Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains.

Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.

Enhancer Features that Drive Formation of Transcriptional Condensates.

Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-separated condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci determined by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process.

Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes.

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-ß, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator ß-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.

CD-CODE: crowdsourcing condensate database and encyclopedia.

The discovery of biomolecular condensates transformed our understanding of intracellular compartmentalization of molecules. To integrate interdisciplinary scientific knowledge about the function and composition of biomolecular condensates, we developed the crowdsourcing condensate database and encyclopedia ( cd-code.org ). CD-CODE is a community-editable platform, which includes a database of biomolecular condensates based on the literature, an encyclopedia of relevant scientific terms and a crowdsourcing web application. Our platform will accelerate the discovery and validation of biomolecular condensates, and facilitate efforts to understand their role in disease and as therapeutic targets.

Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes.

Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma.

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.

Engineering Bioactive Dimeric Transcription Factor Analogs via Palladium Rebound Reagents.

Transcription factors (TF), such as Myc, are proteins implicated in disease pathogenesis, with dysregulation of Myc expression in 50% of all human cancers. Still, targeting Myc remains a challenge due to the lack of small molecule binding pockets in the tertiary structure. Here, we report synthetic covalently linked TF mimetics that inhibit oncogenic Myc-driven transcription by antagonistic binding of the target DNA-binding site. We combined automated flow peptide chemistry with palladium(II) oxidative addition complexes (OACs) to engineer covalent protein dimers derived from the DNA-binding domains of Myc, Max, and Omomyc TF analogs. Palladium-mediated cross-coupling of synthesized protein monomers resulted in milligram quantities of seven different covalent homo- and heterodimers. The covalent helical dimers were found to bind DNA and exhibited improved thermal stability. Cell-based studies revealed the Max-Max covalent dimer is cell-penetrating and interfered with Myc-dependent gene transcription resulting in reduced cancer cell proliferation (EC50 of 6 µM in HeLa). RNA sequencing and gene analysis of extracted RNA from treated cancer cells confirmed that the covalent Max-Max homodimer interferes with Myc-dependent transcription. Flow chemistry, combined with palladium(II) OACs, has enabled a practical strategy to generate new bioactive compounds to inhibit tumor cell proliferation.

Impact of Cancer History on Outcomes Among Hospitalized Patients with COVID-19.

BACKGROUND: Early reports suggested increased mortality from COVID-19 in patients with cancer but lacked rigorous comparisons to patients without cancer. We investigated whether a current cancer diagnosis or cancer history is an independent risk factor for death in hospitalized patients with COVID-19. PATIENTS AND METHODS: We identified patients with a history of cancer admitted to two large hospitals between March 13, 2020, and May 10, 2020, with laboratory-confirmed COVID-19 and matched them 1:2 to patients without a history of cancer. RESULTS: Men made up 56.2% of the population, with a median age of 69 years (range, 30-96). The median time since cancer diagnosis was 35.6 months (range, 0.39-435); 80% had a solid tumor, and 20% had a hematologic malignancy. Among patients with cancer, 27.8% died or entered hospice versus 25.6% among patients without cancer. In multivariable analyses, the odds of death/hospice were similar (odds ratio [OR], 1.09; 95% confidence interval [CI], 0.65-1.82). The odds of intubation (OR, 0.46; 95% CI, 0.28-0.78), shock (OR, 0.54; 95% CI, 0.32-0.91), and intensive care unit admission (OR, 0.51; 95% CI, 0.32-0.81) were lower for patients with a history of cancer versus controls. Patients with active cancer or who had received cancer-directed therapy in the past 6 months had similar odds of death/hospice compared with cancer survivors (univariable OR, 1.31; 95% CI, 0.66-2.60; multivariable OR, 1.47; 95% CI, 0.69-3.16). CONCLUSION: Patients with a history of cancer hospitalized for COVID-19 had similar mortality to matched hospitalized patients with COVID-19 without cancer, and a lower risk of complications. In this population, patients with active cancer or recent cancer treatment had a similar risk for adverse outcomes compared with survivors of cancer. IMPLICATIONS FOR PRACTICE: This study investigated whether a current cancer diagnosis or cancer history is an independent risk factor for death or hospice admission in hospitalized patients with COVID-19. Active cancer, systemic cancer therapy, and a cancer history are not independent risk factors for death from COVID-19 among hospitalized patients, and hospitalized patients without cancer are more likely to have severe COVID-19. These findings provide reassurance to survivors of cancer and patients with cancer as to their relative risk of severe COVID-19, may encourage oncologists to provide standard anticancer therapy in patients at risk of COVID-19, and guide triage in future waves of infection.

53BP1 alters the landscape of DNA rearrangements and suppresses AID-induced B cell lymphoma.

Deficiencies in factors that regulate the DNA damage response enhance the incidence of malignancy by destabilizing the genome. However, the precise influence of the DNA damage response on regulation of cancer-associated rearrangements is not well defined. Here we examine the genome-wide impact of tumor protein P53-binding protein 1 (53BP1) deficiency in lymphoma and translocation. While both activation-induced cytidine deaminase (AID) and 53BP1 have been associated with cancer in humans, neither AID overexpression nor loss of 53BP1 is sufficient to produce malignancy. However, the combination of 53BP1 deficiency and AID deregulation results in B cell lymphoma. Deep sequencing of the genome of 53BP1(-/-) cancer cells and translocation capture sequencing (TC-Seq) of primary 53BP1(-/-) B cells revealed that their chromosomal rearrangements differ from those found in wild-type cells in that they show increased DNA end resection. Moreover, loss of 53BP1 alters the translocatome by increasing rearrangements to intergenic regions.

Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1.

53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.

DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes.

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

AID produces DNA double-strand breaks in non-Ig genes and mature B cell lymphomas with reciprocal chromosome translocations.

Cancer-initiating translocations such as those associated with lymphomas require the formation of paired DNA double-strand breaks (DSBs). Activation-induced cytidine deaminase (AID) produces widespread somatic mutation in mature B cells; however, the extent of "off-target" DSB formation and its role in translocation-associated malignancy is unknown. Here, we show that deregulated expression of AID causes widespread genome instability, which alone is insufficient to induce B cell lymphoma; transformation requires concomitant loss of the tumor suppressor p53. Mature B cell lymphomas arising as a result of deregulated AID expression are phenotypically diverse and harbor clonal reciprocal translocations involving a group of Immunoglobulin (Ig) and non-Ig genes that are direct targets of AID. This group includes miR-142, a previously unknown micro-RNA target that is translocated in human B cell malignancy. We conclude that AID produces DSBs throughout the genome, which can lead to lymphoma-associated chromosome translocations in mature B cells.

Mouse model of endemic Burkitt translocations reveals the long-range boundaries of Ig-mediated oncogene deregulation.

Human Burkitt lymphomas are divided into two main clinical variants: the endemic form, affecting African children infected with malaria and the Epstein-Barr virus, and the sporadic form, distributed across the rest of the world. However, whereas sporadic translocations decapitate Myc from 5' proximal regulatory elements, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances remain unclear. We here recapitulate endemic Burkitt lymphoma-like translocations in plasmacytomas from uracil N-glycosylase and activation-induced cytidine deaminase-deficient mice. Mapping of translocation breakpoints using an acetylated histone H3 lysine 9 chromatin immunoprecipitation sequencing approach reveals Igh fusions up to ∼350 kb upstream of Myc or the related oncogene Mycn. A comprehensive analysis of epigenetic marks, PolII recruitment, and transcription in tumor cells demonstrates that the 3' Igh enhancer (Eα) vastly remodels ∼450 kb of chromatin into translocated sequences, leading to significant polymerase occupancy and constitutive oncogene expression. We show that this long-range epigenetic reprogramming is directly proportional to the physical interaction of Eα with translocated sites. Our studies thus uncover the extent of epigenetic remodeling by Ig 3' enhancers and provide a rationale for the long-range deregulation of translocated oncogenes in endemic Burkitt lymphomas. The data also shed light on the origin of endemic-like chromosomal rearrangements.

High-throughput sequencing reveals principles of adeno-associated virus serotype 2 integration.

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics.

Biomolecular Condensates as Novel Antiviral Targets.

Viral infections pose a significant health risk worldwide. There is a pressing need for more effective antiviral drugs to combat emerging novel viruses and the reemergence of previously controlled viruses. Biomolecular condensates are crucial for viral replication and are promising targets for novel antiviral therapies. Herein, we review the role of biomolecular condensates in the viral replication cycle and discuss novel strategies to leverage condensate biology for antiviral drug discovery. Biomolecular condensates may also provide an opportunity to develop antivirals that are broad-spectrum or less prone to acquired drug resistance.

Rag mutations reveal robust alternative end joining.

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.

Principles and functions of condensate modifying drugs.

Biomolecular condensates are compartmentalized communities of biomolecules, which unlike traditional organelles, are not enclosed by membranes. Condensates play roles in diverse cellular processes, are dysfunctional in many disease states, and are often enriched in classically "undruggable" targets. In this review, we provide an overview for how drugs can modulate condensate structure and function by phenotypically classifying them as dissolvers (dissolve condensates), inducers (induce condensates), localizers (alter localization of the specific condensate community members) or morphers (alter the physiochemical properties). We discuss the growing list of bioactive molecules that function as condensate modifiers (c-mods), including small molecules, oligonucleotides, and peptides. We propose that understanding mechanisms of condensate perturbation of known c-mods will accelerate the discovery of a new class of therapies for difficult-to-treat diseases.

Modulating biomolecular condensates: a novel approach to drug discovery.

In the past decade, membraneless assemblies known as biomolecular condensates have been reported to play key roles in many cellular functions by compartmentalizing specific proteins and nucleic acids in subcellular environments with distinct properties. Furthermore, growing evidence supports the view that biomolecular condensates often form by phase separation, in which a single-phase system demixes into a two-phase system consisting of a condensed phase and a dilute phase of particular biomolecules. Emerging understanding of condensate function in normal and aberrant cellular states, and of the mechanisms of condensate formation, is providing new insights into human disease and revealing novel therapeutic opportunities. In this Perspective, we propose that such insights could enable a previously unexplored drug discovery approach based on identifying condensate-modifying therapeutics (c-mods), and we discuss the strategies, techniques and challenges involved.

The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17.

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.