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Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.
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Sistemas CRISPR-Cas , Resistencia a Medicamentos Antineoplásicos , Genoma Humano , RNA Longo não Codificante/genética , Animais , Citarabina/farmacologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Farmacogenética , Proteínas/genética , RNA/análise , RNA Mensageiro/genética , Transdução de SinaisRESUMO
The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.
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Cromatina/metabolismo , DNA/genética , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Leucemia Promielocítica Aguda/genética , Redes Reguladoras de Genes , Humanos , Leucemia Promielocítica Aguda/patologia , Fatores de Transcrição/metabolismoRESUMO
Severe COVID-19 leads to widespread transcriptomic changes in the human brain, mimicking diminished cognitive performance. As long noncoding RNAs (lncRNAs) play crucial roles in the regulation of gene expression, identification of the lncRNAs differentially expressed upon COVID-19 may nominate key regulatory nodes underpinning cognitive changes. Here we identify hundreds of lncRNAs differentially expressed in the brains of COVID-19 patients relative to uninfected age/sex-matched controls, many of which are associated with decreased cognitive performance and inflammatory cytokine response. Our analyses reveal pervasive transcriptomic changes in lncRNA expression upon severe COVID-19, which may serve as key regulators of neurocognitive changes in the brain.
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COVID-19 , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , COVID-19/genética , Perfilação da Expressão Gênica , Citocinas/genética , RNA Mensageiro/genéticaRESUMO
INTRODUCTION: Cloacal and bladder exstrophy are rare embryological defects that can cause developmental disruption of surrounding organ structures, the pelvis, spinal cord, and small intestines being the most commonly affected. Duplicated appendix is another rare embryological defect that has historically caused confusing clinical presentations. Our case highlights a rare instance of a patient with cloacal exstrophy who presented with a bowel obstruction and an associated inflamed duplicated appendix. CASE PRESENTATION: A newborn male is born with omphalocele-exstrophy-imperforate anus-spinal defects (OEIS) complex. As primary surgical reconstruction was pursued, the patient was found to have a non-inflamed duplicated appendix, which was left unremoved. In the following months, the patient experienced episodes of small bowel obstruction, eventually requiring surgical intervention. During this operation, the duplicated appendix was noted to be inflamed, prompting removal of both appendices. DISCUSSION: This case highlights the increased prevalence of duplicated appendix in a patient with cloacal exstrophy, as well as the utility of prophylactic appendectomy for patients incidentally found to have a duplicated appendix intraoperatively. The duplicated appendix may lead to increased rates of complications and atypical presentation of appendicitis, supporting the practice of prophylactic appendectomy in patients with an incidentally found duplicated appendix. CONCLUSION: We suggest clinicians be aware of the association and potentially atypical presentation of appendicitis in patients with a duplicated appendix, particularly in the setting of cloacal exstrophy. The decision to prophylactically remove an incidentally found, non-inflamed duplicated appendix may be beneficial in preventing confusing clinical presentations and future complications.
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Several microRNAs have emerged as regulators of pathways that control aging. For example, miR-228 is required for normal lifespan and dietary restriction (DR) mediated longevity through interaction with PHA-4 and SKN-1 transcription factors in Caenorhabditis elegans. miR-229,64,65, and 66, a cluster of microRNAs located adjacent to each other on chromosome III, are in the same family as miR-228, albeit with slight differences in the miR-228 seed sequence. We demonstrate that, in contrast to the anti-longevity role of miR-228, the miR-229-66 cluster is required for normal C. elegans lifespan and for the longevity observed in mir-228 mutants. miR-229-66 is also critical for lifespan extension observed under DR and reduced insulin signaling (IIS) and by constitutive nuclear SKN-1. Both DR and low-IIS upregulate the expression of the miRNA cluster, which is dependent on transcription factors PHA-4, SKN-1, and DAF-16. In turn, the expression of SKN-1 and DAF-16 requires mir-229,64,65,66. miR-229-66 targets the odd-skipped-related transcription factor, odd-2 to regulate lifespan. Knockdown of odd-2 increases lifespan, suppresses the short lifespan of mir-229,64,65,66(nDf63) III mutants, and alters levels of SKN-1 in the ASI neurons. Together with SKN-1, the miRNA cluster also indirectly regulates several genes in the xenobiotic detoxification pathway which increases wild-type lifespan and significantly rescues the short lifespan of mir-229,64,65,66(nDf63) III mutants. Thus, by interacting with SKN-1, miR-229-66 transduces the effects of DR and low-IIS in lifespan extension in C. elegans. Given that this pathway is conserved, it is possible that a similar mechanism regulates aging in more complex organisms.
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Proteínas de Caenorhabditis elegans , MicroRNAs , Animais , Caenorhabditis elegans/metabolismo , Longevidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Insulina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.
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Coronavirus disease 2019 (COVID-19) remains a significant public health threat due to the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here, we use our recently developed integrative DNA And Protein Tagging methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.
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COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , Humanos , SARS-CoV-2 , Cromatina , Proteômica , Senescência Celular , Células Gigantes , Proteína Supressora de Tumor p53/genéticaRESUMO
As coronavirus disease 2019 (COVID-19) and aging are both accompanied by cognitive decline, we hypothesized that COVID-19 might lead to molecular signatures similar to aging. We performed whole-transcriptome analysis of the frontal cortex, a critical area for cognitive function, in individuals with COVID-19, age-matched and sex-matched uninfected controls, and uninfected individuals with intensive care unit/ventilator treatment. Our findings indicate that COVID-19 is associated with molecular signatures of brain aging and emphasize the value of neurological follow-up in recovered individuals.
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COVID-19 , Humanos , Envelhecimento/genética , Encéfalo , Lobo Frontal , Perfilação da Expressão GênicaRESUMO
Coronavirus disease 2019 (COVID-19) is predominantly an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and remains a significant threat to public health. COVID-19 is accompanied by neurological symptoms and cognitive decline, but the molecular mechanisms underlying this effect remain unclear. As aging induces distinct molecular signatures in the brain associated with cognitive decline in healthy populations, we hypothesized that COVID-19 may induce molecular signatures of aging. Here, we performed whole transcriptomic analysis of human frontal cortex, a critical area for cognitive function, in 12 COVID-19 cases and age- and sex-matched uninfected controls. COVID-19 induces profound changes in gene expression, despite the absence of detectable virus in brain tissue. Pathway analysis shows downregulation of genes involved in synaptic function and cognition and upregulation of genes involved in immune processes. Comparison with five independent transcriptomic datasets of aging human frontal cortex reveals striking similarities between aged individuals and severe COVID-19 patients. Critically, individuals below 65 years of age exhibit profound transcriptomic changes not observed among older individuals in our patient cohort. Our data indicate that severe COVID-19 induces molecular signatures of aging in the human brain and emphasize the value of neurological follow-up in recovered individuals.
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Chromosomal rearrangements can generate genetic fusions composed of two distinct gene sequences, many of which have been implicated in tumorigenesis and progression. Our study proposes a model whereby oncogenic gene fusions frequently alter the protein stability of the resulting fusion products, via exchanging protein degradation signal (degron) between gene sequences. Computational analyses of The Cancer Genome Atlas (TCGA) identify 2,406 cases of degron exchange events and reveal an enrichment of oncogene stabilization due to loss of degrons from fusion. Furthermore, we identify and experimentally validate that some recurrent fusions, such as BCR-ABL, CCDC6-RET and PML-RARA fusions, perturb protein stability by exchanging internal degrons. Likewise, we also validate that EGFR or RAF1 fusions can be stabilized by losing a computationally-predicted C-terminal degron. Thus, complementary to enhanced oncogene transcription via promoter swapping, our model of degron loss illustrates another general mechanism for recurrent fusion proteins in driving tumorigenesis.
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Motivos de Aminoácidos/genética , Carcinogênese/genética , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Oncogenes/genética , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Knockout , Camundongos Nus , Modelos Genéticos , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Fusão Oncogênica/metabolismo , Proteólise , Transplante HeterólogoRESUMO
The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.
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Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Motivos de Aminoácidos , Sequência de Aminoácidos , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Análise Serial de Proteínas , Ligação Proteica , Análise de Sequência de RNA , Fatores de Transcrição/genéticaRESUMO
Activation of tumor suppressors for the treatment of human cancer has been a long sought, yet elusive, strategy. PTEN is a critical tumor suppressive phosphatase that is active in its dimer configuration at the plasma membrane. Polyubiquitination by the ubiquitin E3 ligase WWP1 (WW domain-containing ubiquitin E3 ligase 1) suppressed the dimerization, membrane recruitment, and function of PTEN. Either genetic ablation or pharmacological inhibition of WWP1 triggered PTEN reactivation and unleashed tumor suppressive activity. WWP1 appears to be a direct MYC (MYC proto-oncogene) target gene and was critical for MYC-driven tumorigenesis. We identified indole-3-carbinol, a compound found in cruciferous vegetables, as a natural and potent WWP1 inhibitor. Thus, our findings unravel a potential therapeutic strategy for cancer prevention and treatment through PTEN reactivation.
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Anticarcinógenos/farmacologia , Indóis/farmacologia , Neoplasias/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Anticarcinógenos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/uso terapêutico , Masculino , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/genética , Multimerização Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacosRESUMO
RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.
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Disceratose Congênita/genética , Epigenômica/métodos , Mutação em Linhagem Germinativa , Proteínas Nucleares/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Ribossômico/genética , Animais , Disceratose Congênita/patologia , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/química , Nucleofosmina , RNA Nucleolar Pequeno , TranscriptomaRESUMO
Women are vulnerable to anxiety during pregnancy and postpartum. However, little is known about antenatal anxiety and neural processing of infant-relevant information. In this experiment, the N170, P300, and LPP (late positive potential) event-related potentials were measured from 43 pregnant women as they viewed infant and adult faces, which were either neutral or distressed in expression. Mother's self-reported anxiety levels were also assessed. The N170 was comparable across face conditions and was not associated with anxiety. However, our central finding was that greater levels of antenatal anxiety were associated with a larger LPP, but only for neutral infant faces. Results suggest that antenatal anxiety may result in deeper processing of neutral, emotionally ambiguous, infant faces during pregnancy. These findings are discussed in light of other work indicating an interpretive bias toward threat in response to neutral stimuli in anxiety.
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Ansiedade/psicologia , Reconhecimento Facial/fisiologia , Complicações na Gravidez/psicologia , Gestantes/psicologia , Adulto , Eletroencefalografia/métodos , Emoções/fisiologia , Potenciais Evocados/fisiologia , Feminino , Humanos , Lactente , Estimulação Luminosa/métodos , GravidezRESUMO
Coronary artery disease (CAD) is the leading cause of mortality and morbidity, driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies have identified >150 loci associated with CAD and myocardial infarction susceptibility in humans. A majority of these variants reside in non-coding regions and are co-inherited with hundreds of candidate regulatory variants, presenting a challenge to elucidate their functions. Herein, we use integrative genomic, epigenomic and transcriptomic profiling of perturbed human coronary artery smooth muscle cells and tissues to begin to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps, we prioritize 64 candidate variants and perform allele-specific binding and expression analyses at seven top candidate loci: 9p21.3, SMAD3, PDGFD, IL6R, BMP1, CCDC97/TGFB1 and LMOD1. We validate our findings in expression quantitative trait loci cohorts, which together reveal new links between CAD associations and regulatory function in the appropriate disease context.
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Cromatina/química , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Genoma Humano , Genômica/métodos , Locos de Características Quantitativas , Alelos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Cultura Primária de Células , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.
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Stents Farmacológicos/efeitos adversos , Artéria Femoral/lesões , Implantação de Prótese/efeitos adversos , Animais , Modelos Animais de Doenças , Hiperplasia/etiologia , Camundongos , Implantação de Prótese/métodos , Túnica Íntima/patologiaRESUMO
While there is a persistent inverse relationship between latitude and species diversity across many taxa and ecosystems, deviations from this norm offer an opportunity to understand the conditions that contribute to large-scale diversity patterns. Marine systems, in particular, provide such an opportunity, as marine diversity does not always follow a strict latitudinal gradient, perhaps because several hypothesized drivers of the latitudinal diversity gradient are uncorrelated in marine systems. We used a large scale public monitoring dataset collected over an eight year period to examine benthic marine faunal biodiversity patterns for the continental shelf (55-183 m depth) and slope habitats (184-1280 m depth) off the US West Coast (47°20'N-32°40'N). We specifically asked whether marine biodiversity followed a strict latitudinal gradient, and if these latitudinal patterns varied across depth, in different benthic substrates, and over ecological time scales. Further, we subdivided our study area into three smaller regions to test whether coast-wide patterns of biodiversity held at regional scales, where local oceanographic processes tend to influence community structure and function. Overall, we found complex patterns of biodiversity on both the coast-wide and regional scales that differed by taxonomic group. Importantly, marine biodiversity was not always highest at low latitudes. We found that latitude, depth, substrate, and year were all important descriptors of fish and invertebrate diversity. Invertebrate richness and taxonomic diversity were highest at high latitudes and in deeper waters. Fish richness also increased with latitude, but exhibited a hump-shaped relationship with depth, increasing with depth up to the continental shelf break, ~200 m depth, and then decreasing in deeper waters. We found relationships between fish taxonomic and functional diversity and latitude, depth, substrate, and time at the regional scale, but not at the coast-wide scale, suggesting that coast-wide patterns can obscure important correlates at smaller scales. Our study provides insight into complex diversity patterns of the deep water soft substrate benthic ecosystems off the US West Coast.