RESUMEN
Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.
Asunto(s)
Proteínas de Ciclo Celular/genética , Silenciador del Gen , Heterocromatina/química , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Proteínas Argonautas/metabolismo , Inmunoprecipitación de Cromatina , ADN/química , Relación Dosis-Respuesta a Droga , Factores Eucarióticos de Iniciación/metabolismo , Regulación Fúngica de la Expresión Génica , Cinética , Metilación , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN/química , Homología de Secuencia de AminoácidoRESUMEN
A woman in her 30s presented to our hospital with the chief complaint of a right breast mass after the birth of her first child. She was diagnosed as having right invasive ductal carcinoma of Luminal-B type and T3N3cM0, stage â ¢c. While undergoing neoadjuvant chemotherapy, she received genetic counseling and underwent genetic testing and was determined to have deleterious BRCA1 and BRCA2 mutations. After completing chemotherapy, she underwent a right total mastectomy and axillary lymph node dissection. Two years postoperatively, she requested to undergo a contralateral risk-reducing mastectomy( CRRM)of her left breast. Therefore, CT and breast MRI were performed to confirm the absence of contralateral lesions and distant metastases, and subsequently, CRRM was performed. Postoperative pathology results showed non-invasive ductal carcinoma lesions at 5 sites. In the case of hereditary breast and ovarian cancer syndrome such as in this study, lesions may be discovered at an early stage by performing risk-reducing mastectomy.
Asunto(s)
Neoplasias de la Mama , Carcinoma Ductal de Mama , Carcinoma Ductal , Carcinoma Intraductal no Infiltrante , Síndrome de Cáncer de Mama y Ovario Hereditario , Neoplasias de la Mama/genética , Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/cirugía , Niño , Femenino , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Síndrome de Cáncer de Mama y Ovario Hereditario/cirugía , Humanos , MastectomíaRESUMEN
In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1(+) mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6(+) reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Interferencia de ARN/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/genética , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina , Metiltransferasas/genética , Metiltransferasas/metabolismo , Microscopía Fluorescente , Mutación , Unión Proteica , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transducción de SeñalRESUMEN
RNA interference (RNAi) is a conserved silencing mechanism that has widespread roles in RNA degradation, translational repression, and the epigenetic control of chromatin structure [1]. In fission yeast, heterochromatin assembly requires RNAi machinery and is initiated by small interference RNAs (siRNAs) derived from heterochromatic regions and by the RNA-induced transcriptional silencing (RITS) complex [2-7]. Although recent studies have been successful in uncovering the functions of effector complexes in the RNAi pathway [4, 5, 8-10], exactly how heterochromatic siRNAs are processed and function in assembling heterochromatin remains unclear. In this study we focused on a conserved ribonuclease, Eri1, which was originally identified as a negative regulator of RNAi in C. elegans [11], and show the importance of the Eri1 protein in RNAi-mediated heterochromatin assembly in fission yeast. Eri1 specifically degrades double-stranded siRNAs through two functional domains and represses the accumulation of cellular siRNAs in vivo. Deletion of eri1(+) causes an increase in siRNAs associated with the RITS complex and enhances heterochromatic silencing, which is accompanied by increased levels of histone H3-K9 methylation and the Swi6 protein. Our findings suggest that the fission yeast Eri1 controls the accumulation of heterochromatic siRNAs and negatively regulates the RNAi-mediated heterochromatin assembly.
Asunto(s)
Exorribonucleasas/fisiología , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Secuencia Conservada , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Eliminación de Gen , Interferencia de ARN/fisiología , ARN Interferente Pequeño/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.
Asunto(s)
Proteínas Arqueales/metabolismo , Chaperoninas/clasificación , Chaperoninas/metabolismo , Glicoproteínas de Membrana , Pliegue de Proteína , Thermococcus/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/clasificación , Proteínas Arqueales/ultraestructura , Proteínas de Unión al Calcio/metabolismo , Chaperonina 10/química , Chaperonina 10/fisiología , Chaperoninas/química , Chaperoninas/ultraestructura , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica , Modelos Biológicos , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Serina Endopeptidasas/metabolismo , Thermococcus/genética , Termolisina/metabolismoRESUMEN
The archaeal chaperonin-mediated folding of green fluorescent protein (GFP) was examined in the presence of various nucleotides. The recombinant alpha- and beta-subunit homo-oligomers and natural chaperonin oligomer from Thermococcus strain KS-1 exhibited folding activity with not only ATP but also with CTP, GTP, or UTP. The ADP-bound form of both recombinant and natural chaperonin had the ability to capture non-native GFP, but could not refold it in the presence of CTP, GTP or UTP until ATP was supplied. The archaeal chaperonin thus utilized ATP, but could not use other nucleoside triphosphates in the cytoplasm where ADP was present.
Asunto(s)
Adenosina Trifosfato/química , Chaperoninas/química , Chaperoninas/metabolismo , Pliegue de Proteína , Thermococcus/química , Adenosina Difosfato/química , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Citidina Trifosfato/química , Proteínas Fluorescentes Verdes , Guanosina Trifosfato/química , Hidrólisis , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Unión Proteica/fisiología , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Uridina Trifosfato/químicaRESUMEN
Heterochromatin protein 1 (HP1) is a conserved chromosomal protein with important roles in chromatin packaging and gene silencing. In fission yeast, two HP1 family proteins, Swi6 and Chp2, are involved in transcriptional silencing at heterochromatic regions, but how they function and whether they act cooperatively or differentially in heterochromatin assembly remain elusive. Here, we show that both Swi6 and Chp2 are required for the assembly of fully repressive heterochromatin, in which they play distinct, nonoverlapping roles. Swi6 is expressed abundantly and plays a dose-dependent role in forming a repressive structure through its self-association property. In contrast, Chp2, expressed at a lower level, does not show a simple dose-dependent repressive activity. However, it contributes to the recruitment of chromatin-modulating factors Clr3 and Epe1 and possesses a novel ability to bind the chromatin-enriched nuclear subfraction that is closely linked with its silencing function. Finally, we demonstrate that a proper balance between Swi6 and Chp2 is critical for heterochromatin assembly. Our findings provide novel insight into the distinct and cooperative functions of multiple HP1 family proteins in the formation of higher-order chromatin structure.