RESUMEN
Stem cells can undergo asymmetric cell division (ACD) giving rise to one new stem cell and one differentiating daughter cell. In Drosophila germline stem cells (GSCs), the centromeric histone CENP-A (CID in flies) is asymmetrically distributed between sister chromatids such that chromosomes that end up in the GSC harbor more CID at centromeres. A model of "mitotic drive" has been proposed in GSCs such that stronger and earlier centromere and kinetochore interactions with microtubules bias sister chromatid segregation. Here we show that in Drosophila males, centromere proteins CID, CAL1, and CENP-C are asymmetrically distributed in newly divided GSCs and daughter cells in S phase. We find that overexpression of CID (either with or without CAL1) or CENP-C depletion disrupts CID asymmetry, with an increased pool of GSCs relative to daughter cells detectable in the niche. This result suggests a shift toward GSC self-renewal rather than differentiation, important for maintaining tissue homeostasis. Overexpression of CAL1 does not disrupt asymmetry, but instead drives germ cell proliferation in the niche. Our results in male GSCs are comparable to female GSCs, indicating that despite differences in signaling, organization, and niche composition, the effects of centromere proteins on GSC maintenance are conserved between the sexes.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Masculino , Femenino , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Centrómero/metabolismo , Células Germinativas/metabolismoRESUMEN
Bulls used in artificial insemination, with apparently normal semen quality, can vary significantly in their field fertility. This study aimed to characterize the transcriptome of spermatozoa from high (HF) and low (LF) fertility bulls at the mRNA and miRNA level in order to identify potential novel markers of fertility. Holstein-Friesian bulls were assigned to either the HF or LF group (n = 10 per group) based on an adjusted national fertility index from a minimum of 500 inseminations. Total RNA was extracted from a pool of frozen-thawed spermatozoa from three different ejaculates per bull, following which mRNA-seq and miRNA-seq were performed. Six mRNAs and 13 miRNAs were found differentially expressed (P < 0.05, FC > 1.5) between HF and LF bulls. Of particular interest, the gene pathways targeted by the 13 differentially expressed miRNAs were related to embryonic development and gene expression regulation. Previous studies reported that disruptions to protamine 1 mRNA (PRM1) had deleterious consequences for sperm chromatin structure and fertilizing ability. Notably, PRM1 exhibited a higher expression in spermatozoa from LF than HF bulls. In contrast, Western Blot analysis revealed a decrease in PRM1 protein abundance for spermatozoa from LF bulls; this was not associated with increased protamine deficiency (measured by the degree of chromatin compaction) or DNA fragmentation, as assessed by flow cytometry analyses. However, protamine deficiency was positively and moderately correlated with the percentage of spermatozoa with DNA fragmentation, irrespective of fertility group. This study has identified potential biomarkers that could be used for improving semen quality assessments of bull fertility.
RESUMEN
The centromere is the constricted chromosomal region required for the correct separation of the genetic material at cell division. The kinetochore protein complex assembles at the centromere and captures microtubules emanating from the centrosome to orchestrate chromosome segregation in mitosis and meiosis. Asymmetric cell division (ACD) is a special type of mitosis that generates two daughter cells with different fates. Epigenetic mechanisms operating at the centromere have been proposed to contribute to ACD. Recent studies have shown that an asymmetric distribution of CENP-A-the centromere-specific histone H3 variant-between sister chromatids can bias chromosome segregation in ACD. In stem cells, this leads to non-random sister chromatid segregation, which can affect cell fate. These findings support the 'silent sister' hypothesis, according to which the mechanisms of ACD are epigenetically regulated through centromeres. Here, we review the recent data implicating centromeres in ACDs and cell fate in Drosophila melanogaster female and male germline stem cells.
Asunto(s)
Células Madre Germinales Adultas/citología , Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas de Drosophila/metabolismo , Ovario/citología , Células Madre Germinales Adultas/metabolismo , Animales , División Celular Asimétrica , Drosophila melanogaster , Epigénesis Genética , Femenino , Masculino , Células Madre/citología , Células Madre/metabolismoRESUMEN
Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)-the epigenetic determinant of centromere identity-is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of 'mitotic drive' that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Epigénesis Genética , Células Germinativas/metabolismo , Células Madre/metabolismo , Animales , Autorrenovación de las Células , Senescencia Celular , Proteínas Cromosómicas no Histona/deficiencia , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/metabolismo , Femenino , Fase G2 , Masculino , Profase , Fase SRESUMEN
Murillo-Pineda and colleagues (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202007210) use CRISPR-Cas9-based genetic engineering in human cells to induce a new functional centromere at a naive chromosomal site. Long-read DNA sequencing at the neocentromere provides firm evidence that centromere establishment is a truly epigenetic event.
Asunto(s)
Centrómero , Epigenómica , Centrómero/genética , Proteína A Centromérica/genética , Epigénesis Genética , HumanosRESUMEN
Asymmetric cell division (ACD) produces daughter cells with separate distinct cell fates and is critical for the development and regulation of multicellular organisms. Epigenetic mechanisms are key players in cell fate determination. Centromeres, epigenetically specified loci defined by the presence of the histone H3-variant, centromere protein A (CENP-A), are essential for chromosome segregation at cell division. ACDs in stem cells and in oocyte meiosis have been proposed to be reliant on centromere integrity for the regulation of the non-random segregation of chromosomes. It has recently been shown that CENP-A is asymmetrically distributed between the centromeres of sister chromatids in male and female Drosophila germline stem cells (GSCs), with more CENP-A on sister chromatids to be segregated to the GSC. This imbalance in centromere strength correlates with the temporal and asymmetric assembly of the mitotic spindle and potentially orientates the cell to allow for biased sister chromatid retention in stem cells. In this essay, we discuss the recent evidence for asymmetric sister centromeres in stem cells. Thereafter, we discuss mechanistic avenues to establish this sister centromere asymmetry and how it ultimately might influence cell fate.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Cromátides/metabolismo , Segregación Cromosómica , Proteínas de Drosophila/metabolismo , Células Madre/citología , Animales , Drosophila , Femenino , Masculino , Huso Acromático/metabolismo , Células Madre/metabolismoRESUMEN
Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. We show that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. Our work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis.
Asunto(s)
Centrómero/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Epigénesis Genética/genética , Células Madre/citología , Células Madre/metabolismo , Animales , Centrómero/metabolismoRESUMEN
Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes 'primes' post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.
Asunto(s)
Drosophila melanogaster/genética , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Repeticiones de Microsatélite , ARN Mensajero/genética , Maduración del Esperma/genética , Espermatogénesis/genética , Animales , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Evolución Molecular , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Histonas/metabolismo , Masculino , Protaminas/metabolismo , ARN Mensajero/biosíntesis , Espermatocitos/citología , Espermatocitos/crecimiento & desarrollo , Espermatocitos/metabolismo , Transcripción GenéticaRESUMEN
This chapter describes a method used to assay the cell cycle dynamics of the centromeric histone H3 variant CENP-A in meiosis using Drosophila males as the experimental system. Specifically, we describe a method that combines Immunofluorescence (IF) and Fluorescence in-situ Hybridization (FISH) protocols, performed on fixed Drosophila testes. An advantage of this protocol is the ability to localize individual centromeres on the four Drosophila homologous chromosomes that form distinct nuclear territories in spermatocytes. We also describe a method to quantify CENP-A focal intensities using Image J software.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Drosophila melanogaster/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Histonas/metabolismo , Imagenología Tridimensional , Hibridación Fluorescente in Situ/métodos , Espermatocitos/metabolismo , Animales , Disección , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Masculino , Permeabilidad , Fijación del TejidoRESUMEN
The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. Here we report an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, we report that knockdown of CENP-A, as well as subunits ATPsyn-α, -ßlike (a testis-specific paralogue of ATPsyn-ß) and -γ disrupts sister centromere cohesion in meiotic prophase I. We find that this disruption is likely independent of reduced ATP levels. We identify that ATPsyn-α and -ßlike localise to meiotic centromeres and that this localisation is dependent on the presence of CENP-A. We show that ATPsyn-α directly interacts with the N-terminus of CENP-A in vitro and that truncation of its N terminus perturbs sister centromere cohesion in prophase I. We propose that the CENP-A N-terminus recruits ATPsyn-α and -ßlike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation.
Asunto(s)
Proteína A Centromérica/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiosis , ATPasas de Translocación de Protón Mitocondriales/genética , Subunidades de Proteína/genética , Espermatocitos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Centrómero/metabolismo , Centrómero/ultraestructura , Proteína A Centromérica/metabolismo , Segregación Cromosómica , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epigénesis Genética , Fertilidad/genética , Histonas/genética , Histonas/metabolismo , Masculino , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Subunidades de Proteína/metabolismo , Espermatocitos/citologíaRESUMEN
In sexually reproducing organisms the germ line is the cellular lineage that gives rise to gametes. All germ cells originate from germline stem cells that divide asymmetrically to generate gonial pre-cursors, which are amplified in number by mitotic divisions, undergo meiosis and eventually differentiate into mature gametes (haploid eggs and sperm). Information transmitted with gametes is inherited by offspring, and potentially by subsequent generations, instructing in organismal development and beyond. Meiosis comprises one round of DNA replication, followed by two rounds of chromosome segregation; homologous chromosomes segregate in the first division (meiosis I) and sister chromatids segregate in the second division (meiosis II). Important mechanistic features of meiosis occur in substages of prophase I and are critical for genetic recombination, including pairing and synapsis of homologous chromosomes (at leptotene and zygotene), crossing-over (at pachytene), and the appearance of chiasmata (at diplotene/diakinesis). Another unique feature of meiosis is the altered centromere/kinetochore geometry at metaphase I, such that sister kinetochores face the same spindle pole (mono-orientation) and stay together at anaphase I. This chapter reviews centromere dynamics in germ cells, focusing on centromere function and assembly in meiotic cell cycles, as well as centromere inheritance in zygotes. Centromeres are functionally defined by the presence of the histone H3 variant CENP-A, the epigenetic determinant of centromere identity. In most eukaryotes, it is well established that CENP-A function is essential for chromosome segregation in mitosis. CENP-A function in meiosis is less well understood and emerging insights into the differential regulation of meiotic and mitotic CENP-A are discussed.
Asunto(s)
Centrómero/metabolismo , Células Germinativas/citología , Segregación Cromosómica , Femenino , Humanos , Cinetocoros , Masculino , MeiosisRESUMEN
The centromere-specific histone CENP-A is the key epigenetic determinant of centromere identity. Whereas most histones are removed from mature sperm, CENP-A is retained to mark paternal centromeres. In Drosophila males we show that the centromere assembly factors CAL1 and CENP-C are required for meiotic chromosome segregation, CENP-A assembly and maintenance on sperm, as well as fertility. In meiosis, CENP-A accumulates with CAL1 in nucleoli. Furthermore, we show that CENP-C normally limits the release of CAL1 and CENP-A from nucleoli for proper centromere assembly in meiotic prophase I. Finally, we show that RNA polymerase I transcription is required for efficient CENP-A assembly in meiosis, as well as centromere tethering to nucleoli.
Asunto(s)
Nucléolo Celular/fisiología , Centrómero/fisiología , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Histonas/fisiología , Meiosis , Animales , Proteína A Centromérica , Drosophila melanogaster , Fertilidad , Masculino , Profase Meiótica I , Mutación , ARN Polimerasa I/metabolismo , Espermatogénesis , Espermatozoides/citología , Transcripción GenéticaAsunto(s)
Autoantígenos/análisis , Autoantígenos/metabolismo , Centrómero/química , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Multimerización de Proteína , Proteína A Centromérica , HumanosRESUMEN
CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.
Asunto(s)
Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Meiosis , Mitosis , Animales , Encéfalo/citología , Proteína A Centromérica , Segregación Cromosómica , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Fase G1 , Histonas/metabolismo , Larva/citología , Masculino , Modelos Biológicos , Transporte de Proteínas , Espermátides/citología , Espermátides/metabolismo , Telofase , Factores de TiempoRESUMEN
Centromeres are key regions of eukaryotic chromosomes that ensure proper chromosome segregation at cell division. In most eukaryotes, centromere identity is defined epigenetically by the presence of a centromeric histone H3 variant CenH3, called CENP-A in humans. How CENP-A is incorporated and reproducibly transmitted during the cell cycle is at the heart of this fundamental epigenetic mechanism. Centromeric DNA is replicated during S phase; however unlike replication-coupled assembly of canonical histones during S phase, newly synthesized CENP-A deposition at centromeres is restricted to a discrete time in late telophase/early G(1). These observations raise an important question: when 'old' CENP-A nucleosomes are segregated at the replication fork, are the resulting 'gaps' maintained until the next G(1), or are they filled by H3 nucleosomes during S phase and replaced by CENP-A in the following G(1)? Understanding such molecular mechanisms is important to reveal the composition/organization of centromeres in mitosis, when the kinetochore forms and functions. Here we investigate centromeric chromatin status during the cell cycle, using the SNAP-tag methodology to visualize old and new histones on extended chromatin fibers in human cells. Our results show that (1) both histone H3 variants H3.1 and H3.3 are deposited at centromeric domains in S phase and (2) there is reduced H3.3 (but not reduced H3.1) at centromeres in G(1) phase compared to S phase. These observations are consistent with a replacement model, where both H3.1 and H3.3 are deposited at centromeres in S phase and 'placeholder' H3.3 is replaced with CENP-A in G(1).
Asunto(s)
Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fase G1 , Histonas/metabolismo , Fase S , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Proteína A Centromérica , Cromatina/metabolismo , HumanosRESUMEN
The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.
Asunto(s)
Autoantígenos/metabolismo , Ciclo Celular/fisiología , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Autoantígenos/genética , Secuencia de Bases , Línea Celular , Centrómero/ultraestructura , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Histonas/metabolismo , Humanos , Unión ProteicaRESUMEN
Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication.
Asunto(s)
Centrómero , ADN Polimerasa I/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Schizosaccharomyces/ultraestructura , Acetilación , Inmunoprecipitación de Cromatina , Cromosomas Fúngicos , Clonación Molecular , Silenciador del Gen , Histona Desacetilasas/genética , Histonas/metabolismo , Microscopía Fluorescente , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.