A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

DARESOME enables concurrent profiling of multiple DNA modifications with restriction enzymes in single cells and cell-free DNA.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these different epigenetic marks aimed at understanding their combined effects and dynamic interconversion are, however, hampered by the inability of current methods to simultaneously measure both modifications, particularly in samples with limited quantities. We present DNA analysis by restriction enzyme for simultaneous detection of multiple epigenomic states (DARESOME), an assay based on modification-sensitive restriction digest and sequential tag ligation that can concurrently perform quantitative profiling of unmodified cytosine, 5mC, and 5hmC in CCGG sites genome-wide. DARESOME reveals the opposing roles of 5mC and 5hmC in gene expression regulation as well as their interconversion during aging in mouse brain. Implementation of DARESOME in single cells demonstrates pronounced 5hmC strand bias that reflects the semiconservative replication of DNA. Last, we showed that DARESOME enables integrative genomic, 5mC, and 5hmC profiling of cell-free DNA that uncovered multiomics cancer signatures in liquid biopsy.

Melt-Encoded-Tags for Expanded Optical Readout in Digital PCR (METEOR-dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling.

Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.

Inhibition of USP14 promotes TNFα-induced cell death in head and neck squamous cell carcinoma (HNSCC).

TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.

Combined Inhibition of IAPs and WEE1 Enhances TNFα- and Radiation-Induced Cell Death in Head and Neck Squamous Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK-NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC.

Telomerase Inhibition by MST-312 Sensitizes Breast Cancer Cells to the Anti-cancer Properties of Plumbagin.

Breast cancer is the most common cause of malignancy and the second most common cause of death due to cancer in women. This heterogeneous disease is currently broadly classified as estrogen receptor (ER), progesterone receptor (PR) positive luminal tumors, human epidermal growth factor receptor 2 (HER2) amplified tumors and triple-negative breast cancers (TNBC). Phytochemicals are proven to be promising anti-cancer chemotherapeutics agents with minimal cytotoxic effects on normal cells. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is a phytochemical derived from the roots of Plumbago zeylanica and it is known to possess anti-cancer properties similar to other compounds of naphthoquinones. In about 90% of cancer cells, the telomerase enzyme activity is revived to add telomeric repeats to evade apoptosis. In this study, a combinatorial approach of combining the anti-cancer compound plumbagin to induce genotoxicity and a potent telomerase inhibitor, MST-312 (synthetic derivative of tea catechins), was used to determine the combinational treatment-induced lethality in breast cancer cells such as MDA-MB-231 (TNBC) and MCF-7 (lumina) cells. MDA-MB-231 cells were responsive to combination treatment in both short-term (48 h) and long-term treatment (14 days) in a synergistic manner, whereas in MCF-7, the combination treatment was more effective in the long-term regimen. Furthermore, the cytotoxic effects of the plumbagin and MST-312 combination treatment were not recoverable after the short-term treatment. In conclusion, a combination treatment of MST-312 and plumbagin is proven to be more effective than a single plumbagin compound treatment in inducing DNA damage and telomere dysfunction leading to greater genome instability, cell cycle arrest and eventually cell death in cancer cells.

Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients.

Genome-wide analysis of cell-free DNA methylation profile is a promising approach for sensitive and specific detection of many cancers. However, scaling such assays for clinical translation is impractical because of the high cost of whole-genome bisulfite sequencing. We show that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors most of the cancer-specific methylation signature. Here, we report on the simple and effective heat enrichment of CpG-rich regions for bisulfite sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation and quantitative tracking of colorectal cancer patient's response to treatment at much reduced sequencing cost suitable for frequent monitoring. We also show tumor epigenetic subtyping using Heatrich-BS, which could enable patient stratification. Heatrich-BS holds great potential for highly scalable screening and monitoring of cancer using liquid biopsy.

Inhibiting WEE1 and IKK-RELA Crosstalk Overcomes TNFα Resistance in Head and Neck Cancers.

TNFα is a key mediator of immune and radiotherapy-induced cytotoxicity, but many cancers, including head and neck squamous cell carcinomas (HNSCC), display TNF resistance due to activation of the canonical IKK-NF-κB/RELA pro-survival pathway. However, toxicities associated with direct targeting of the canonical pathway point to the need to identify mechanism(s) contributing to TNFα resistance and synthetic lethal targets to overcome such resistance in cancer cells. Here, RNAi screening for modulators of TNFα-NF-κB reporter activity and cell survival unexpectedly implicated the WEE1 and CDC2 G2-M checkpoint kinases. The IKKα/ß-RELA and WEE1-CDC2 signaling pathways are activated by TNFα and form a complex in cell lines derived from both human papillomavirus (-) and (+) subtypes of HNSCC. WEE1 inhibitor AZD1775 reduced IKK/RELA phosphorylation and the expression of NF-κB-dependent pro-survival proteins Cyclin D1 and BCL2. Combination of TNFα and AZD1775 enhanced caspase-mediated apoptosis in vitro, and combination treatment with radiotherapy and AZD1775 potentiated inhibition of HNSCC tumor xenograft growth in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insight into the interplay between NF-κB signaling and WEE1-mediated regulation of the G2-M cell-cycle checkpoint in HNSCC. IMPLICATIONS: Inhibiting WEE1 and IKK-RELA crosstalk could potentially enhance the effects of therapies mediated by TNFα with less systemic immune suppression and toxicity than observed with direct interruption of IKK-NF-κB/RELA signaling.

AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines.

Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

Massively parallel single-molecule telomere length measurement with digital real-time PCR.

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP-DNA dissociation.

The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1's ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1's catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.

Single-cell multimodal profiling reveals cellular epigenetic heterogeneity.

Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme.

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection.

The RSC chromatin remodeler slides and ejects nucleosomes, utilizing a catalytic subunit (Sth1) with DNA translocation activity, which can pump DNA around the nucleosome. A central question is whether and how DNA translocation is regulated to achieve sliding versus ejection. Here, we report the regulation of DNA translocation efficiency by two domains residing on Sth1 (Post-HSA and Protrusion 1) and by actin-related proteins (ARPs) that bind Sth1. ARPs facilitated sliding and ejection by improving "coupling"-the amount of DNA translocation by Sth1 relative to ATP hydrolysis. We also identified and characterized Protrusion 1 mutations that promote "coupling," and Post-HSA mutations that improve ATP hydrolysis; notably, the strongest mutations conferred efficient nucleosome ejection without ARPs. Taken together, sliding-to-ejection involves a continuum of DNA translocation efficiency, consistent with higher magnitudes of ATPase and coupling activities (involving ARPs and Sth1 domains), enabling the simultaneous rupture of multiple histone-DNA contacts facilitating ejection.

Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.

Measuring chromatin interaction dynamics on the second time scale at single-copy genes.

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins, but it is unknown how stable most native interactions are. Although live-cell imaging suggests short-lived interactions at tandem gene arrays, current methods cannot measure rapid binding dynamics at single-copy genes. We show, by using a modified ChIP assay with subsecond temporal resolution, that the time dependence of formaldehyde cross-linking can be used to extract in vivo on and off rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. By using the method, we show that a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation. This assay provides an approach for systematic, quantitative analyses of chromatin binding dynamics in vivo.

Two-step mechanism for modifier of transcription 1 (Mot1) enzyme-catalyzed displacement of TATA-binding protein (TBP) from DNA.

The TATA box binding protein (TBP) is a central component of the transcription preinitiation complex, and its occupancy at a promoter is correlated with transcription levels. The TBP-promoter DNA complex contains sharply bent DNA and its interaction lifetime is limited by the ATP-dependent TBP displacement activity of the Snf2/Swi2 ATPase Mot1. Several mechanisms for Mot1 action have been proposed, but how it catalyzes TBP removal from DNA is unknown. To better understand the Mot1 mechanism, native gel electrophoresis and FRET were used to determine how Mot1 affects the trajectory of DNA in the TBP-DNA complex. Strikingly, in the absence of ATP, Mot1 acts to unbend DNA, whereas TBP remains closely associated with the DNA in a stable Mot1-TBP-DNA ternary complex. Interestingly, and in contrast to full-length Mot1, the isolated Mot1 ATPase domain binds DNA, and its affinity for DNA is nucleotide-dependent, suggesting parallels between the Mot1 mechanism and DNA translocation-based mechanisms of chromatin remodeling enzymes. Based on these findings, a model is presented for Mot1 that links a DNA conformational change with ATP-induced DNA translocation.

Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP.

Swi2/Snf2-type ATPases regulate genome-associated processes such as transcription, replication and repair by catalysing the disruption, assembly or remodelling of nucleosomes or other protein-DNA complexes. It has been suggested that ATP-driven motor activity along DNA disrupts target protein-DNA interactions in the remodelling reaction. However, the complex and highly specific remodelling reactions are poorly understood, mostly because of a lack of high-resolution structural information about how remodellers bind to their substrate proteins. Mot1 (modifier of transcription 1 in Saccharomyces cerevisiae, denoted BTAF1 in humans) is a Swi2/Snf2 enzyme that specifically displaces the TATA box binding protein (TBP) from the promoter DNA and regulates transcription globally by generating a highly dynamic TBP pool in the cell. As a Swi2/Snf2 enzyme that functions as a single polypeptide and interacts with a relatively simple substrate, Mot1 offers an ideal system from which to gain a better understanding of this important enzyme family. To reveal how Mot1 specifically disrupts TBP-DNA complexes, we combined crystal and electron microscopy structures of Mot1-TBP from Encephalitozoon cuniculi with biochemical studies. Here we show that Mot1 wraps around TBP and seems to act like a bottle opener: a spring-like array of 16 HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats grips the DNA-distal side of TBP via loop insertions, and the Swi2/Snf2 domain binds to upstream DNA, positioned to weaken the TBP-DNA interaction by DNA translocation. A 'latch' subsequently blocks the DNA-binding groove of TBP, acting as a chaperone to prevent DNA re-association and ensure efficient promoter clearance. This work shows how a remodelling enzyme can combine both motor and chaperone activities to achieve functional specificity using a conserved Swi2/Snf2 translocase.

One small step for Mot1; one giant leap for other Swi2/Snf2 enzymes?

The TATA-binding protein (TBP) is a major target for transcriptional regulation. Mot1, a Swi2/Snf2-related ATPase, dissociates TBP from DNA in an ATP dependent process. The experimental advantages of this relatively simple reaction have been exploited to learn more about how Swi2/Snf2 ATPases function biochemically. However, many unanswered questions remain and fundamental aspects of the Mot1 mechanism are still under debate. Here, we review the available data and integrate the results with structural and biochemical studies of related enzymes to derive a model for Mot1's catalytic action consistent with the broad literature on enzymes in this family. We propose that the Mot1 ATPase domain is tethered to TBP by a flexible, spring-like linker of alpha helical hairpins. The linker juxtaposes the ATPase domain such that it can engage duplex DNA on one side of the TBP-DNA complex. This allows the ATPase to employ short-range, nonprocessive ATP-driven DNA tracking to pull or push TBP off its DNA site. DNA translocation is a conserved property of ATPases in the broader enzyme family. As such, the model explains how a structurally and functionally conserved ATPase domain has been put to use in a very different context than other enzymes in the Swi2/Snf2 family. This article is part of a Special Issue entitled:Snf2/Swi2 ATPase structure and function.

The RSC chromatin remodelling ATPase translocates DNA with high force and small step size.

ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase 'motor' on bare DNA, using high-resolution optical tweezers and a 'tethered' translocase system. We observe on dsDNA a processivity of ∼35 bp, a speed of ∼25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA 'buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force.