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Successful plant reproduction depends on the adequate development of flower organs controlled by cell proliferation and other processes. The SCI1 gene regulates cell proliferation and affects the final size of the female reproductive organ. To unravel the molecular mechanism exerted by SCI1 in cell proliferation control, we searched for its interaction partners through semi-in vivo pulldown experiments, uncovering a cyclin-dependent kinase, NtCDKG;2. Bimolecular fluorescence complementation (BiFC) and co-localization experiments showed that SCI1 interacts with NtCDKG;2 and its cognate NtCyclin L in nucleoli and splicing speckles. The screening of a yeast two-hybrid (Y2H) cDNA library using SCI1 as bait revealed a novel DEAD-box RNA helicase (NtRH35). The interaction between the NtCDKG;2-NtCyclin L complex, and NtRH35 was also shown. Subcellular localization experiments showed that SCI1, NtRH35, and the NtCDKG;2-NtCyclin L complex associate with each other within splicing speckles. The Y2H screening of NtCDKG;2 and NtRH35 identified the conserved spliceosome components U2a', NKAP, and CACTIN. This work presents SCI1 and its interactors NtCDKG;2-NtCyclin L complex, and NtRH35 as new spliceosome-associated proteins. Our findings reveal a network of interactions and suggest that SCI1 may regulate cell proliferation through the splicing process. This study provides new valuable insights into the intricate molecular pathways governing plant development.
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Vectors incorporating the human H1 (hH1) promoter are being applied for RNA interference (RNAi) experiments and genome editing. Although extensive studies have been conducted on the hH1 promoter, our understanding of the mouse H1 promoter remains limited. In this study, we predicted the 163 bp mouse H1 (mH1) promoter and 84 bp mouse H1 core (mH1 core) promoter through global alignment and detected its RNA polymerase II (Pol II) and III activities through the expression of the EGFP and the abundance of artificial sequence, which were generally slightly weaker than those of the hH1 promoter. Furthermore, to boost its Pol III activity, we engineered various promoter mutants by introducing mutations or systematically swapping elements. Surprisingly, the Pol II activity of mH1 core mut5 with AT stretch was at least 2-fold greater than that of the wild type, making it a potential candidate for target protein expression purposes. Fortunately, the Pol III activities of mH1 mut1 and mH1 core mut5 were at least 1.5 times stronger than those of the parental promoters in human and mouse cell lines on account of AT stretch, as did the mH1 mut4 with AT stretch and proximal sequence element (PSE) and TATA box insertion mutations. We highly recommend these three promoters as valuable supplements to the type 3 Pol III promoter toolbox.
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Neurodegeneration associated with ageing is closely linked to oxidative stress (OS) and disrupted calcium homeostasis. Some areas of the brain, like the hippocampus - particularly the CA1 region - have shown a high susceptibility to age-related changes, displaying early signs of pathology and neuronal loss. Antioxidants such as α-tocopherol (αT) have been effective in mitigating the impact of OS during ageing. αT homeostasis is primarily regulated by the α-tocopherol transfer protein (αTTP), which is widely distributed throughout the brain - where it plays a crucial role in maintaining αT levels within neuronal cells. This study investigates the distribution of αTTP in the hippocampus of 4- and 24-month-old Pol µ knockout mice (Pol µ-/-), a delayed-ageing model, and the wild type (Pol µ+/+). We also examine the colocalisation in the stratum oriens (st.or) of CA1 region with the primary interneuron populations expressing calcium-binding proteins (CBPs) (calbindin (CB), parvalbumin (PV), and calretinin (CR)). Our findings reveal that αTTP immunoreactivity (-IR) in the st.or of Pol µ mice is significantly reduced. The density of PV-expressing interneurons (INs) increased in aged mice in both Pol µ genotypes (Pol µ-/- and Pol µ+/+), although the density of PV-positive INs was lower in the aged Pol µ-/- mice compared to wild-type mice. By contrast, CR- and CB-positive INs in Pol µ mice remained unchanged during ageing. Furthermore, double immunohistochemistry reveals the colocalisation of αTTP with CBPs in INs of the CA1 st.or. Our study also shows that the PV/αTTP-positive IN population remains unchanged in all groups. A significant decrease of CB/αTTP-positive INs in young Pol µ-/- mice has been detected, as well as a significant increase in CR/αTTP-IR in older Pol µ-/- animals. These results suggest that the differential expression of αTTP and CBPs could have a crucial effect in aiding the survival and maintenance of the different IN populations in the CA1 st.or, and their coexpression could contribute to the enhancement of their resistance to OS-related damage and neurodegeneration associated with ageing.
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RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers.
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Recycling histone proteins from parental chromatin, a process known as parental histone transfer, is an important component in chromosome replication and is essential for epigenetic inheritance. We review recent advances in our understanding of the recycling mechanism of parental histone H3-H4 tetramers (parH3:H4tet), emphasizing the pivotal role of the DNA replisome. In particular, we highlight the function of the MCM2-7 helicase subunit Mcm2 as a histone H3-H4 tetramer chaperone. Disruption of this histone chaperone's functions affects mouse embryonic stem cell differentiation and can lead to embryonic lethality in mice, underscoring the crucial role of the replisome in maintaining epigenomic stability.
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Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirusï¼APVï¼ has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus.
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OBJECTIVE: To determine the effect of an adapted white-coat and warm-heart intervention (AWWI) among nurses. BACKGROUND: HIV discrimination among the medical staff hinders the progress in HIV prevention. METHODS: Seven hundred seventy-nine nurses were randomized into intervention and control groups. The intervention group was provided AWWI training. The control group did not receive AWWI training. HIV-related knowledge, attitudes, and behaviors of participants were assessed. RESULTS: Participants in the intervention group had better HIV-related knowledge and less stigmatizing attitudes and work avoidance behavior levels than participants in the control group after the 1-, 3-, and 6-month interventions (P<0.05). The main effects of group and time factors were highly significant in the intervention group. There were significant interaction effects in group and time factors. CONCLUSIONS: AWWI effectively improved the level of HIV-related knowledge and reduced general stigmatizing attitudes and work avoidance behaviors among nurses based on self-reported data in a tertiary hospital of China during a 6-month period.
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The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.
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Proteasa del VIH , Transcriptasa Inversa del VIH , VIH-1 , Multimerización de Proteína , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Transcriptasa Inversa del VIH/genética , Proteasa del VIH/química , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , VIH-1/química , Humanos , Modelos Moleculares , DimerizaciónRESUMEN
In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed DNA Methylation requires the activity of the RNA Polymerases IV and V, which produce small RNA precursors and noncoding targets of small RNAs, respectively. These polymerases are distinguished from Polymerase II by multiple plant-specific paralogous subunits. Most RNA-directed DNA Methylation components are present in all land plants, and some have been found in the charophytic green algae, a paraphyletic group that is sister to land plants. However, the evolutionary origin of key RNA-directed DNA Methylation components, including the two largest subunits of Polymerase IV and Polymerase V, remains unclear. Here, we show that multiple lineages of charophytic green algae encode a single-copy precursor of the largest subunits of Polymerase IV and Polymerase V, resolving the two presumed duplications in this gene family. We further demonstrate the presence of a Polymerase V-like C-terminal domain, suggesting that the earliest form of RNA-directed DNA Methylation utilized a single Polymerase V-like polymerase. Finally, we reveal that charophytic green algae encode a single CLSY/DRD1-type chromatin remodeling protein, further supporting the presence of a single specialized polymerase in charophytic green algae.
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Metilación de ADN , ARN Polimerasas Dirigidas por ADN , Evolución Molecular , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Filogenia , Carofíceas/genética , Carofíceas/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Chlorophyta/genética , Chlorophyta/enzimología , Subunidades de Proteína/genéticaRESUMEN
A large body of work in the last four decades has revealed the key pillars of HIV-1 transcription control at the initiation and elongation steps. Here, I provide a recount of this collective knowledge starting with the genomic elements (DNA and nascent TAR RNA stem-loop) and transcription factors (cellular and the viral transactivator Tat), and later transitioning to the assembly and regulation of transcription initiation and elongation complexes, and the role of chromatin structure. Compelling evidence support a core HIV-1 transcriptional program regulated by the sequential and concerted action of cellular transcription factors and Tat to promote initiation and sustain elongation, highlighting the efficiency of a small virus to take over its host to produce the high levels of transcription required for viral replication. I summarize new advances including the use of CRISPR-Cas9, genetic tools for acute factor depletion, and imaging to study transcriptional dynamics, bursting and the progression through the multiple phases of the transcriptional cycle. Finally, I describe current challenges to future major advances and discuss areas that deserve more attention to both bolster our basic knowledge of the core HIV-1 transcriptional program and open up new therapeutic opportunities.
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The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81-kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR-Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expressions were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the KMT2A/MLL1 complex. Myrlin CRISPRi compromised KMT2A occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting KMT2A.
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Elementos de Facilitación Genéticos , N-Metiltransferasa de Histona-Lisina , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Proto-Oncogénicas c-myb , Transcripción Genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Animales , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Ratones , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Elementos de Facilitación Genéticos/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Proto-Oncogenes Mas , Unión Proteica , Diferenciación Celular/genética , ARN PotenciadoresRESUMEN
Genome compaction is a common evolutionary feature of parasites. The unicellular, obligate intracellular parasite Encephalitozoon cuniculi has one of smallest known eukaryotic genomes, and is nearly four times smaller than its distant fungi relative, the budding yeast Saccharomyces cerevisiae. Comparison of the proteins encoded by compacted genomes to those encoded by larger genomes can reveal the most highly conserved features of the encoded proteins. In this study, we identified the proteins comprising the RNA polymerases and their corresponding general transcription factors by using several bioinformatic approaches to compare the transcription machinery of E. cuniculi and S. cerevisiae. Surprisingly, our analyses revealed an overall reduction in the size of the proteins comprising transcription machinery of E. cuniculi, which includes the loss of entire regions or functional domains from proteins, as well as the loss of entire proteins and complexes. Unexpectedly, we found that the E. cuniculi ortholog of Rpc37 (a RNA Polymerase III subunit) more closely resembles the H. sapiens ortholog of Rpc37 than the S. cerevisiae ortholog of Rpc37, in both size and structure. Overall, our findings provide new insight into the minimal core eukaryotic transcription machinery and help define the most critical features of Pol components and general transcription factors.
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Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-ß, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.
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Estrés Fisiológico , Humanos , Estrés Fisiológico/genética , Regulación de la Expresión Génica , Iniciación de la Transcripción Genética , Animales , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Terminal differentiation requires massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4,000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identify dynamic occupancy of RNA polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II occupancy and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, chromatin immunoprecipitation sequencing (ChIP-seq), and immunoprecipitation (IP) mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II occupancy at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms that drive differentiation gene expression and find that pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in renewing adult tissue.
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Diferenciación Celular , Factor Nuclear 4 del Hepatocito , ARN Polimerasa II , Animales , Ratones , Elementos de Facilitación Genéticos , Factor Nuclear 4 del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Intestinos , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismoRESUMEN
BACKGROUND: DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS: To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS: Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.
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Replicación del ADN , ARN Polimerasa II , Transcripción Genética , ARN Polimerasa II/metabolismo , Humanos , Fase S/genética , Daño del ADN , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Genoma Humano , Origen de RéplicaRESUMEN
RNA Polymerase II (Pol II) transcriptional elongation pausing is an integral part of the dynamic regulation of gene transcription in the genome of metazoans. It plays a pivotal role in many vital biological processes and disease progression. However, experimentally measuring genome-wide Pol II pausing is technically challenging and the precise governing mechanism underlying this process is not fully understood. Here, we develop RP3 (RNA Polymerase II Pausing Prediction), a network regularized logistic regression machine learning method, to predict Pol II pausing events by integrating genome sequence, histone modification, gene expression, chromatin accessibility, and protein-protein interaction data. RP3 can accurately predict Pol II pausing in diverse cellular contexts and unveil the transcription factors that are associated with the Pol II pausing machinery. Furthermore, we utilize a forward feature selection framework to systematically identify the combination of histone modification signals associated with Pol II pausing. RP3 is freely available at https://github.com/AMSSwanglab/RP3.
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Código de Histonas , ARN Polimerasa II , ARN Polimerasa II/metabolismo , Humanos , Elongación de la Transcripción Genética , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Aprendizaje Automático , AnimalesRESUMEN
Pol II pause release is a rate-limiting step in gene transcription, influencing various cell fate alterations. Numerous proteins orchestrate Pol II pause release, thereby playing pivotal roles in the intricate process of cellular fate modulation. Super elongation complex (SEC), a large assembly comprising diverse protein components, has garnered attention due to its emerging significance in orchestrating physiological and pathological cellular identity changes by regulating the transcription of crucial genes. Consequently, SEC emerges as a noteworthy functional complex capable of modulating cell fate alterations. Therefore, a comprehensive review is warranted to systematically summarize the core roles of SEC in different types of cell fate alterations. This review focuses on elucidating the current understanding of the structural and functional basis of SEC. Additionally, we discuss the intricate regulatory mechanisms governing SEC in various models of cell fate alteration, encompassing both physiological and pathological contexts. Furthermore, leveraging the existing knowledge of SEC, we propose some insightful directions for future research, aiming to enhance our mechanistic and functional comprehension of SEC within the diverse landscape of cell fate alterations.
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Diferenciación Celular , Humanos , Animales , Diferenciación Celular/fisiología , Transcripción GenéticaRESUMEN
In the longitudinal, retrospective study, the ability of the FRAX, Garvan, and POL-RISK algorithms to predict osteoporotic fractures was compared in a group of 457 women. Using the rigid threshold of 10% showed a significant discrepancy in sensitivity and specificity of all tools. New thresholds for high risk of fractures were established for each calculator separately: 6.3% for FRAX major fracture, 20.0% for Garvan any fracture, and 18.0% for POL-RISK any fracture. Such thresholds allow for improving the diagnostic accuracy of all three calculators. INTRODUCTION: The aim of the longitudinal, retrospective study was to compare three tools designed to assess fracture risk: FRAX, Garvan, and POL-RISK in their prediction of fracture incidence. MATERIAL: The study group consisted of 457 postmenopausal women with a mean age of 64.21 ± 5.94 years from the Gliwice Osteoporosis (GO) Study. Comprehensive data on clinical factors related to fractures were collected for all participants. Bone densitometry was performed at the proximal femur using the Prodigy device (GE, USA). Fracture risk was established using the FRAX, Garvan, and POL-RISK algorithms. Data on the incidence of osteoporotic fractures were collected over the last 10 years. RESULTS: During the period of observation 72, osteoporotic fractures occurred in 63 subjects. For a preliminary comparison of the predictive value of analyzed diagnostic tools, the fracture risk threshold of 10% was used. For FRAX, the fracture probability exceeding 10% was observed only in 11 subjects who experienced fractures; thus, the fracture was properly predicted only in 22.9% of women. For Garvan, the respective value was 90.5%, and for POL-RISK, it was 98.4%. That gave a very low true positive value for FRAX and a very high false positive value for Garvan and POL-RISK. Based on ROC curves, new thresholds for high risk of fractures were established for each calculator separately: 6.3% for FRAX major fracture, 20.0% for Garvan any fracture, and 18.0% for POL-RISK any fracture. Such thresholds improve the diagnostic accuracy of all compared fracture prediction tools. CONCLUSION: The current study showed that different fracture risk assessment tools, although having similar clinical purposes, require different cut-off thresholds for making therapeutic decisions. Better identification of patients requiring therapy based on such an approach may help reduce the number of new fractures.
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Algoritmos , Osteoporosis Posmenopáusica , Fracturas Osteoporóticas , Humanos , Femenino , Fracturas Osteoporóticas/epidemiología , Persona de Mediana Edad , Medición de Riesgo/métodos , Anciano , Estudios Retrospectivos , Osteoporosis Posmenopáusica/epidemiología , Osteoporosis Posmenopáusica/complicaciones , Estudios Longitudinales , Densidad Ósea , Posmenopausia , Factores de Riesgo , Incidencia , Sensibilidad y Especificidad , Absorciometría de Fotón/estadística & datos numéricosRESUMEN
Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.