Interaction modules that impart specificity to disordered protein.

Intrinsically disordered regions (IDRs) are especially enriched among proteins that regulate chromatin and transcription. As a result, mechanisms that influence specificity of IDR-driven interactions have emerged as exciting unresolved issues for understanding gene regulation. We review the molecular elements frequently found within IDRs that confer regulatory specificity. In particular, we summarize the differing roles of disordered low-complexity regions (LCRs) and short linear motifs (SLiMs) towards selective nuclear regulation. Examination of IDR-driven interactions highlights SLiMs as organizers of selectivity, with widespread roles in gene regulation and integration of cellular signals. Analysis of recurrent interactions between SLiMs and folded domains suggests diverse avenues for SLiMs to influence phase-separated condensates and highlights opportunities to manipulate these interactions for control of biological activity.

Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF.

Several cancer core regulatory circuitries (CRCs) depend on the sustained generation of DNA accessibility by SWI/SNF chromatin remodelers. However, the window when SWI/SNF is acutely essential in these settings has not been identified. Here we used neuroblastoma (NB) cells to model and dissect the relationship between cell-cycle progression and SWI/SNF ATPase activity. We find that SWI/SNF inactivation impairs coordinated occupancy of non-pioneer CRC members at enhancers within 1 hour, rapidly breaking their autoregulation. By precisely timing inhibitor treatment following synchronization, we show that SWI/SNF is dispensable for survival in S and G2/M, but becomes acutely essential only during G1 phase. We furthermore developed a new approach to analyze the oscillating patterns of genome-wide DNA accessibility across the cell cycle, which revealed that SWI/SNF-dependent CRC binding sites are enriched at enhancers with peak accessibility during G1 phase, where they activate genes involved in cell-cycle progression. SWI/SNF inhibition strongly impairs G1-S transition and potentiates the ability of retinoids used clinically to induce cell-cycle exit. Similar cell-cycle effects in diverse SWI/SNF-addicted settings highlight G1-S transition as a common cause of SWI/SNF dependency. Our results illustrate that deeper knowledge of the temporal patterns of enhancer-related dependencies may aid the rational targeting of addicted cancers.

Cancer cells driven by runaway transcription factor networks frequently depend on the cellular machinery that promotes DNA accessibility. For this reason, recently developed small molecules that impair SWI/SNF (or BAF) chromatin remodeling activity have been under active evaluation as anti-cancer agents. However, exactly when SWI/SNF activity is essential in dependent cancers has remained unknown. By combining live-cell imaging and genome-wide profiling in neuroblastoma cells, Cermakova et al. discover that SWI/SNF activity is needed for survival only during G1 phase of the cell cycle. The authors reveal that in several cancer settings, dependency on SWI/SNF arises from the need to reactivate factors involved in G1-S transition. Because of this role, authors find that SWI/SNF inhibition potentiates cell-cycle exit by retinoic acid.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

The TFIIS N-terminal domain (TND): a transcription assembly module at the interface of order and disorder.

Interaction scaffolds that selectively recognize disordered protein strongly shape protein interactomes. An important scaffold of this type that contributes to transcription is the TFIIS N-terminal domain (TND). The TND is a five-helical bundle that has no known enzymatic activity, but instead selectively reads intrinsically disordered sequences of other proteins. Here, we review the structural and functional properties of TNDs and their cognate disordered ligands known as TND-interacting motifs (TIMs). TNDs or TIMs are found in prominent members of the transcription machinery, including TFIIS, super elongation complex, SWI/SNF, Mediator, IWS1, SPT6, PP1-PNUTS phosphatase, elongin, H3K36me3 readers, the transcription factor MYC, and others. We also review how the TND interactome contributes to the regulation of transcription. Because the TND is the most significantly enriched fold among transcription elongation regulators, TND- and TIM-driven interactions have widespread roles in the regulation of many transcriptional processes.

CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation.

The chromatin remodeler CHD8 is among the most frequently mutated genes in autism spectrum disorder (ASD). CHD8 has a dosage-sensitive role in ASD, but when and how it becomes critical to human social function is unclear. Here, we conducted genomic analyses of heterozygous and homozygous Chd8 mouse embryonic stem cells and differentiated neural progenitors. We identify dosage-sensitive CHD8 transcriptional targets, sites of regulated accessibility, and an unexpected cooperation with SOX transcription factors. Collectively, our findings reveal that CHD8 negatively regulates expression of neuronal genes to maintain pluripotency and also during differentiation. Thus, CHD8 is essential for both the maintenance of pluripotency and neural differentiation, providing mechanistic insight into its function with potential implications for ASD.

Switching of the folding-energy landscape governs the allosteric activation of protein kinase A.

Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of PKA harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.

Affinity switching of the LEDGF/p75 IBD interactome is governed by kinase-dependent phosphorylation.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Next-Generation Drugs and Probes for Chromatin Biology: From Targeted Protein Degradation to Phase Separation.

Chromatin regulation is a critical aspect of nuclear function. Recent advances have provided detailed information about dynamic three-dimensional organization of chromatin and its regulatory factors. Mechanisms crucial for normal nuclear function and epigenetic control include compartmentalization of biochemical reactions by liquid-phase separated condensates and signal-dependent regulation of protein stability. Synthetic control of these phenomena by small molecules provides deep insight into essential activities such as histone modification, BAF (SWI/SNF) and PBAF remodeling, Polycomb repression, enhancer looping by cohesin and CTCF, as well as many other processes that contribute to transcription. As a result, a complete understanding of the spatiotemporal mechanisms that underlie chromatin regulation increasingly requires the use of fast-acting drugs and chemical probes. Here, we provide a comprehensive review of next-generation chemical biology tools to interrogate the chromatin regulatory landscape, including selective PROTAC E3 ubiquitin ligase degraders, degrons, fluorescent ligands, dimerizers, inhibitors, and other drugs. These small molecules provide important insights into the mechanisms that govern gene regulation, DNA repair, development, and diseases like cancer.

Pharmacological blockade of a pioneer transcription factor.

Cancers frequently co-opt lineage-specific transcription factors (TFs) utilized in normal development to sustain proliferation. However, the effects of these TFs on tumor development depend considerably on where in the genome they bind. A new paper by Taylor and colleagues expands on previously developed diamidine compounds that obstruct the DNA binding sites of the pioneer TF PU.1 (SPI1) in acute myeloid leukemia (AML). Immobilization and sequencing of genomic DNA targeted by these compounds revealed that these inhibitors alter the genomic binding patterns of PU.1. The authors report that their strategy constrains the genomic binding preferences of PU.1, leading to redistribution of PU.1 to promoters and other gene-proximal regions with elevated G/C content. Here we discuss recent developments for targeting PU.1 in hematologic malignancies. We also explore the shared functional roles of PU.1 and SWI/SNF ATP-dependent chromatin remodeling complexes, which work together to sustain the enhancer landscape needed for tumor cell proliferation but also have key roles in non-tumor settings.

The IL6/JAK/STAT3 signaling axis is a therapeutic vulnerability in SMARCB1-deficient bladder cancer.

SMARCB1 loss has long been observed in many solid tumors. However, there is a need to elucidate targetable pathways driving growth and metastasis in SMARCB1-deficient tumors. Here, we demonstrate that SMARCB1 deficiency, defined as genomic SMARCB1 copy number loss associated with reduced mRNA, drives disease progression in patients with bladder cancer by engaging STAT3. SMARCB1 loss increases the chromatin accessibility of the STAT3 locus in vitro. Orthotopically implanted SMARCB1 knockout (KO) cell lines exhibit increased tumor growth and metastasis. SMARCB1-deficient tumors show an increased IL6/JAK/STAT3 signaling axis in in vivo models and patients. Furthermore, a pSTAT3 selective inhibitor, TTI-101, reduces tumor growth in SMARCB1 KO orthotopic cell line-derived xenografts and a SMARCB1-deficient patient derived xenograft model. We have identified a gene signature generated from SMARCB1 KO tumors that predicts SMARCB1 deficiency in patients. Overall, these findings support the clinical evaluation of STAT3 inhibitors for the treatment of SMARCB1-deficient bladder cancer.

SWI/SNF Blockade Disrupts PU.1-Directed Enhancer Programs in Normal Hematopoietic Cells and Acute Myeloid Leukemia.

In acute myeloid leukemia (AML), SWI/SNF chromatin remodeling complexes sustain leukemic identity by driving high levels of MYC. Previous studies have implicated the hematopoietic transcription factor PU.1 (SPI1) as an important target of SWI/SNF inhibition, but PU.1 is widely regarded to have pioneer-like activity. As a result, many questions have remained regarding the interplay between PU.1 and SWI/SNF in AML as well as normal hematopoiesis. Here we found that PU.1 binds to most of its targets in a SWI/SNF-independent manner and recruits SWI/SNF to promote accessibility for other AML core regulatory factors, including RUNX1, LMO2, and MEIS1. SWI/SNF inhibition in AML cells reduced DNA accessibility and binding of these factors at PU.1 sites and redistributed PU.1 to promoters. Analysis of nontumor hematopoietic cells revealed that similar effects also impair PU.1-dependent B-cell and monocyte populations. Nevertheless, SWI/SNF inhibition induced profound therapeutic response in an immunocompetent AML mouse model as well as in primary human AML samples. In vivo, SWI/SNF inhibition promoted leukemic differentiation and reduced the leukemic stem cell burden in bone marrow but also induced leukopenia. These results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. SIGNIFICANCE: Disruption of PU.1-directed enhancer programs upon SWI/SNF inhibition causes differentiation of AML cells and induces leukopenia of PU.1-dependent B cells and monocytes, revealing the on- and off-tumor effects of SWI/SNF blockade.

Therapeutic Synergy in Esophageal Cancer and Mesothelioma Is Predicted by Dynamic BH3 Profiling.

Approximately 20,000 patients per year are diagnosed with esophageal adenocarcinoma (EAC) and malignant pleural mesothelioma (MPM); fewer than 20% survive 5 years. Effective therapeutic strategies are limited although patients receive a combination of chemotherapeutics. These tumors harbor thousands of mutations that contribute to tumor development. Downstream of oncogenic driving mutations, altered tumor mitochondria promote resistance to apoptosis. Dynamic Bcl-2 homology-3 profiling (DBP) is a functional assay of live cells that identifies the mitochondrial proteins responsible for resistance to apoptosis. We hypothesized that DBP will predict which protein to target to overcome resistance thereby enhancing combinatorial therapy.DBP predicted that targeting either Mcl-1 or Bcl-xL increases the efficacy of the chemotherapeutic agent, cisplatin, whereas targeting Bcl-2 does not. We performed these assays by treating EAC and MPM cells with a combination of Bcl-2 homology-3 (BH3) mimetics and cisplatin. Following treatments, we performed efficacy assessments including apoptosis assays, IC50 calculations, and generation of a combinatorial index. DBP confirmed that targeting mitochondria with BH3 mimetics alters the threshold of apoptosis. These apoptotic effects were abolished when the mitochondrial pathway was disrupted. We validated our findings by developing knockdown models of antiapoptotic proteins Mcl-1, Bcl-xL, and the mitochondrial effector proteins Bax/Bak. Knockdown of Mcl-1 or Bcl-xL recapitulated the results of BH3 mimetics. In addition, we report an approach for BH3 profiling directly from patient tumor samples. We demonstrate that the DBP assay on living tumor cells measures the dynamic changes of resistance mechanisms, assesses response to combinatorial therapy, and provides results in a clinically feasible time frame.

A ubiquitous disordered protein interaction module orchestrates transcription elongation.

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

A cytoskeletal function for PBRM1 reading methylated microtubules.

Epigenetic effectors "read" marks "written" on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.

The surface topography of silicone breast implants mediates the foreign body response in mice, rabbits and humans.

Silicone is widely used in chronic implants and is generally perceived to be safe. However, textured breast implants have been associated with immune-related complications, including malignancies. Here, by examining for up to one year the foreign body response and capsular fibrosis triggered by miniaturized or full-scale clinically approved breast implants with different surface topography (average roughness, 0-90 µm) placed in the mammary fat pads of mice or rabbits, respectively, we show that surface topography mediates immune responses to the implants. We also show that the surface surrounding human breast implants collected during revision surgeries also differentially alters the individual's immune responses to the implant. Moreover, miniaturized implants with an average roughness of 4 µm can largely suppress the foreign body response and fibrosis (but not in T-cell-deficient mice), and that tissue surrounding these implants displayed higher levels of immunosuppressive FOXP3+ regulatory T cells. Our findings suggest that, amongst the topographies investigated, implants with an average roughness of 4 µm provoke the least amount of inflammation and foreign body response.

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma.

More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.

The Spatial and Genomic Hierarchy of Tumor Ecosystems Revealed by Single-Cell Technologies.

Many malignancies display heterogeneous features that support cancer progression. Emerging high-resolution methods provide a view of heterogeneity that recognizes the influence of diverse cell types and cell states of the tumor microenvironment. Here we outline a hierarchical organization of tumor heterogeneity from a genomic perspective, summarize the origins of spatially patterned metabolic features, and review recent developments in single-cell and spatially resolved techniques for genome-wide study of multicellular tissues. We also discuss how integrating these approaches can yield new insights into human cancer and emerging immune therapies. Applying these technologies for the analysis of primary tumors, patient-derived xenografts, and in vitro systems holds great promise for understanding the hierarchical structure and environmental influences that underlie tumor ecosystems.

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains.

Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation.

Updated Recommendations on the Diagnosis, Management, and Clinical Trial Eligibility Criteria for Patients With Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is one of the most aggressive renal cell carcinomas. It predominantly afflicts young adults and adolescents with sickle cell trait and other sickle hemoglobinopathies, and is refractory to targeted and antiangiogenic therapies used in patients with clear-cell renal cell carcinoma. Platinum-based cytotoxic chemotherapy is the mainstay for RMC treatment. On the basis of recent advances in the diagnosis, management, and clinical trial development for RMC, a panel of experts met in October 2017 and developed updated consensus recommendations to inform clinicians, researchers, and patients. Because RMC often aggressively recurs while patients are still recovering from nephrectomy, upfront chemotherapy should be considered for most patients, including those with localized disease. After safety and dosing information has been established in adults, phase II and III trials enrolling patients with RMC should allow patients aged 12 years and older to be accrued. Patients with the very rare unclassified renal cell carcinoma with medullary phenotype variant should be included in RMC trials. Medical providers should be aware that RMC can afflict subjects of all races, and not only those of African descent, and that the presence of sickle cell trait, or of other sickle hemoglobinopathies, can affect drug responses and toxicity.