RESUMO
Activation of calcitonin gene-related peptide (CGRP)-positive sensory neurons in the tumor microenvironment has been shown to be involved in tumor growth. However, how CGRP-positive sensory neurons are activated requires elucidation. In this study, we focused on transient receptor potential vanilloid 1 (TRPV1) and examined the contribution of TRPV1 to tumor growth and cancer pain in a mouse cancer model in which Lewis lung carcinoma was subcutaneously inoculated in the left plantar region. Tumor inoculation gradually increased the volumes of the hind paws of wild type (WT) mice over time, but those of both αCGRP knockout mice and TRPV1 knockout mice were significantly smaller than those of WT mice after tumor inoculation. Both TRPV1 and CGRP are therefore suggested to be involved in tumor growth. In an immunohistochemical study, the percentage of phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB)-positive profiles in CGRP-positive dorsal root ganglion (DRG) neurons in WT mice was significantly increased after tumor inoculation. The percentage of p-CREB-positive profiles in CGRP-positive DRG neurons in TRPV1 knockout mice was also increased after tumor inoculation, but was significantly lower than that in WT mice, indicating the contribution of TRPV1 to activation of CGRP-positive DRG neurons. Cancer pain in TRPV1 knockout mice was significantly lower than that in WT mice. In conclusion, TRPV1 is involved in both tumor growth and cancer pain, potentially leading to a novel strategy for the treatment of cancer pain and cancer development. Cancer pain is also suggested to facilitate tumor growth.
Assuntos
Antineoplásicos , Dor do Câncer , Neoplasias , Camundongos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dor/metabolismo , Modelos Animais de Doenças , Células Receptoras Sensoriais/metabolismo , Neoplasias/patologia , Camundongos Knockout , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Gânglios Espinais/metabolismo , Microambiente TumoralRESUMO
BACKGROUND: Lymphedema is an intractable disease that can be caused by injury to lymphatic vessels, such as by surgical treatments for cancer. It can lead to impaired joint mobility in the extremities and reduced quality of life. Chronic inflammation due to infiltration of various immune cells in an area of lymphedema is thought to lead to local fibrosis, but the molecular pathogenesis of lymphedema remains unclear. Development of effective therapies requires elucidation of the immunological mechanisms involved in the progression of lymphedema. The complement system is part of the innate immune system which has a central role in the elimination of invading microbes and acts as a scavenger of altered host cells, such as apoptotic and necrotic cells and cellular debris. Complement-targeted therapies have recently been clinically applied to various diseases caused by complement overactivation. In this context, we aimed to determine whether complement activation is involved in the development of lymphedema. RESULTS: Our mouse tail lymphedema models showed increased expression of C3, and that the classical or lectin pathway was locally activated. Complement activation was suggested to be involved in the progression of lymphedema. In comparison of the C3 knockout (KO) mouse lymphedema model and wild-type mice, there was no difference in the degree of edema at three weeks postoperatively, but the C3 KO mice had a significant increase of TUNEL+ necrotic cells and CD4+ T cells. Infiltration of macrophages and granulocytes was not significantly elevated in C3 KO or C5 KO mice compared with in wild-type mice. Impaired opsonization and decreased migration of macrophages and granulocytes due to C3 deficiency should therefore induce the accumulation of dead cells and may lead to increased infiltration of CD4+ T cells. CONCLUSIONS: Vigilance for exacerbation of lymphedema is necessary when surgical treatments have the potential to injure lymphatic vessels in patients undergoing complement-targeted therapies or with complement deficiency. Future studies should aim to elucidate the molecular mechanism of CD4+ T cell infiltration by accumulated dead cells.
Assuntos
Vasos Linfáticos , Linfedema , Humanos , Animais , Camundongos , Qualidade de Vida , Linfedema/etiologia , Linfedema/metabolismo , Linfedema/patologia , Linfócitos T CD4-Positivos , Inflamação , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure.
Assuntos
Leucemia , Complexo de Proteínas Formadoras de Poros Nucleares , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia/metabolismo , Cromatina , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Corpos NuclearesRESUMO
Polycomb group proteins (PcG), polycomb repressive complexes 1 and 2 (PRC1 and 2), repress lineage inappropriate genes during development to maintain proper cellular identities. It has been recognized that PRC1 localizes at the replication fork, however, the precise functions of PRC1 during DNA replication are elusive. Here, we reveal that a variant PRC1 containing PCGF1 (PCGF1-PRC1) prevents overloading of activators and chromatin remodeling factors on nascent DNA and thereby mediates proper deposition of nucleosomes and correct downstream chromatin configurations in hematopoietic stem and progenitor cells (HSPCs). This function of PCGF1-PRC1 in turn facilitates PRC2-mediated repression of target genes such as Hmga2 and restricts premature myeloid differentiation. PCGF1-PRC1, therefore, maintains the differentiation potential of HSPCs by linking proper nucleosome configuration at the replication fork with PcG-mediated gene silencing to ensure life-long hematopoiesis.
Assuntos
Cromatina , Replicação do DNA , Cromatina/genética , Linhagem da Célula/genética , Nucleossomos/genética , Proteínas do Grupo Polycomb , Complexo Repressor Polycomb 2RESUMO
The murine penile erectile tissues including corpus cavernosum (CC) are composed of blood vessels, smooth muscle, and connective tissue, showing marked sexual differences. It has been known that the androgens are required for sexually dimorphic organogenesis. It is however unknown about the features of androgen signaling during mouse CC development. It is also unclear how androgen-driven downstream factors are involved such processes. In the current study, we analyzed the onset of sexually dimorphic CC formation based on histological analyses, the dynamics of androgen receptor (AR) expression, and regulation of cell proliferation. Of note, we identified Dickkopf-related protein 2 (Dkk2), an inhibitor of ß-catenin signaling, was predominantly expressed in female CC compared with male. Furthermore, administration of androgens resulted in activation of ß-catenin signaling. We have found the Sox9 gene, one of the essential markers for chondrocyte, was specifically expressed in the developing CC. Hence, we utilized CC-specific, Sox9 CreERT2 , ß-catenin conditional mutant mice. Such mutant mice showed defective cell proliferation. Furthermore, introduction of activated form of ß-catenin mutation (gain of function mutation for Wnt/ß-catenin signaling) in CC induced augmented cell proliferation. Altogether, we revealed androgen-Wnt/ß-catenin signal dependent cell proliferation was essential for sexually dimorphic CC formation. These findings open new avenues for understanding developmental mechanisms of androgen-dependent cell proliferation during sexual differentiation.
Assuntos
Androgênios , beta Catenina , Androgênios/genética , Androgênios/farmacologia , Animais , Proliferação de Células , Feminino , Masculino , Camundongos , Pênis , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore, there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1 domain of the viral L protein to the Na+/taurocholate cotransporting polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/hepatitis D virus (HDV) infection and has been approved as Hepcludex in Europe for the treatment of patients with chronic HDV infection. We established a monoclonal antibody (MAb), N6HB426-20, that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo, administration of the N6HB426-20 MAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84 to 87 in ECL1 and aa 157 to 165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope mapping and the three-dimensional (3D) model of the NTCP structure suggested that the N6HB426-20 MAb may recognize aa 276/277 at the tip of ECL4 and interfere with binding of HBV to the region from aa 84 to 87. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleoside/nucleotide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic MAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these MAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations, and the presence of subviral particles. Although N6HB426-20 requires a higher dose than myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time postadministration in proportion to the half-life of an IgG MAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.
Assuntos
Vírus da Hepatite B , Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Simportadores , Internalização do Vírus , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Células Hep G2 , Hepatite B/tratamento farmacológico , Hepatite B/prevenção & controle , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos , Humanos , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
BACKGROUND: Because of the high frequency of chronic edema formation in the current "aged" society, analyses and detailed observation of post-surgical edema are getting more required. Post-surgical examination of the dynamic vasculature including L.V. (Lymphatic Vasculature) to monitor edema formation has not been efficiently performed. Hence, procedures for investigating such vasculature are essential. By inserting transparent sheet into the cutaneous layer of mouse tails as a novel surgery model (the Tail Edema by Silicone sheet mediated Transparency protocol; TEST), the novel procedures are introduced and analyzed by series of histological analyses including video-based L.V. observation and 3D histological reconstruction of vasculatures in mouse tails. RESULTS: The dynamic generation of post-surgical main and fine (neo) L.V. connective structure during the edematous recovery process was visualized by series of studies with a novel surgery model. Snapshot images taken from live binocular image recording for TEST samples suggested the presence of main and elongating fine (neo) L.V. structure. After the ligation of L.V., the enlargement of main L.V. was confirmed. In the case of light sheet fluorescence microscopy (LSFM) observation, such L.V. connections were also suggested by using transparent 3D samples. Finally, the generation of neo blood vessels particularly in the region adjacent to the silicone sheet and the operated boundary region was suggested in 3D reconstruction images. However, direct detection of elongating fine (neo) L.V. was not suitable for analysis by such LSFM and 3D reconstruction procedures. Thus, such methods utilizing fixed tissues are appropriate for general observation for the operated region including of L.V. CONCLUSIONS: The current surgical procedures and analysis on the post-surgical status are the first case to observe vasculatures in vivo with a transparent sheet. Systematic analyses including the FITC-dextran mediated snap shot images observation suggest the elongation of fine (neo) lymphatic vasculature. Post-surgical analyses including LSFM and 3D histological structural reconstruction, are suitable to reveal the fixed structures of blood and lymphatic vessels formation.
RESUMO
Testicular androgen is a master endocrine factor in the establishment of external genital sex differences. The degree of androgenic exposure during development is well known to determine the fate of external genitalia on a spectrum of female- to male-specific phenotypes. However, the mechanisms of androgenic regulation underlying sex differentiation are poorly defined. Here, we show that the genomic environment for the expression of male-biased genes is conserved to acquire androgen responsiveness in both sexes. Histone H3 at lysine 27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1) are enriched at the enhancer of male-biased genes in an androgen-independent manner. Specificity protein 1 (Sp1), acting as a collaborative transcription factor of androgen receptor, regulates H3K27ac enrichment to establish conserved transcriptional competency for male-biased genes in both sexes. Genetic manipulation of MafB, a key regulator of male-specific differentiation, and Sp1 regulatory MafB enhancer elements disrupts male-type urethral differentiation. Altogether, these findings demonstrate conservation of androgen responsiveness in both sexes, providing insights into the regulatory mechanisms underlying sexual fate during external genitalia development.
Assuntos
Genitália Masculina/metabolismo , Diferenciação Sexual , Acetilação , Androgênios , Animais , Sistemas CRISPR-Cas , Feminino , Regulação da Expressão Gênica , Histonas/metabolismo , Fator de Transcrição MafB , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Receptores Androgênicos , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Coatomer subunit alpha (COPA) syndrome, also known as autoinflammatory interstitial lung, joint, and kidney disease, is caused by heterozygous mutations in COPA. We identified a novel COPA variant in 4 patients in one family. We undertook this study to elucidate whether and how the variant causes manifestations of COPA syndrome by studying these 4 patients and by analyzing results from a gene-targeted mouse model. METHODS: We performed whole-exome sequencing in 7 family members and measured the type I interferon (IFN) signature of the peripheral blood cells. We analyzed the effects of COPA variants in in vitro experiments and in Copa mutant mice that were generated. RESULTS: We identified a heterozygous variant of COPA (c.725T>G, p.Val242Gly) in the 4 affected members of the family. The IFN score was high in the members carrying the variant. In vitro analysis revealed that COPA V242G, as well as the previously reported disease-causing variants, augmented stimulator of interferon genes (STING)-induced type I IFN promoter activities. CopaV242G/+ mice manifested interstitial lung disease and STING-dependent elevation of IFN-stimulated gene expression. In CopaV242G/+ dendritic cells, the STING pathway was not constitutively activated but was hyperactivated upon stimulation, leading to increased type I IFN production. CONCLUSION: V242G, a novel COPA variant, was found in 4 patients from one family. In gene-targeted mice with the V242G variant, interstitial lung disease was recapitulated and augmented responses of the STING pathway, leading to an increase in type I IFN production, were demonstrated.
Assuntos
Proteína Coatomer/genética , Interferon Tipo I/genética , Artropatias/genética , Nefropatias/genética , Doenças Pulmonares Intersticiais/genética , Mutação de Sentido Incorreto , Alelos , Análise Mutacional de DNA , Feminino , Heterozigoto , Humanos , Artropatias/imunologia , Nefropatias/imunologia , Doenças Pulmonares Intersticiais/imunologia , Masculino , Linhagem , Sequenciamento do ExomaRESUMO
The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation of Phc2 in mice causes a severe defect in HSPC mobilization through the derepression of Vcam1 in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1 in Phc2-deficient mice reverses the symptoms. We further determine that Phc2-dependent Vcam1 repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub. Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche.
Assuntos
Movimento Celular/genética , Repressão Epigenética , Células-Tronco Hematopoéticas/imunologia , Complexo Repressor Polycomb 2/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Animais , Transplante de Medula Óssea/efeitos adversos , Movimento Celular/imunologia , Células Cultivadas , Metilação de DNA/imunologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Complexo Repressor Polycomb 2/genética , Cultura Primária de Células , Molécula 1 de Adesão de Célula Vascular/antagonistas & inibidoresRESUMO
Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.
Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Espermátides/crescimento & desenvolvimento , Espermatogênese , Transativadores/metabolismo , Acetilação , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Histona Acetiltransferases/genética , Lisina Acetiltransferase 5 , Masculino , Camundongos , Proteínas Repressoras/genética , Espermátides/metabolismo , Transativadores/genéticaRESUMO
In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.
Assuntos
Linfócitos B/citologia , Transformação Celular Neoplásica/genética , Epigênese Genética/genética , Inativação Gênica , Proteínas do Grupo Polycomb/metabolismo , Linfócitos T/citologia , Animais , Linhagem da Célula , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos Endogâmicos C57BL , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The CCR4-NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre-B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4-NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA.
Assuntos
Linfócitos B/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B/imunologia , Estabilidade de RNA/imunologia , RNA Mensageiro/imunologia , Fatores de Transcrição/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Camundongos , Camundongos Transgênicos , Estabilidade de RNA/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Oxirredutases do Álcool , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Proteínas Correpressoras , DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição Kruppel-Like/química , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Ativação TranscricionalRESUMO
BACKGROUND: The presence of somatic mutations in splicing factor 3b subunit 1 (SF3B1) in patients with Myelodysplastic syndromes with ring sideroblasts (MDS-RS) highlights the importance of the RNA-splicing machinery in MDS. We previously reported the presence of bone marrow (BM) RS in Sf3b1 heterozygous (Sf3b1 (+/-)) mice which are rarely found in mouse models of MDS. Sf3b1 (+/-) mice were originally engineered to study the interaction between polycomb genes and other proteins. METHODS: We used routine blood tests and histopathologic analysis of BM, spleen, and liver to evaluate the hematologic and morphologic characteristics of Sf3b1 (+/-) mice in the context of MDS by comparing the long term follow-up (15 months) of Sf3b1 (+/-) and Sf3b1 (+/+) mice. We then performed a comprehensive RNA-sequencing analysis to evaluate the transcriptome of BM cells from Sf3b1 (+/-) and Sf3b1 (+/+) mice. RESULTS: Sf3b1 (+/-) exhibited macrocytic anemia (MCV: 49.5 ± 1.6 vs 47.2 ± 1.4; Hgb: 5.5 ± 1.7 vs 7.2 ± 1.0) and thrombocytosis (PLTs: 911.4 ± 212.1 vs 878.4 ± 240.9) compared to Sf3b1 (+/+) mice. BM analysis showed dyserythropoiesis and occasional RS in Sf3b1 (+/-) mice. The splenic architecture showed increased megakaryocytes with hyperchromatic nuclei, and evidence of extramedullary hematopoiesis. RNA-sequencing showed higher expression of a gene set containing Jak2 in Sf3b1 (+/-) compared to Sf3b1 (+/+). CONCLUSIONS: Our study indicates that Sf3b1 (+/-) mice manifest features of low risk MDS-RS and may be relevant for preclinical therapeutic studies.
Assuntos
Anemia Sideroblástica/genética , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Anemia Sideroblástica/sangue , Animais , Modelos Animais de Doenças , Feminino , Genótipo , Haploinsuficiência/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fosfoproteínas/sangue , Splicing de RNA , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/sangue , Fatores de RiscoRESUMO
In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.
Assuntos
Diferenciação Celular , Proliferação de Células , Complexo Repressor Polycomb 2/fisiologia , Animais , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Estrutura Terciária de ProteínaRESUMO
Numerous studies have recently reported mutations involving multiple components of the messenger RNA (mRNA) splicing machinery in patients with myelodysplastic syndrome (MDS). SF3B1 is mutated in 70% to 85% of refractory anemia with ringed sideroblasts (RARS) patients and is highly associated with the presence of RARS, although the pathological role of SF3B1 mutations in MDS-RARS has not been elucidated yet. Here, we analyzed the function of pre-mRNA splicing factor Sf3b1 in hematopoiesis. Sf3b1(+/-) mice maintained almost normal hematopoiesis and did not develop hematological malignancies during a long observation period. However, Sf3b1(+/-) cells had a significantly impaired capacity to reconstitute hematopoiesis in a competitive setting and exhibited some enhancement of apoptosis, but they did not show any obvious defects in differentiation. Additional depletion of Sf3b1 with shRNA in Sf3b1(+/-) hematopoietic stem cells (HSCs) severely compromised their proliferative capacity both in vitro and in vivo. Finally, we unexpectedly found no changes in the frequencies of sideroblasts in either Sf3b1(+/-) erythroblasts or cultured Sf3b1(+/-) erythroblasts expressing shRNA against Sf3b1. Our findings indicate that the level of Sf3b1 expression is critical for the proliferative capacity of HSCs, but the haploinsufficiency for Sf3b1 is not sufficient to induce a RARS-like phenotype.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Anemia Refratária/genética , Anemia Refratária/patologia , Animais , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Haploidia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Precursores de RNA/genética , Splicing de RNA , Fatores de Processamento de RNA , RNA Interferente Pequeno/genéticaRESUMO
UNLABELLED: Polycomb-group (PcG) proteins play crucial roles in self-renewal of stem cells by suppressing a host of genes through histone modifications. Identification of the downstream genes of PcG proteins is essential for elucidation of the molecular mechanisms of stem cell self-renewal. However, little is known about the PcG target genes in tissue stem cells. We found that the PcG protein, Ring1B, which regulates expression of various genes through monoubiquitination of histone H2AK119, is essential for expansion of hepatic stem/progenitor cells. In mouse embryos with a conditional knockout of Ring1B, we found that the lack of Ring1B inhibited proliferation and differentiation of hepatic stem/progenitor cells and thereby inhibited hepatic organogenesis. These events were characterized by derepression of cyclin-dependent kinase inhibitors (CDKIs) Cdkn1a and Cdkn2a, known negative regulators of cell proliferation. We conducted clonal culture experiments with hepatic stem/progenitor cells to investigate the individual genetic functions of Ring1B, Cdkn1a, and Cdkn2a. The data showed that the cell-cycle inhibition caused by Ring1B depletion was reversed when Cdkn1a and Cdkn2a were suppressed simultaneously, but not when they were suppressed individually. CONCLUSION: Our results show that expansion of hepatic stem/progenitor cells requires Ring1B-mediated epigenetic silencing of Cdkn1a and Cdkn2a, demonstrating that Ring1B simultaneously regulates multiple CDKIs in tissue stem/progenitor cells.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células-Tronco Embrionárias/citologia , Fígado/citologia , Complexo Repressor Polycomb 1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Epigênese Genética/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fígado/embriologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos Knockout , Organogênese/fisiologia , Complexo Repressor Polycomb 1/genética , Gravidez , Ubiquitina-Proteína Ligases/genéticaRESUMO
Polycomb-group (PcG) proteins mediate repression of developmental regulators in a reversible manner, contributing to their spatiotemporally regulated expression. However, it is poorly understood how PcG-repressed genes are activated by developmental cues. Here, we used the mouse Meis2 gene as a model to identify a role of a tissue-specific enhancer in removing PcG from the promoter. Meis2 repression in early development depends on binding of RING1B, an essential E3 component of PcG, to its promoter, coupled with its association with another RING1B-binding site (RBS) at the 3' end of the Meis2 gene. During early midbrain development, a midbrain-specific enhancer (MBE) transiently associates with the promoter-RBS, forming a promoter-MBE-RBS tripartite interaction in a RING1-dependent manner. Subsequently, RING1B-bound RBS dissociates from the tripartite complex, leaving promoter-MBE engagement to activate Meis2 expression. This study therefore demonstrates that PcG and/or related factors play a role in Meis2 activation by regulating the topological transition of cis-regulatory elements.
Assuntos
Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/metabolismo , Mesencéfalo/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Prosencéfalo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos , Especificidade de Órgãos , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas , Ligação Proteica , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
The Polycomb-group (PcG) repressive complex-1 (PRC1) forms microscopically visible clusters in nuclei; however, the impact of this cluster formation on transcriptional regulation and the underlying mechanisms that regulate this process remain obscure. Here, we report that the sterile alpha motif (SAM) domain of a PRC1 core component Phc2 plays an essential role for PRC1 clustering through head-to-tail macromolecular polymerization, which is associated with stable target binding of PRC1/PRC2 and robust gene silencing activity. We propose a role for SAM domain polymerization in this repression by two distinct mechanisms: first, through capturing and/or retaining PRC1 at the PcG targets, and second, by strengthening the interactions between PRC1 and PRC2 to stabilize transcriptional repression. Our findings reveal a regulatory mechanism mediated by SAM domain polymerization for PcG-mediated repression of developmental loci that enables a robust yet reversible gene repression program during development.