homeostasis. In this issue of Developmental Cell, Ji et al. (2019) now describe an enterohepatic feedback loop that balances tissue size and function in the mammalian liver.


Assuntos
Carcinogênese , Transdução de Sinais , Animais , Ácidos e Sais Biliares , Fígado , Tamanho do Órgão
9.
Immunity ; 49(1): 178-193.e7, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29958801

RESUMO

The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.


Assuntos
Adenocarcinoma/terapia , Fatores Imunológicos/imunologia , Imunoterapia , Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/terapia , Microambiente Tumoral/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos T CD8-Positivos/imunologia , Epigenômica , Feminino , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Cultura Primária de Células , Neoplasias Pancreáticas
10.
Nat Rev Mol Cell Biol ; 17(7): 413-25, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26979497

RESUMO

Biologists have long been intrigued by the possibility that cells can change their identity, a phenomenon known as cellular plasticity. The discovery that terminally differentiated cells can be experimentally coaxed to become pluripotent has invigorated the field, and recent studies have demonstrated that changes in cell identity are not limited to the laboratory. Specifically, certain adult cells retain the capacity to de-differentiate or transdifferentiate under physiological conditions, as part of an organ's normal injury response. Recent studies have highlighted the extent to which cell plasticity contributes to tissue homeostasis, findings that have implications for cell-based therapy.


Assuntos
Células-Tronco Adultas/fisiologia , Desdiferenciação Celular , Transdiferenciação Celular , Animais , Transformação Celular Neoplásica , Humanos , Neoplasias/patologia , Regeneração , Transdução de Sinais
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