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Specific multivalent molecules boost CRISPR-mediated transcriptional activation via optimal cis-trans interaction and functional chromatin looping [RNA-seq]

ABSTRACT: Synthetic transcriptional activators based on CRISPR/Cas technology can be enhanced by intrinsically disordered regions (IDRs). However, the potential of all IDRs in this regard remains uncertain, and the mechanisms underlying their influence are a subject of debate. Here, we examined 12 well-known IDRs by fusing them to the dCas9-VP64 activator. Our findings reveal that seven IDRs could augment activation, albeit independently of their phase separation properties. Moreover, modular domains (MDs), which facilitate multivalent interactions but are ineffective in enhancing dCas9-VP64 activity on their own, showed substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and combining dCas9-VP64 with the IDRs and MDs of different multivalent capabilities, we uncovered that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs with our IDR-enhanced activation system yielded synergistic effects, potentially further amplifiable by induced functional chromatin looping.

ORGANISM(S): Homo sapiens

PROVIDER: GSE248523 | GEO | 2024/06/01

REPOSITORIES: GEO

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Specific multivalent molecules boost CRISPR-mediated transcriptional activation via optimal cis-trans interaction and functional chromatin looping [ChIA-PET]

Project description:Synthetic transcriptional activators based on CRISPR/Cas technology can be enhanced by intrinsically disordered regions (IDRs). However, the potential of all IDRs in this regard remains uncertain, and the mechanisms underlying their influence are a subject of debate. Here, we examined 12 well-known IDRs by fusing them to the dCas9-VP64 activator. Our findings reveal that seven IDRs could augment activation, albeit independently of their phase separation properties. Moreover, modular domains (MDs), which facilitate multivalent interactions but are ineffective in enhancing dCas9-VP64 activity on their own, showed substantial enhancement in transcriptional activation when combined with dCas9-VP64-IDR. By varying the number of gRNA binding sites and combining dCas9-VP64 with the IDRs and MDs of different multivalent capabilities, we uncovered that optimal, rather than maximal, cis-trans cooperativity enables the most robust activation. Finally, targeting promoter-enhancer pairs with our IDR-enhanced activation system yielded synergistic effects, potentially further amplifiable by induced functional chromatin looping.

2024-06-01 | GSE248522 | GEO

Functional partitioning of transcriptional regulators by patterned charge blocks

Project description:Components of the transcriptional machinery are dynamically compartmentalized by weak multivalent interactions, yet we know little about how specificity or selectivity of partitioning is achieved. Here we show that condensates composed of the intrinsically disordered region (IDR) of MED1 (MED1_IDR) selectively partition positive regulators of transcription(SPT6, CTR9, IWS1) with RNA Pol II while excluding negative regulators of transcription (NELF and HP1a).This selective compartmentalization by MED1_IDR is sufficient to activate transcription and is required for gene activation during a cell state transition. Surprisingly, the IDRs of partitioned proteins are sufficient to recapitulate selective compartmentalization. We find that IDR-mediated selective partitioning requires multivalent blocks of positive and negative charge on MED1_IDR and IDRs of partitioned proteins. The charge patterns required for partitioning are also required for gene activation. These findings demonstrate that proteins can be functionally compartmentalized by specific multivalent interactions driven by the charge patterning within disordered regions.

2023-01-03 | GSE210875 | GEO

Potent and targeted activation of HIV-1 using the CRISPR/Cas9 activator Complex

Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The â??shock and killâ?? strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). Â We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a â??hotspotâ?? for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple differentÂ in vitroÂ latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT).Â While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs.Â In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus.Â In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a â??functional cureâ?? of HIV/AIDS. Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes

2016-08-20 | E-GEOD-72259 | biostudies-arrayexpress

Transcriptome-wide analysis of IL2RA enhancer activation by CRISPRa

Project description:We previously performed a tiling CRISPR activation (CRISPRa) screen in Jurkat T cells to discover enhancers controlling IL2RA expression. This screen identified six regions where recruitment of dCas9-VP64 was sufficient to drive increased expression of IL2RA. We named these putative enhancers CRISPRa Responsive Elements (CaREs). To examine transcriptome-wide consequences of dCas9-VP64 recruitment to IL2RA CaREs, we performed RNA-Seq on HuT78 cells stably expressing dCas9-VP64 and gRNAs targeting the IL2RA TSS, CaRE3, or CaRE4, or stimulated with anti-CD3/CD28 antibodies.

2017-04-26 | GSE98177 | GEO

Potent and targeted activation of HIV-1 using the CRISPR/Cas9 activator Complex

Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a “hotspot” for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple different in vitro latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT). While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs. In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus. In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a “functional cure” of HIV/AIDS.

2016-08-20 | GSE72259 | GEO

Evolutional heterochromatin condensation delineates chromocenter formation and retrotransposon constraint in plants

Project description:Heterochromatic condensates (chromocenters) are critical for maintaining the silencing of heterochromatin. However, it is a riddle why the presence of chromocenters is variable across species. Here, we reveal that variations in the plant heterochromatin protein ADCP1 confer a diversity in chromocenter formation via phase separation. ADCP1 physically interacts with the high mobility group protein HMGA to form a complex and mediates heterochromatin condensation by multivalent interactions. The loss of intrinsically disordered regions (IDRs) in ADCP1 homologues during evolution has led to the absence of prominent chromocenter in various plant species, and introduction of IDR-containing ADCP1 with HMGA promotes heterochromatin condensation and retrotransposon silencing. Moreover, plants in the Cucurbitaceae group have evolved an IDR-containing chimera of ADCP1 and HMGA, which enables formation of chromocenters remarkably. Together, our work uncovers a coevolved mechanism of phase separation in packing heterochromatin and constraining retrotransposons.

2024-06-22 | GSE233265 | GEO

TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing

Project description:TFIID is an essential eukaryotic transcription factor, which is required for RNA polymerase II promoter recognition and activation. As a multiprotein complex, TFIID contains several intrinsically disordered regions (IDRs), but the functions of these IDRs are unknown. Here, we show that a conserved IDR drives the TFIID subunit TAF2 to nuclear speckles, where it forms biomolecular condensates, separate from the TFIID complex. Quantitative mass spectrometry analyses reveal that the TAF2 IDR is required for the interaction with the nuclear speckle and spliceosome-associated protein SRRM2, which is thereby recruited to TFIID. The formation of SRRM2-free TFIID complexes elicits a set of unique alternative splicing events. These include events in RNAs coding for proteins involved in transcription and transmembrane transport. Together, these data identify an IDR of the basal transcription machinery as a molecular switch between nuclear compartments to control protein complex composition and pre-mRNA splicing.

2024-04-26 | PXD049117 | Pride

Intrinsically disordered regions of transcription factor encode multiple functions using interwoven sequence grammars (ChEC-seq)

Project description:Intrinsically disordered regions (IDRs) are abundant within eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as exemplified by the budding-yeast TF- Msn2. To examine how low-complexity IDRs encode multiple functions, we compared genomic binding preferences, gene induction, and coactivator recruitment amongst a large set of designed Mns2-IDR mutants. We show that multiple regions across the >500AA IDR contribute to both functions. Yet, transcription activity was readily disrupted by variants having no consequences on Msn2 binding. Our data attribute this differential sensitivity to the integration of relaxed, composition-based code directing binding preferences with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Interwoven sequence grammar may present a general paradigm through which low-complexity IDRs encode multiple functions.

2023-11-19 | GSE239882 | GEO

Intrinsically disordered regions of transcription factor encode multiple functions using interwoven sequence grammars (RNA-Seq)

2023-11-19 | GSE239883 | GEO

Hierarchical antagonistic regulatory elements in the TRPV4 intrinsically disordered N-terminus modulate lipid binding and channel activity through transient long-range interactions

Project description:Intrinsically disordered regions (IDRs) are important for the functional regulation of membrane receptors. Among the TRP Vanilloid channels involved in thermo- and osmosensation, TRPV4 features the largest N-terminal IDR of ~150 residues. Here, we elucidate the structural ensemble of the TRPV4 IDR through an integrated structural biology approach. Novel structural and functional properties can be assigned to distinct IDR regions. Along the length of the entire IDR, a hierarchical interaction network of structurally and functionally coupled antagonistic regulatory modules can be mapped. Long-range transient crosstalk between these regulatory elements mediates channel responses to osmotic stimuli. Most notably, a highly conserved regulatory patch in the N-terminal IDR asserts dominant negative control over lipid binding and channel activity by acting on a sensitizing PIP2 binding site in the C-terminal IDR. This work demonstrates the importance of IDRs and “IDR cartography” for understanding TRP channel function and regulation.

2023-08-01 | PXD038153 | Pride

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