RESUMO
BACKGROUND: Mining the vast pool of biomedical literature to extract accurate responses and relevant references is challenging due to the domain's interdisciplinary nature, specialized jargon, and continuous evolution. Early natural language processing (NLP) approaches often led to incorrect answers as they failed to comprehend the nuances of natural language. However, transformer models have significantly advanced the field by enabling the creation of large language models (LLMs), enhancing question-answering (QA) tasks. Despite these advances, current LLM-based solutions for specialized domains like biology and biomedicine still struggle to generate up-to-date responses while avoiding "hallucination" or generating plausible but factually incorrect responses. RESULTS: Our work focuses on enhancing prompts using a retrieval-augmented architecture to guide LLMs in generating meaningful responses for biomedical QA tasks. We evaluated two approaches: one relying on text embedding and vector similarity in a high-dimensional space, and our proposed method, which uses explicit signals in user queries to extract meaningful contexts. For robust evaluation, we tested these methods on 50 specific and challenging questions from diverse biomedical topics, comparing their performance against a baseline model, BM25. Retrieval performance of our method was significantly better than others, achieving a median Precision@10 of 0.95, which indicates the fraction of the top 10 retrieved chunks that are relevant. We used GPT-4, OpenAI's most advanced LLM to maximize the answer quality and manually accessed LLM-generated responses. Our method achieved a median answer quality score of 2.5, surpassing both the baseline model and the text embedding-based approach. We developed a QA bot, WeiseEule ( https://github.com/wasimaftab/WeiseEule-LocalHost ), which utilizes these methods for comparative analysis and also offers advanced features for review writing and identifying relevant articles for citation. CONCLUSIONS: Our findings highlight the importance of prompt enhancement methods that utilize explicit signals in user queries over traditional text embedding-based approaches to improve LLM-generated responses for specialized queries in specialized domains such as biology and biomedicine. By providing users complete control over the information fed into the LLM, our approach addresses some of the major drawbacks of existing web-based chatbots and LLM-based QA systems, including hallucinations and the generation of irrelevant or outdated responses.
Assuntos
Mineração de Dados , Processamento de Linguagem Natural , Mineração de Dados/métodos , Armazenamento e Recuperação da Informação/métodosRESUMO
Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Nucleossomos , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Arginina/química , Arginina/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Conformação Proteica , Homologia de SequênciaRESUMO
Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.
Assuntos
Autofagia/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Autofagia/genética , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Edição de Genes , Expressão Gênica , Síndrome de Hallermann/genética , Humanos , Mutação , FenótipoRESUMO
CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Coração/embriologia , Trifosfato de Adenosina/metabolismo , Alelos , Animais , Síndrome CHARGE/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Cardiopatias Congênitas/genética , Camundongos , Camundongos Knockout , Mutação , Crista Neural/embriologia , Crista Neural/metabolismo , Organogênese/fisiologiaRESUMO
BACKGROUND: Maintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown. RESULTS: Given that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation. CONCLUSIONS: We propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.
Assuntos
DNA Helicases/metabolismo , Nucleossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Reparo do DNA , Histonas/metabolismo , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
We report a high-affinity photoswitchable DNA binder, which displays different nucleosome-binding capacities upon visible-light irradiation. Both photochemical and DNA-recognition properties were examined by UV-Vis, HPLC, CD spectroscopy, NMR, FID assays, EMSA and DLS. Our probe sets the basis for developing new optoepigenetic tools for conditional modulation of nucleosomal DNA accessibility.
Assuntos
Compostos Azo/química , DNA/química , Substâncias Intercalantes/química , Luz , Nucleossomos/química , Compostos Azo/síntese química , Substâncias Intercalantes/síntese química , Estrutura Molecular , Processos FotoquímicosRESUMO
Gene regulation by steroid hormones plays important roles in health and disease. In Drosophila, the hormone ecdysone governs transitions between key developmental stages. Ecdysone-regulated genes are bound by a heterodimer of ecdysone receptor (EcR) and Ultraspiracle. According to the bimodal switch model, steroid hormone receptors recruit corepressors in the absence of hormone and coactivators in its presence. Here we show that the nucleosome remodeller dMi-2 is recruited to ecdysone-regulated genes to limit transcription. Contrary to the prevalent model, recruitment of the dMi-2 corepressor increases upon hormone addition to constrain gene activation through chromatin remodelling. Furthermore, EcR and dMi-2 form a complex that is devoid of Ultraspiracle. Unexpectedly, EcR contacts the dMi-2 ATPase domain and increases the efficiency of dMi-2-mediated nucleosome remodelling. This study identifies a non-canonical EcR-corepressor complex with the potential for a direct regulation of ATP-dependent nucleosome remodelling by a nuclear hormone receptor.
Assuntos
Adenosina Trifosfatases/fisiologia , Autoantígenos/fisiologia , Proteínas de Drosophila/fisiologia , Ecdisona/fisiologia , Regulação da Expressão Gênica/fisiologia , Receptores de Esteroides/fisiologia , Transcrição Gênica/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Cromatina/metabolismo , Drosophila/genética , Ecdisona/metabolismo , Cinética , Ativação TranscricionalRESUMO
Biochemical assays reveal that nucleosome maturation and chromatin remodelling by the motor protein Chd1 are distinct, separable enzymatic activities.
Assuntos
Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Animais , HumanosRESUMO
DNA damage is linked to multiple human diseases, such as cancer, neurodegeneration, and aging. Little is known about the role of chromatin accessibility in DNA repair. Here, we find that the deacetylase sirtuin 6 (SIRT6) is one of the earliest factors recruited to double-strand breaks (DSBs). SIRT6 recruits the chromatin remodeler SNF2H to DSBs and focally deacetylates histone H3K56. Lack of SIRT6 and SNF2H impairs chromatin remodeling, increasing sensitivity to genotoxic damage and recruitment of downstream factors such as 53BP1 and breast cancer 1 (BRCA1). Remarkably, SIRT6-deficient mice exhibit lower levels of chromatin-associated SNF2H in specific tissues, a phenotype accompanied by DNA damage. We demonstrate that SIRT6 is critical for recruitment of a chromatin remodeler as an early step in the DNA damage response, indicating that proper unfolding of chromatin plays a rate-limiting role. We present a unique crosstalk between a histone modifier and a chromatin remodeler, regulating a coordinated response to prevent DNA damage.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Instabilidade Genômica , Sirtuínas/metabolismo , Sirtuínas/fisiologia , Adenosina Trifosfatases/genética , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Hipocampo/citologia , Hipocampo/metabolismo , Histonas/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Nucleossomos/metabolismo , Sirtuínas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Mutations in the CHD7 gene cause human developmental disorders including CHARGE syndrome. Genetic studies in model organisms have further established CHD7 as a central regulator of vertebrate development. Functional analysis of the CHD7 protein has been hampered by its large size. We used a dual-tag system to purify intact recombinant CHD7 protein and found that it is an ATP-dependent nucleosome remodeling factor. Biochemical analyses indicate that CHD7 has characteristics distinct from SWI/SNF- and ISWI-type remodelers. Further investigations show that CHD7 patient mutations have consequences that range from subtle to complete inactivation of remodeling activity, and that mutations leading to protein truncations upstream of amino acid 1899 of CHD7 are likely to cause a hypomorphic phenotype for remodeling. We propose that nucleosome remodeling is a key function for CHD7 during developmental processes and provide a molecular basis for predicting the impact of disease mutations on that function.
Assuntos
Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/genética , Mutação Puntual/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/genética , Síndrome CHARGE/genética , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Hipogonadismo/genética , Proteínas Mutantes/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Methylation-sensitive single-molecule analysis of chromatin structure is a high-resolution method for studying nucleosome positioning. As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro-remodeled nucleosomes by treatment with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by bisulfite sequencing of individual progeny DNA molecules. Unlike nuclease-based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population.
Assuntos
Cromatina/química , Metilação de DNA , Técnicas Genéticas , Animais , Biocatálise , Ilhas de CpG , HumanosRESUMO
Chromatin remodeling enzymes use the energy of ATP hydrolysis to alter histone-DNA contacts and regulate DNA-based processes in eukaryotes. Whether different subfamilies of remodeling complexes generate distinct products remains uncertain. We have developed a protocol to analyze nucleosome remodeling on individual products formed in vitro. We used a DNA methyltransferase to examine DNA accessibility throughout nucleosomes that had been remodeled by the ISWI and SWI/SNF families of enzymes. We confirmed that ISWI-family enzymes mainly created patterns of accessibility consistent with canonical nucleosomes. In contrast, SWI/SNF-family enzymes generated widespread DNA accessibility. The protection patterns created by these enzymes were usually located at the extreme ends of the DNA and showed no evidence for stable loop formation on individual molecules. Instead, SWI/SNF family proteins created extensive accessibility by generating heterogeneous products that had fewer histone-DNA contacts than a canonical nucleosome, consistent with models in which a canonical histone octamer has been 'pushed' off of the end of the DNA.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/química , Trifosfato de Adenosina/metabolismo , DNA/química , DNA Helicases/metabolismo , DNA-Citosina Metilases , Histonas/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The regulation of chromatin structure is of fundamental importance for many DNA-based processes in eukaryotes. Activation or repression of gene transcription or DNA replication depends on enzymes which can generate the appropriate chromatin environment. Several of these enzymes utilize the energy of ATP hydrolysis to alter nucleosome structure. In recent years our understanding of the multisubunit complexes within which they function, their mechanisms of action, their regulation and their in-vivo roles has increased. Much of what we have learned has been gleaned from studies in Drosophila melanogaster. Here we will review what we know about the main classes of ATP-dependent chromatin remodelers in Drosophila.
Assuntos
Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/fisiologia , Animais , Autoantígenos , Montagem e Desmontagem da Cromatina/fisiologia , DNA Helicases , Dineínas , Proteínas de HomeodomínioRESUMO
A plethora of ATP-dependent chromatin-remodeling enzymes have been identified during the last decade. Many have been shown to play pivotal roles in the organization and expression of eukaryotic genomes. It is clear that their activities need to be tightly regulated to ensure their coordinated action. However, little is known about how ATP-dependent remodelers are regulated at the molecular level. Here, we have investigated the ATP-dependent chromatin remodeling enzyme Mi-2 of Drosophila melanogaster. Radioactive labeling of S2 cells reveals that dMi-2 is a phosphoprotein in vivo. dMi-2 phosphorylation is constitutive, and we identify dCK2 as a major dMi-2 kinase in cell extracts. dCK2 binds to and phosphorylates a dMi-2 N-terminal region. Dephosphorylation of recombinant dMi-2 increases its affinity for the nucleosome substrate, nucleosome-stimulated ATPase, and ATP-dependent nucleosome mobilization activities. Our results reveal a potential mechanism for regulation of the dMi-2 enzyme and point toward CK2 phosphorylation as a common feature of CHD family ATPases.
Assuntos
Adenosina Trifosfatases/metabolismo , Autoantígenos/metabolismo , Cromatina/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia de Afinidade , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/isolamento & purificação , Drosophila melanogaster , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Homologia de Sequência de AminoácidosAssuntos
Mecanismo Genético de Compensação de Dose , Drosophila/genética , Acetiltransferases/genética , Animais , Proteínas Cromossômicas não Histona , Cromossomos Humanos X/genética , Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Alemanha , Histona Acetiltransferases , Humanos , Masculino , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Cromossomo X/genéticaRESUMO
Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. We have tested a panel of dMi-2 deletion mutants in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to dHP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. Our results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, we identify DNA binding as a novel chromodomain-associated activity.