RESUMEN
Myelin sheath formation in the peripheral nervous system and the ensuing saltatory conduction rely on differentiated Schwann cells. We have previously shown that transition of Schwann cells from an immature into a differentiated state requires Brg1 that serves as the central energy generating subunit in two related SWI/SNF-type chromatin remodelers, the BAF and the PBAF complex. Here we used conditional deletion of Pbrm1 to selectively interfere with the PBAF complex in Schwann cells. Despite efficient loss of Pbrm1 early during lineage progression, we failed to detect any substantial alterations in the number, proliferation or survival of immature Schwann cells as well as in their rate and timing of terminal differentiation. As a consequence, postnatal myelin formation in peripheral nerves appeared normal. There were no inflammatory alterations in the nerve or other signs of a peripheral neuropathy. We conclude from our study that Pbrm1 and very likely the PBAF complex are dispensable for proper Schwann cell development and that Schwann cell defects previously observed upon Brg1 deletion are mostly attributable to altered or absent function of the BAF complex.
Asunto(s)
Diferenciación Celular/genética , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Células de Schwann/fisiología , Factores de Transcripción/fisiología , Animales , Linaje de la Célula/genética , Proliferación Celular/genética , Supervivencia Celular/genética , ADN Helicasas/genética , Eliminación de Gen , Ratones , Vaina de Mielina/fisiología , Proteínas Nucleares/genética , Nervios Periféricos/fisiología , Factores de Transcripción/genéticaRESUMEN
PURPOSE: To investigate the effect of microRNA-543 (miR-543) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of triple-negative breast cancer (TNBC) cells, and the associated mechanism. METHODS: Human breast cancer cells (MDA-MB-231, HCC1937, and MCF-7, ZR-75-1) and normal human breast epithelial cell line (MCF10A) were transfected with miR-543 mimics or inhibitor using lipofectamine 2000. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the mRNA and protein expression levels of miR-543, actin-like protein 6A (ACTL6A), vimentin, Snail, and E-cadherin in breast cancer cells/tissue. Cell counting kit-8 (CCK-8), wound-healing, and Transwell assays were used to measure the effect of miR-543 on TNBC cell proliferation, invasion, and migration. Overall survival was determined using data from Gene Expression Omnibus (GEO) and Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis and luciferase reporter gene assay were used to determine the regulatory effect of miR-543 on ACTL6A. RESULTS: The level of expression of miR-543 was significantly lower in breast cancer cells/tissue than in normal human breast epithelial cell/tissue (p < 0.05). MicroRNA-543 expression level was significantly reduced in TNBC cells/tissue, relative to the other breast cancer cells/normal breast tissue (p < 0.05). MicroRNA-543 significantly suppressed tumor growth and the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells, in mouse xenograft model (p < 0.05). CONCLUSIONS: miR-543 influences the biological behavior of TNBC cells by directly targeting ACTL6A gene. miR-543 could serve as a novel diagnostic and therapeutic target for TNBC.
Asunto(s)
Actinas/fisiología , Movimiento Celular , Proliferación Celular , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , MicroARNs/fisiología , Neoplasias de la Mama Triple Negativas/patología , Animales , Humanos , Ratones , Invasividad Neoplásica , Células Tumorales CultivadasRESUMEN
Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain-deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína Quinasa CDC2/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Histonas/metabolismo , Interfase , Cinetocoros/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Mitosis , Fosforilación , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologíaRESUMEN
Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/fisiología , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Cromatina/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Cromosomas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Epigénesis Genética , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
STAG2, a component of the mitotically essential cohesin complex, is highly mutated in several different tumour types, including glioblastoma and bladder cancer. Whereas cohesin has roles in many cancer-related pathways, such as chromosome instability, DNA repair and gene expression, the complex nature of cohesin function has made it difficult to determine how STAG2 loss might either promote tumorigenesis or be leveraged therapeutically across divergent cancer types. Here, we have performed whole-genome CRISPR-Cas9 screens for STAG2-dependent genetic interactions in three distinct cellular backgrounds. Surprisingly, STAG1, the paralog of STAG2, was the only negative genetic interaction that was shared across all three backgrounds. We also uncovered a paralogous synthetic lethal mechanism behind a genetic interaction between STAG2 and the iron regulatory gene IREB2 Finally, investigation of an unusually strong context-dependent genetic interaction in HAP1 cells revealed factors that could be important for alleviating cohesin loading stress. Together, our results reveal new facets of STAG2 and cohesin function across a variety of genetic contexts.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Carcinogénesis , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Humanos , Proteína 2 Reguladora de Hierro/genética , Proteína 2 Reguladora de Hierro/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mutaciones Letales Sintéticas , CohesinasRESUMEN
Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Regulación de la Expresión Génica , Heterocromatina/fisiología , Células Cultivadas , Secuencia Conservada , Células Hep G2 , Histonas/metabolismo , HumanosRESUMEN
Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/metabolismo , Heterocromatina/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Animales , Quinasa de la Caseína II/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Cricetinae , Células Madre Embrionarias/citología , Técnicas de Inactivación de Genes , Humanos , Ratones , Fosforilación , Serina/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , Proteína 28 que Contiene Motivos Tripartito/fisiologíaRESUMEN
To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Meiosis/fisiología , Separasa/metabolismo , Animales , Animales no Consanguíneos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , División Celular/fisiología , División del Núcleo Celular , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Femenino , Células HeLa , Humanos , Cinetocoros/metabolismo , Ratones , Oocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Separasa/fisiología , Quinasa Tipo Polo 1RESUMEN
During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1-/- tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Oogénesis/fisiología , Espermatogénesis/fisiología , Pez Cebra/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Emparejamiento Cromosómico , Femenino , Genes p53 , Gónadas/anatomía & histología , Masculino , Mutación , Pez Cebra/fisiología , CohesinasRESUMEN
Transcription elongation can be affected by numerous types of obstacles, such as nucleosome, pausing sequences, DNA lesions and non-B-form DNA structures. Spt4/5 and Elf1 are conserved transcription elongation factors that promote RNA polymerase II (Pol II) bypass of nucleosome and pausing sequences. Importantly, genetic studies have shown that Spt4/5 plays essential roles in the transcription of expanded nucleotide repeat genes associated with inherited neurological diseases. Here, we investigate the function of Spt4/5 and Elf1 in the transcription elongation of CTGâ¢CAG repeat using an in vitro reconstituted yeast transcription system. We found that Spt4/5 helps Pol II transcribe through the CTGâ¢CAG tract duplex DNA, which is in good agreement with its canonical roles in stimulating transcription elongation. In sharp contrast, surprisingly, we revealed that Spt4/5 greatly inhibits Pol II transcriptional bypass of CTG and CAG slip-out structures. Furthermore, we demonstrated that transcription elongation factor Elf1 individually and cooperatively with Spt4/5 inhibits Pol II bypass of the slip-out structures. This study uncovers the important functional interplays between template DNA structures and the function of transcription elongation factors. This study also expands our understanding of the functions of Spt4/5 and Elf1 in transcriptional processing of trinucleotide repeat DNA.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , ADN Forma B/química , ADN/química , Proteínas Nucleares/fisiología , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Transcripción Genética , Factores de Elongación Transcripcional/fisiología , Repeticiones de TrinucleótidosRESUMEN
Similar to other core biological processes, the vast majority of cell division components are essential for viability across human cell lines. However, recent genome-wide screens have identified a number of proteins that exhibit cell line-specific essentiality. Defining the behaviors of these proteins is critical to our understanding of complex biological processes. Here, we harness differential essentiality to reveal the contributions of the four-subunit centromere-localized CENP-O complex, whose precise function has been difficult to define. Our results support a model in which the CENP-O complex and BUB1 act in parallel pathways to recruit a threshold level of PLK1 to mitotic kinetochores, ensuring accurate chromosome segregation. We demonstrate that targeted changes to either pathway sensitizes cells to the loss of the other component, resulting in cell-state dependent requirements. This approach also highlights the advantage of comparing phenotypes across diverse cell lines to define critical functional contributions and behaviors that could be exploited for the targeted treatment of disease.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Centrómero/metabolismo , Centrómero/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Histonas/genética , Histonas/fisiología , Humanos , Cinetocoros/fisiología , Mitosis/fisiología , Unión Proteica , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Quinasa Tipo Polo 1RESUMEN
The curiosity to understand the mechanisms regulating transcription in pluripotent cells resulted in identifying a unique transcription factor named Undifferentiated embryonic cell transcription factor 1 (UTF1). This proline-rich, nuclear protein is highly conserved among placental mammals with prominent expression observed in pluripotent, germ, and cancer cells. In pluripotent and germ cells, its role has been implicated primarily in proper cell differentiation, whereas in cancer, it shows tissue-specific function, either as an oncogene or a tumor suppressor gene. Furthermore, UTF1 is crucial for germ cell development, spermatogenesis, and maintaining male fertility in mice. In addition, recent studies have demonstrated the importance of UTF1 in the generation of high quality induced Pluripotent Stem Cells (iPSCs) and as an excellent biomarker to identify bona fide iPSCs. Functionally, UTF1 aids in establishing a favorable chromatin state in embryonic stem cells, reducing "transcriptional noise" and possibly functions similarly in re-establishing this state in differentiated cells upon their reprogramming to generate mature iPSCs. This review highlights the multifaceted roles of UTF1 and its implication in development, spermatogenesis, stem, and cancer cells.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Células Madre Embrionarias , Neoplasias , Transactivadores , Animales , Femenino , Masculino , Ratones , Neoplasias/genética , Proteínas Nucleares , Placenta , Embarazo , Transactivadores/fisiología , Factores de TranscripciónRESUMEN
UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor that regulates UV-B photomorphogenesis in plants. UV-B photon perception promotes UVR8 homodimer dissociation into monomer, which is reverted to homodimer post UV-B, forming a complete photocycle. UVR8 monomer interacts with CONSTITUTIVELY PHOTOMORPHOGENEIC 1 (COP1) to initiate UV-B signaling. The function and mechanism of Arabidopsis UVR8 (AtUVR8) are extensively investigated, however, little is known about UVR8 and its signaling mechanisms in other plant species. Tomato is a widely used model plant for horticulture research. In this report we tested whether an ortholog of AtUVR8 in Tomato (SIUVR8) can complement Arabidopsis uvr8 mutant and whether the above-mentioned key signaling mechanisms of UVR8 are conserved. Heterologous expressed SIUVR8 in an Arabidopsis uvr8 null mutant rescued the uvr8 mutant in the tested UV-B responses including hypocotyl elongation, UV-B target gene expression and anthocyanin accumulation, demonstrating that the SIUVR8 is a putative UV-B photoreceptor. Moreover, in response to UV-B, SIUVR8 forms a protein complex with Arabidopsis COP1 in plants, suggesting conserved signaling mechanism. SIUVR8 exhibits similar photocycle as AtUVR8 in plants, which highlights conserved photoreceptor activation and inactivation mechanisms.
Asunto(s)
Fotorreceptores de Plantas/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Antocianinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Secuencia Conservada/genética , Luz , Solanum lycopersicum/metabolismo , Fotorreceptores de Plantas/metabolismo , Fotorreceptores de Plantas/fisiología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Cytokine activation of cells induces gene networks involved in inflammation and immunity. Transient gene activation can have a lasting effect even in the absence of ongoing transcription, known as long-term transcriptional memory. Here we explore the nature of the establishment and maintenance of interferon γ (IFNγ)-induced priming of human cells. We find that, although ongoing transcription and local chromatin signatures are short-lived, the IFNγ-primed state stably propagates through at least 14 cell division cycles. Single-cell analysis reveals that memory is manifested by an increased probability of primed cells to engage in target gene expression, correlating with the strength of initial gene activation. Further, we find that strongly memorized genes tend to reside in genomic clusters and that long-term memory of these genes is locally restricted by cohesin. We define the duration, stochastic nature, and molecular mechanisms of IFNγ-induced transcriptional memory, relevant to understanding enhanced innate immune signaling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Interferón gamma/metabolismo , Activación Transcripcional/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular , Cromatina/genética , Proteínas Cromosómicas no Histona/fisiología , Regulación de la Expresión Génica/inmunología , Células HeLa , Humanos , Inflamación , Interferón gamma/fisiología , Unión Proteica/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/genética , Transcripción Genética/genética , Activación Transcripcional/fisiología , CohesinasRESUMEN
While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Cinetocoros/metabolismo , Meiosis , Animales , Centrómero/metabolismo , Drosophila , Células HeLa , Humanos , RatonesRESUMEN
Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromatina/fisiología , Proteínas Cromosómicas no Histona/fisiología , Técnicas de Transferencia Nuclear , Cigoto/fisiología , Animales , Línea Celular , Núcleo Celular , Ensamble y Desensamble de Cromatina , Biología Computacional/métodos , Conjuntos de Datos como Asunto , Desarrollo Embrionario , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , CohesinasRESUMEN
The conserved kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments and 2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to kinetochores and for generation of kinetochore-microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates kinetochore-microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex--microtubule interface.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Cinetocoros/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Proteínas del Citoesqueleto/fisiología , Células HeLa , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiología , Mitosis , Proteínas Nucleares/metabolismo , FosforilaciónRESUMEN
Histones are highly basic proteins involved in packaging DNA into chromatin, and histone modifications are fundamental in epigenetic regulation in eukaryotes. Among the numerous chromatin modifiers identified in Arabidopsis (Arabidopsis thaliana), MORF-RELATED GENE (MRG)1 and MRG2 have redundant functions in reading histone H3 lysine 36 trimethylation (H3K36me3). Here, we show that MRG2 binds histone chaperones belonging to the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) family, including NAP1-RELATED PROTEIN (NRP)1 and NRP2. Characterization of the loss-of-function mutants mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 revealed that MRG1/MRG2 and NRP1/NRP2 regulate flowering time through fine-tuning transcription of floral genes by distinct molecular mechanisms. In particular, the physical interaction between NRP1/NRP2 and MRG1/MRG2 inhibited the binding of MRG1/MRG2 to the transcription factor CONSTANS (CO), leading to a transcriptional repression of FLOWERING LOCUS T (FT) through impeded H4K5 acetylation (H4K5ac) within the FT chromatin. By contrast, NRP1/NRP2 and MRG1/MRG2 act together, likely in a multiprotein complex manner, in promoting the transcription of FLOWERING LOCUS C (FLC) via an increase of both H4K5ac and H3K9ac in the FLC chromatin. Because the expression pattern of FLC represents the major category of differentially expressed genes identified by genome-wide RNA-sequencing analysis in the mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 mutants, it is reasonable to speculate that the NRP1/NRP2-MRG1/MRG2 complex may be involved in transcriptional activation of genes beyond FLC and flowering time control.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Proteínas Cromosómicas no Histona/fisiología , Flores/crecimiento & desarrollo , Chaperonas de Histonas/fisiología , Chaperonas Moleculares/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Flores/metabolismo , Flores/fisiología , Genes de Plantas/genética , Genes de Plantas/fisiología , Estudio de Asociación del Genoma Completo , Chaperonas de Histonas/metabolismo , Código de Histonas , Chaperonas Moleculares/metabolismoRESUMEN
Chromosome alignment and biorientation is essential for mitotic progression and genomic stability. Most chromosomes align at the spindle equator in a motor-independent manner. However, a subset of polar kinetochores fail to bi-orient and require a microtubule motor-based transport mechanism to move to the cell equator. Centromere Protein E (CENP-E/KIF10) is a kinesin motor from the Kinesin-7 family, which localizes to unattached kinetochores during mitosis and utilizes plus-end directed microtubule motility to slide mono-oriented chromosomes to the spindle equator. Recent work has revealed how CENP-E cooperates with chromokinesins and dynein to mediate chromosome congression and highlighted its role at aligned chromosomes. Additionally, we have gained new mechanistic insights into the targeting and regulation of CENP-E motor activity at the kinetochore. Here, we will review the function of CENP-E in chromosome congression, the pathways that contribute to CENP-E loading at the kinetochore, and how CENP-E activity is regulated during mitosis.
Asunto(s)
Centrómero/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitosis , Huso Acromático/metabolismo , Animales , HumanosRESUMEN
Spindle and kinetochore associated complex subunit 2 (SKA2) is a part of the spindle and kinetochore associated (SKA) complex, which has been reported in various cancers, including the breast cancer, lung cancer, and glioma. However, its role remains unclear in hepatocellular carcinoma (HCC). Our study found that SKA2 mRNA levels and immunohistochemistry staining were significantly increased in HCC tissues compared with normal tissues. The role of SKA2 in cell proliferation and invasion was also determined. Overexpression of SKA2 significantly promoted cell proliferation and invasion, while knocking down of SKA2 expression inhibited the growth and invasion of HCC cells. In experiments investigating the underlying mechanism, overexpression of SKA2 may increase the expression levels of total ß-catenin, and knockdown of SKA2 may decrease the expression levels of total ß-catenin. Our studies thus suggest that SKA2 may promote proliferation and invasion of hepatocellular carcinoma cells by activating the ß-catenin signaling pathway, which can serve as a potential target in the diagnosis and/or treatment of HCC.