RESUMEN
The mammalian SWI/SNF (mSWI/SNF or BAF) family of chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression. The three final-form subcomplexes-cBAF, PBAF, and ncBAF-are distinct in biochemical componentry, chromatin targeting, and roles in disease; however, the contributions of their constituent subunits to gene expression remain incompletely defined. Here, we performed Perturb-seq-based CRISPR-Cas9 knockout screens targeting mSWI/SNF subunits individually and in select combinations, followed by single-cell RNA-seq and SHARE-seq. We uncovered complex-, module-, and subunit-specific contributions to distinct regulatory networks and defined paralog subunit relationships and shifted subcomplex functions upon perturbations. Synergistic, intra-complex genetic interactions between subunits reveal functional redundancy and modularity. Importantly, single-cell subunit perturbation signatures mapped across bulk primary human tumor expression profiles both mirror and predict cBAF loss-of-function status in cancer. Our findings highlight the utility of Perturb-seq to dissect disease-relevant gene regulatory impacts of heterogeneous, multi-component master regulatory complexes.
Asunto(s)
Ensamble y Desensamble de Cromatina , Neoplasias , Animales , Humanos , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cromatina/genética , Mamíferos/metabolismoRESUMEN
Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Cromosómicas no Histona/química , Código de Histonas , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Mutación , Nucleosomas/química , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Factores de Transcripción/químicaRESUMEN
STEM training of college-bound and college students has reliably employed hands-on experiential learning by placing students in on-campus research settings. Dana-Farber/Harvard Cancer Center's Young Empowered Scientists for ContinUed Research Engagement (DF/HCC's YES for CURE) program introduces Massachusetts high school and college students from underrepresented populations to cancer research by immersing them in scientific and nursing research environments. Amidst the COVID-19 pandemic, the 2020 summer program was re-designed and delivered virtually for 45 students. Because the program spans three years, we could evaluate the experiences of 18 students (cohort 2) who completed the 2019 (pre-pandemic) and 2020 (pandemic) summer programs. Analysis of cohort 2 data revealed three areas where students felt their competence improved with virtual programming (i.e., effective communication of ideas, access to high caliber speakers, engagement with program leaders) and two areas where it declined significantly (i.e., engaging other students, learning lab material). Additionally, student-reported competence to perform 21 scientific research and seven critical thinking processes were not negatively impacted by the virtual transition. Herein, we describe the adaptation of DF/HCC's YES for CURE program to a virtual format and the impact on students as a resource for institutions interested in enhancing their STEM training programs with virtual programming.
RESUMEN
SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Discapacidades del Desarrollo/genética , Mutación Missense , Fenotipo , Proteínas Supresoras de Tumor/genética , Adolescente , Animales , Niño , Preescolar , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Genes Dominantes , Variación Genética , Haploinsuficiencia , Humanos , Lactante , Masculino , Microscopía Confocal , Neuroglía/metabolismo , Neuronas/metabolismo , Unión Proteica , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
SF3B1 is the most commonly mutated RNA splicing factor in cancer1-4, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L5-7. Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Neoplasias/genética , Empalme del ARN , Empalmosomas/metabolismo , Animales , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias/patología , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , Empalmosomas/genética , Factores de Transcripción/metabolismoRESUMEN
Mammalian SWI/SNF chromatin remodelling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF and a newly characterized non-canonical complex (ncBAF). However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma and malignant rhabdoid tumours, which both exhibit cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the ncBAF subunit, BRD9, rapidly attenuates synovial sarcoma and malignant rhabdoid tumour cell proliferation. Importantly, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites and function in a manner distinct from fusion oncoprotein-bound complexes. Together, these findings unmask the unique targeting and functional roles of ncBAF complexes and present new cancer-specific therapeutic targets.
Asunto(s)
Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Tumor Rabdoide/genética , Sarcoma Sinovial/genética , Factores de Transcripción/genética , Línea Celular Tumoral , Proliferación Celular/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Tumor Rabdoide/metabolismo , Sarcoma Sinovial/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción/metabolismo , Animales , Cromatina/química , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/genética , Drosophila/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Espectrometría de Masas , Mutagénesis , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.
Asunto(s)
Aptitud Genética/genética , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Células A549 , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Genes Esenciales/genética , Pruebas Genéticas/métodos , Células HEK293 , Humanos , Mamíferos/genética , Complejos Multiproteicos/genética , Interferencia de ARNRESUMEN
Mammalian SWI/SNF complexes have critical roles in development and differentiation, and are implicated in the pathogenesis of several diseases; however, the mechanisms underpinning disease manifestation and the specificity of the subunits mutated are incompletely understood. Newly identified loss-of-function mutations in the SMARCD2 gene (part of the SMARCD1, SMARCD2 and SMARCD3 paralog family) reveal an evolutionarily conserved role specifically for the SMARCD2 subunit in granulopoiesis, and further investigation implicates the CEBPÉ transcription factor as a key effector of this specific function.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Granulocitos/fisiología , Hematopoyesis/fisiología , Factores de Transcripción/metabolismo , Proteínas Cromosómicas no Histona/genética , Humanos , Factores de Transcripción/genéticaRESUMEN
Alp7A is a bacterial actin from Bacillus subtilis plasmid pLS20 that functions in plasmid segregation. Alp7A's function requires that it assemble into filaments that treadmill and exhibit dynamic instability. These dynamic properties require the two other components of the alp7A operon, the downstream alp7R gene and the upstream alp7C sequence, as does the ability of Alp7A to form filaments at its physiological concentration in the cell. Here, we show that these two other components of the operon also determine the amount of Alp7A that is produced in the cell. The deletion of alp7R leads to overproduction of Alp7A, which assembles into large, amorphous, static filaments that disrupt chromosome segregation and cell division. The product of the alp7R gene is a DNA-binding protein that represses transcription of the alp7A operon. Purified Alp7R protein binds specifically to alp7C, which contains two σ(A) promoters embedded within a series of near-repeats of a 10-mer. Alp7R also shows the typical nonspecific binding activity of a DNA-binding protein: Alp7R-GFP (green fluorescent protein) associates with the chromosomes of cells that lack alp7C. When Alp7A-GFP is produced in B. subtilis along with untagged Alp7R, Alp7A-GFP also colocalizes with the chromosome, indicating that Alp7R associates with Alp7A. Hence Alp7R, determines both the activity and the cellular concentration of Alp7A, and it can associate with Alp7A even if it is not bound to alp7C.