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1.
Cell ; 186(1): 80-97.e26, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608661

RESUMO

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.


Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Humanos
2.
Nat Methods ; 19(8): 959-968, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35927480

RESUMO

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.


Assuntos
Cromatina , DNA , Biotinilação , Cromatina/genética , DNA/genética , DNA/metabolismo , Plasmídeos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Cancer Res ; 82(17): 3143-3157, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-35705526

RESUMO

Epithelial squamous cell carcinomas (SCC) most commonly originate in the skin, where they display disruptions in the normally tightly regulated homeostatic balance between keratinocyte proliferation and terminal differentiation. We performed a transcriptome-wide screen for genes of unknown function that possess inverse expression patterns in differentiating keratinocytes compared with cutaneous SCC (cSCC), leading to the identification of MAB21L4 (C2ORF54) as an enforcer of terminal differentiation that suppresses carcinogenesis. Loss of MAB21L4 in human cSCC organoids increased expression of RET to enable malignant progression. In addition to transcriptional upregulation of RET, deletion of MAB21L4 preempted recruitment of the CacyBP-Siah1 E3 ligase complex to RET and reduced its ubiquitylation. In SCC organoids and in vivo tumor models, genetic disruption of RET or selective inhibition of RET with BLU-667 (pralsetinib) suppressed SCC growth while inducing concomitant differentiation. Overall, loss of MAB21L4 early during SCC development blocks differentiation by increasing RET expression. These results suggest that targeting RET activation is a potential therapeutic strategy for treating SCC. SIGNIFICANCE: Downregulation of RET mediated by MAB21L4-CacyBP interaction is required to induce epidermal differentiation and suppress carcinogenesis, suggesting RET inhibition as a potential therapeutic approach in squamous cell carcinoma.


Assuntos
Carcinoma de Células Escamosas , Neoplasias Cutâneas , Humanos , Proteínas de Ligação ao Cálcio/metabolismo , Carcinogênese/patologia , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Queratinócitos/patologia , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias Cutâneas/patologia
4.
Nat Genet ; 53(11): 1564-1576, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34650237

RESUMO

Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.


Assuntos
Epiderme/fisiologia , Modelos Genéticos , Elementos Reguladores de Transcrição , Diferenciação Celular/genética , Cromatina/genética , Epigenoma , Regulação da Expressão Gênica , Genes Reporter , Estudo de Associação Genômica Ampla , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Redes Neurais de Computação , Dermatopatias/genética , Fatores de Transcrição/genética
5.
Hum Mol Genet ; 29(4): 541-553, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-31628467

RESUMO

Missense mutations in the RNA exosome component exosome component 2 (EXOSC2), also known as ribosomal RNA-processing protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of the SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes and Drosophila. We determined that human EXOSC2 is an essential gene and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3'UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas de Ligação a RNA/genética , Animais , Autofagia/genética , Modelos Animais de Doenças , Drosophila/genética , Nanismo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Feminino , Genômica/métodos , Células HEK293 , Perda Auditiva/genética , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Fenótipo , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Retinose Pigmentar/genética , Síndrome
7.
Nat Genet ; 49(10): 1522-1528, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28805829

RESUMO

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer-promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer-promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation.


Assuntos
Linhagem da Célula/genética , Cromossomos Humanos/ultraestrutura , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Queratinócitos/citologia , Regiões Promotoras Genéticas/genética , Acetilação , Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Cromossomos Humanos/genética , Células Epidérmicas , Biblioteca Gênica , Código das Histonas , Histonas/metabolismo , Humanos , Queratinócitos/metabolismo , Masculino , Processamento de Proteína Pós-Traducional , RNA/genética , Interferência de RNA , Fatores de Transcrição/metabolismo
8.
Cell Cycle ; 15(11): 1405-9, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27097296

RESUMO

Numerous regulatory factors in epidermal differentiation and their role in regulating different cell states have been identified in recent years. However, the genetic interactions between these regulators over the dynamic course of differentiation have not been studied. In this Extra-View article, we review recent work by Lopez-Pajares et al. that explores a new regulatory network in epidermal differentiation. They analyze the changing transcriptome throughout epidermal regeneration to identify 3 separate gene sets enriched in the progenitor, early and late differentiation states. Using expression module mapping, MAF along with MAFB, are identified as transcription factors essential for epidermal differentiation. Through double knock-down of MAF:MAFB using siRNA and CRISPR/Cas9-mediated knockout, epidermal differentiation was shown to be impaired both in-vitro and in-vivo, confirming MAF:MAFB's role to activate genes that drive differentiation. Lopez-Pajares and collaborators integrated 42 published regulator gene sets and the MAF:MAFB gene set into the dynamic differentiation gene expression landscape and found that lncRNAs TINCR and ANCR act as upstream regulators of MAF:MAFB. Furthermore, ChIP-seq analysis of MAF:MAFB identified key transcription factor genes linked to epidermal differentiation as downstream effectors. Combined, these findings illustrate a dynamically regulated network with MAF:MAFB as a crucial link for progenitor gene repression and differentiation gene activation.


Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição MafB/genética , Proteínas Proto-Oncogênicas c-maf/genética , Células-Tronco/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epidérmicas , Epiderme/metabolismo , Células Epiteliais/citologia , Redes Reguladoras de Genes , Humanos , Fator de Transcrição MafB/antagonistas & inibidores , Fator de Transcrição MafB/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-maf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-maf/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Células-Tronco/citologia
9.
Pflugers Arch ; 468(6): 971-81, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26996975

RESUMO

Transcriptome analysis of mammalian genomes has revealed widespread transcription, much of which does not encode protein. Long non-coding RNAs (lncRNAs) are a subset of the non-coding transcriptome that are emerging as critical regulators of various cellular processes. Differentiation of stem and progenitor cells requires a careful execution of specific genetic programs, and recent studies have revealed that lncRNA expression contributes to specification of cell identity. LncRNAs participate in regulating differentiation at multiple levels of gene expression through various mechanisms of action. In this review, functional roles of lncRNAs in regulating cellular differentiation of blood, muscle, skin, cardiomyocytes, adipocytes, and neurons are discussed.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Animais , Humanos , RNA Longo não Codificante/metabolismo
10.
Dev Cell ; 35(4): 444-57, 2015 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-26609959

RESUMO

Current gene expression network approaches commonly focus on transcription factors (TFs), biasing network-based discovery efforts away from potentially important non-TF proteins. We developed proximity analysis, a network reconstruction method that uses topological constraints of scale-free, small-world biological networks to reconstruct relationships in eukaryotic systems, independent of subcellular localization. Proximity analysis identified MPZL3 as a highly connected hub that is strongly induced during epidermal differentiation. MPZL3 was essential for normal differentiation, acting downstream of p63, ZNF750, KLF4, and RCOR1, each of which bound near the MPZL3 gene and controlled its expression. MPZL3 protein localized to mitochondria, where it interacted with FDXR, which was itself also found to be essential for differentiation. Together, MPZL3 and FDXR increased reactive oxygen species (ROS) to drive epidermal differentiation. ROS-induced differentiation is dependent upon promotion of FDXR enzymatic activity by MPZL3. ROS induction by the MPZL3 and FDXR mitochondrial proteins is therefore essential for epidermal differentiation.


Assuntos
Diferenciação Celular , Células Epidérmicas , Ferredoxina-NADP Redutase/metabolismo , Redes Reguladoras de Genes , Queratinócitos/citologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Epiderme/metabolismo , Ferredoxina-NADP Redutase/genética , Ferredoxinas/metabolismo , Regulação da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Fator 4 Semelhante a Kruppel , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Metabolômica , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição/metabolismo
11.
Genes Dev ; 29(21): 2225-30, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26545810

RESUMO

Outward migration of epidermal progenitors occurs with induction of hundreds of differentiation genes, but the identities of all regulators required for this process are unknown. We used laser capture microdissection followed by RNA sequencing to identify calmodulin-like 5 (CALML5) as the most enriched gene in differentiating outer epidermis. CALML5 mRNA was up-regulated by the ZNF750 transcription factor and then stabilized by the long noncoding RNA TINCR. CALML5 knockout impaired differentiation, abolished keratohyalin granules, and disrupted epidermal barrier function. Mass spectrometry identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein. CALML5 interacts with SFN in suprabasal epidermis, cocontrols 13% of late differentiation genes, and modulates interaction of SFN to some of its binding partners. A ZNF750-TINCR-CALML5-SFN network is thus essential for epidermal differentiation.


Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/genética , Células Epidérmicas , Exorribonucleases/metabolismo , RNA não Traduzido/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fosfoproteínas/metabolismo , Ligação Proteica , Transporte Proteico , Células-Tronco/citologia , Proteínas Supressoras de Tumor , Proteínas de Sinalização YAP
12.
Dev Cell ; 32(6): 693-706, 2015 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-25805135

RESUMO

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.


Assuntos
Diferenciação Celular/genética , Células Epidérmicas , Fator de Transcrição MafB/genética , Proteínas Proto-Oncogênicas c-maf/genética , RNA Longo não Codificante/genética , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Organogênese/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo , Interferência de RNA , RNA Interferente Pequeno , Proteínas Repressoras/biossíntese , Fatores de Transcrição/biossíntese , Proteínas Supressoras de Tumor
13.
Cell Stem Cell ; 12(2): 193-203, 2013 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-23395444

RESUMO

Somatic progenitors suppress differentiation to maintain tissue self-renewal. The mammalian SWI/SNF chromatin-remodeling complex regulates nucleosome packaging to control differentiation in embryonic and adult stem cells. Catalytic Brg1 and Brm subunits are required for these processes; however, the roles of SWI/SNF regulatory subunits are not fully understood. Here, we show that ACTL6a/BAF53A modulates the SWI/SNF complex to suppress differentiation in epidermis. Conditional loss of ACTL6a resulted in terminal differentiation, cell-cycle exit, and hypoplasia, whereas ectopic expression of ACTL6a promoted the progenitor state. A significant portion of genes regulated by ACTL6a were found to also be targets of KLF4, a known activator of epidermal differentiation. Mechanistically, we show that ACTL6a prevents SWI/SNF complex binding to promoters of KLF4 and other differentiation genes and that SWI/SNF catalytic subunits are required for full induction of KLF4 targets. Thus, ACTL6a controls the epidermal progenitor state by sequestering SWI/SNF to prevent activation of differentiation programs.


Assuntos
Actinas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Actinas/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Trends Genet ; 29(1): 31-40, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23141808

RESUMO

More than 100 human genetic skin diseases, impacting over 20% of the population, are characterized by disrupted epidermal differentiation. A significant proportion of the 90 genes identified in these disorders to date are concentrated within several functional pathways, suggesting the emergence of organizing themes in epidermal differentiation. Among these are the Notch, transforming growth factor ß (TGFß), IκB kinase (IKK), Ras/mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), p63, and Wnt signaling pathways, as well as core biological processes mediating calcium homeostasis, tissue integrity, cornification, and lipid biogenesis. Here, we review recent results supporting the central role of these pathways in epidermal differentiation, highlighting the integration of genetic information with functional studies to illuminate the biological actions of these pathways in humans as well as to guide development of future therapeutics to correct their dysfunction.


Assuntos
Diferenciação Celular/genética , Epiderme/fisiologia , Transdução de Sinais/genética , Dermatopatias/genética , Dermatopatias/fisiopatologia , Animais , Epiderme/metabolismo , Redes Reguladoras de Genes/fisiologia , Humanos , Modelos Biológicos , Dermatopatias/etiologia
15.
Transl Cancer Res ; 1(2): 88-89, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23002429

RESUMO

p53, a critical tumor suppressor, is activated by various cellular stresses to prevent and repair damages that can lead to tumor development. In response to these stresses, p53 activation can cause very serious cellular effects including permanent cell cycle arrest and cell death. p53 must therefore be very tightly regulated to avoid unnecessary pathological effects. The homologs MDM2 and MDMX have been shown to be the major, essential negative regulators of p53. In normal cells, MDM2 and MDMX suppress p53 activity, but in the event of cellular stress, they themselves must be inhibited so that p53 may respond to the stress. MDM2 and MDMX are known to bind together, and play multifaceted, non-redundant roles in modulating p53 protein activity. Recently, evidence has emerged showing that MDM2 and MDMX most effectively inhibit p53 as a complex, and possibly play non-redundant roles because they must function as one to control p53. In this review, we give an overview of MDM2 and MDMX and discuss a few ways in which they are modified so that p53 may be activated. Lastly, we discuss the non-redundant roles of MDM2 and MDMX and how it is important to investigate the effect on the complex as a whole when investigating either protein.

16.
Am J Hum Genet ; 91(3): 435-43, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22922031

RESUMO

The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Displasia Ectodérmica/genética , Anormalidades do Olho/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Diferenciação Celular/genética , Epiderme/metabolismo , Pálpebras/anormalidades , Humanos , Fator 4 Semelhante a Kruppel , Mutação , Técnicas de Cultura de Órgãos/métodos , Transcriptoma
17.
Genes Dev ; 26(4): 338-43, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22302877

RESUMO

Long noncoding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti-differentiation ncRNA) as an 855-base-pair lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.


Assuntos
Diferenciação Celular , Queratinócitos/citologia , RNA não Traduzido/metabolismo , Células-Tronco/citologia , Células Cultivadas , Células Epidérmicas , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , RNA Longo não Codificante , RNA não Traduzido/genética , Transcriptoma
18.
Cancer Res ; 68(22): 9131-6, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19010883

RESUMO

Although epidemiologic studies have linked arsenic exposure to the development of human cancer, the mechanisms underlying the tumorigenic role of arsenic remain largely undefined. We report here that treatment of cells with sodium arsenite at the concentrations close to environmental exposure is associated with the up-regulation of Hdm2 and the accumulation of p53 in the cytoplasm. Through the mitogen-activated protein kinase pathway, arsenite stimulates the P2 promoter-mediated expression of Hdm2, which then promotes p53 nuclear export. As a consequence, the p53 response to genotoxic stress is compromised, as evidenced by the impaired p53 activation and apoptosis in response to UV irradiation or 5FU treatment. The ability of arsenite to impede p53 activation is further demonstrated by a significantly blunted p53-dependent tissue response to 5FU treatment when mice were fed with arsenite-containing water. Together, our data suggests that arsenic compounds predispose cells to malignant transformation by up-regulation of Hdm2 and subsequent p53 inactivation.


Assuntos
Arsênio/toxicidade , Transformação Celular Neoplásica/efeitos dos fármacos , Citoplasma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Dano ao DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fluoruracila/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/fisiologia
19.
J Biol Chem ; 283(20): 13707-13, 2008 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-18356162

RESUMO

The critical tumor suppressor p53 is mutated or functionally inactivated in nearly all cancers. We have shown previously that the MDM2-MDMX complex functions as an integral unit in targeting p53 for degradation. Here we identify the small protein 14-3-3 as a binding partner of MDMX, which binds at the C terminus (Ser367) in a phosphorylation-dependent manner. Importantly, we demonstrate that the serine/threonine kinase Akt mediates phosphorylation of MDMX at Ser367. This phosphorylation leads to stabilization of MDMX and consequent stabilization of MDM2. Previous studies have shown that Akt phosphorylates and stabilizes MDM2. Our data suggest that stabilization of MDMX by Akt may be an alternative mechanism by which Akt up-regulates MDM2 protein levels and exerts its oncogenic effects on p53 in tumor cells.


Assuntos
Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Linhagem Celular , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/química
20.
Cancer Res ; 67(13): 6026-30, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17616658

RESUMO

The RING domain of MDM2 that is essential for its E3 ligase activity mediates binding to itself and its structural homologue MDMX. Whereas it has been reported that RING domain interactions are critical, it is not well understood how they affect the E3 ligase activity of MDM2. We report that the E3 ligase activity requires the RING domain-dependent complex formation. In vivo, MDM2 and MDMX hetero-RING complexes are the predominant form versus the MDM2 homo-RING complex. Importantly, the MDM2/MDMX hetero-RING complexes exhibit a greater E3 ligase activity than the MDM2 homo-RING complexes. Disruption of the binding between MDM2 and MDMX resulted in a marked increase in both abundance and activity of p53, emphasizing the functional importance of this heterocomplex in p53 control.


Assuntos
Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Motivos de Aminoácidos , Animais , Linhagem Celular , Cruzamentos Genéticos , Fibroblastos/metabolismo , Humanos , Camundongos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transfecção , Proteína Supressora de Tumor p53/metabolismo
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