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Regulation of biological processes typically incorporate mechanisms that both initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism wherein low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from both in vitro and in vivo experiments support the model that RNAs produced during early steps in transcription initiation stimulate condensate formation whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process.

Pleiotropy—when a single mutation affects multiple traits—is a controversial topic with far-reaching implications. Pleiotropy plays a central role in debates about how complex traits evolve and whether biological systems are modular or are organized such that every gene has the potential to affect many traits. Pleiotropy is also critical to initiatives in evolutionary medicine that seek to trap infectious microbes or tumors by selecting for mutations that encourage growth in some conditions at the expense of others. Research in these fields, and others, would benefit from understanding the extent to which pleiotropy reflects inherent relationships among phenotypes that correlate no matter the perturbation (vertical pleiotropy). Alternatively, pleiotropy may result from genetic changes that impose correlations between otherwise independent traits (horizontal pleiotropy). We distinguish these possibilities by using clonal populations of yeast cells to quantify the inherent relationships between single-cell morphological features. Then, we demonstrate how often these relationships underlie vertical pleiotropy and how often these relationships are modified by genetic variants (quantitative trait loci [QTL]) acting via horizontal pleiotropy. Our comprehensive screen measures thousands of pairwise trait correlations across hundreds of thousands of yeast cells and reveals ample evidence of both vertical and horizontal pleiotropy. Additionally, we observe that the correlations between traits can change with the environment, genetic background, and cell-cycle position. These changing dependencies suggest a nuanced view of pleiotropy: biological systems demonstrate limited pleiotropy in any given context, but across contexts (e.g., across diverse environments and genetic backgrounds) each genetic change has the potential to influence a larger number of traits. Our method suggests that exploiting pleiotropy for applications in evolutionary medicine would benefit from focusing on traits with correlations that are less dependent on context., Pleiotropy has been studied since the dawn of genetics, yet diametrically opposite views of it persist. This study presents the first massive-scale quantification of the inherent relationships between traits and of how genetic and nongenetic perturbations alter these relationships.

Cellular transformation is accompanied by extensive re-wiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T-cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress response machinery by inducing the expression of HSF1 and its downstream effectors. The NOTCH1 signaling status controls the levels of chaperone/co-chaperone complexes and predicts the response of T-ALL patient samples to HSP90 inhibition. Our data demonstrate an integral crosstalk between mediators of oncogene and non-oncogene addiction and reveal critical nodes of the heat shock response pathway that can be targeted therapeutically.

The metazoan genome is compartmentalized in areas of highly interacting chromatin known as topologically associating domains (TADs). TADs are demarcated by boundaries mostly conserved across cell types and even across species. However, a genome-wide characterization of TAD boundary strength in mammals is still lacking. In this study, we first use fused two-dimensional lasso as a machine learning method to improve Hi-C contact matrix reproducibility, and, subsequently, we categorize TAD boundaries based on their insulation score. We demonstrate that higher TAD boundary insulation scores are associated with elevated CTCF levels and that they may differ across cell types. Intriguingly, we observe that super-enhancers are preferentially insulated by strong boundaries. Furthermore, we demonstrate that strong TAD boundaries and super-enhancer elements are frequently co-duplicated in cancer patients. Taken together, our findings suggest that super-enhancers insulated by strong TAD boundaries may be exploited, as a functional unit, by cancer cells to promote oncogenesis., Topologically associating domains (TADs) detected by Hi-C technologies are megabase-scale areas of highly interacting chromatin. Here Gong, Lazaris et al. develop a computational approach to improve the reproducibility of Hi-C contact matrices and stratify TAD boundaries based on their insulating strength.

Maintenance of stem cell plasticity is determined by the ability to balance opposing forces that control gene expression. Regulation of transcriptional networks, signaling cues and chromatin-modifying mechanisms constitute crucial determinants of tissue equilibrium. Histone modifications can affect chromatin compaction, therefore co-transcriptional events that influence their deposition determine the propensities toward quiescence, self-renewal, or cell specification. The Paf1 complex (Paf1C) is a critical regulator of RNA PolII elongation that controls gene expression and deposition of histone modifications, however few studies have focused on its role affecting stem cell fate decisions. Here we delineate the functions of Paf1C in pluripotency and characterize its impact in deposition of H2B ubiquitylation (H2BK120-ub) and H3K79 methylation (H3K79me), 2 fundamental histone marks that shape transcriptional regulation. We identify that H2BK120-ub is increased in the absence of Paf1C on its embryonic stem cell targets, in sharp contrast to H3K79me, suggesting opposite functions in the maintenance of self-renewal. Furthermore, we found that core pluripotency genes are characterized by a dual gain of H2BK120-ub and loss of H3K79me on their gene bodies. Our findings elucidate molecular mechanisms of cellular adaptation and reveal novel functions of Paf1C in the regulation of the self-renewal network.

Summary: Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis. : In a human neural stem cell model of low-grade astrocytoma, Modrek et al. show that mutant IDH1 and loss of P53 and ATRX together block differentiation via disassociation of SOX2 from putative enhancers. This occurs because of disruption of chromatin looping secondary to hypermethylation at CTCF motifs. Keywords: low-grade glioma, astrocytoma, IDH, P53, ATRX, neural stem cells, SOX2, chromatin looping, CTCF, DNA methylation

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming, and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by the Phf5a in stem cells, which directs their transcriptional program ultimately regulating maintenance of pluripotency and cellular reprogramming.

e14006 Background: Next generation sequencing (NGS) allows for reliable, comprehensive and cost-effective identification of clinically actionable genetic and genomic alterations. The increasing adoption of NGS in clinical oncology has increased our ability to identify germline alterations predisposing to cancer development as well as somatic changes enabling prescription of individualized cancer treatment and enhanced clinical trial participation. Here we summarize implementation of an NGS-based precision medicine initiative involving oncology patients from a single institution cancer center. Methods: IRB-approved NGS matched whole exome (WES) germline and solid tumor somatic tumor sequencing together with somatic tumor RNA sequencing (RNA-seq) were performed using germline DNA extracted from peripheral blood lymphocytes and nucleic acids for tumor DNA and RNA sequencing obtained from formalin-fixed, paraffin-embedded tumor specimens. Results of sequencing and analyses were presented to a multi-disciplinary tumor board to establish recommendations for management of germline pathogenic variation, therapeutic drug matching, clinical trials eligibility and molecularly informed patient prognosis. Results: A total of 1,005 patients completed sequencing. Germline and somatic WES exceeded 100X and 250X mean target coverage, respectively; somatic RNA-seq exceeded 200 million mean reads. Patients ranged in age from 17 to 90 years. The study cohort comprised comparable numbers of female (51%) and male (49%) patients. Ethnicities and races were broadly represented with 22% of participants identifying as Hispanic, 14% as Asian, 4% as Black, 55% as Non-Hispanic White and 5% as other. The most common solid tumor histological classification was colorectal (18%), followed by breast (16%), prostate (7%), head and neck (7%), sarcoma (7%), ovarian (5%), melanoma (4%) and lung (3%). Bioinformatic analyses and precision medicine tumor board review established that 12% of patients harbored a germline pathogenic variant and 43% carried clinically actionable genetic/genomic alterations; a majority of patients met molecular requirements for participation in a clinical trial. Conclusions: This study confirms the feasibility and utility of clinical NGS and precision medicine tumor board review in clinical oncology to identify germline genetic pathology, deliver personalized cancer therapeutics, increase clinical trial enrollment and clarify diagnosis and prognosis.

Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer–promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP–seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD ‘fusion’ event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia. Analysis of 3D chromatin architecture in T-ALL identifies differences in intra-TAD interactions and TAD boundary insulation. Inhibition of oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions.

Cutaneous T-cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic, and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome showed a highly heterogeneous landscape of genetic perturbations, and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway previously implicated in cutaneous T-cell lymphoma pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of cutaneous T-cell lymphoma. Using this mouse model, we show that T-cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

The metazoan genome is compartmentalized in megabase-scale areas of highly interacting chromatin known as topologically associating domains (TADs), typically identified by computational analyses of Hi-C sequencing data. TADs are demarcated by boundaries that are largely conserved across cell types and even across species, although, increasing evidence suggests that the seemingly invariant TAD boundaries may exhibit plasticity and their insulating strength can vary. However, a genome-wide characterization of TAD boundary strength in mammals is still lacking. A systematic classification and characterization of TAD boundaries may generate new insights into their function. In this study, we first use fused two-dimensional lasso as a machine learning method to improve Hi-C contact matrix reproducibility, and, subsequently, we categorize TAD boundaries based on their insulation score. We demonstrate that higher TAD boundary insulation scores are associated with elevated CTCF levels and that they may differ across cell types. Intriguingly, we observe that super-enhancer elements are preferentially insulated by strong boundaries, i.e. boundaries of higher insulation score. Furthermore, we perform a pan-cancer analysis to demonstrate that strong TAD boundaries and super-enhancer elements are frequently co-duplicated in cancer patients. Taken together, our findings suggest that super-enhancers insulated by strong TAD boundaries may be exploited, as a functional unit, by cancer cells to promote oncogenesis.

BackgroundChromatin conformation capture techniques have evolved rapidly over the last few years and have provided new insights into genome organization at an unprecedented resolution. Analysis of Hi-C data is complex and computationally intensive involving multiple tasks and requiring robust quality assessment. This has led to the development of several tools and methods for processing Hi-C data. However, most of the existing tools do not cover all aspects of the analysis and only offer few quality assessment options. Additionally, availability of a multitude of tools makes scientists wonder how these tools and associated parameters can be optimally used, and how potential discrepancies can be interpreted and resolved. Most importantly, investigators need to be ensured that slight changes in parameters and/or methods do not affect the conclusions of their studies.ResultsTo address these issues (compare, explore and reproduce), we introduce HiC-bench, a configurable computational platform for comprehensive and reproducible analysis of Hi-C sequencing data. HiC-bench performs all common Hi-C analysis tasks, such as alignment, filtering, contact matrix generation and normalization, identification of topological domains, scoring and annotation of specific interactions using both published tools and our own. We have also embedded various tasks that perform quality assessment and visualization. HiC-bench is implemented as a data flow platform with an emphasis on analysis reproducibility. Additionally, the user can readily perform parameter exploration and comparison of different tools in a combinatorial manner that takes into account all desired parameter settings in each pipeline task. This unique feature facilitates the design and execution of complex benchmark studies that may involve combinations of multiple tool/parameter choices in each step of the analysis. To demonstrate the usefulness of our platform, we performed a comprehensive benchmark of existing and new TAD callers exploring different matrix correction methods, parameter settings and sequencing depths. Users can extend our pipeline by adding more tools as they become available.ConclusionsHiC-bench consists an easy-to-use and extensible platform for comprehensive analysis of Hi-C datasets. We expect that it will facilitate current analyses and help scientists formulate and test new hypotheses in the field of three-dimensional genome organization.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of pediatric and 25% of adult ALL cases. Despite improved prognosis driven by the significant advances in the molecular understanding of T-ALL, the outcome of T-ALL patients with primary resistant and relapsed leukemia remains poor. Recent evidences have highlighted a role for changes in 3D chromatin architecture in cancer progression. Herein, for the first time we explored genomewide changes in chromatin organization in primary T-ALL samples relative to T cells from healthy donors. We show how the organization of topologically associated domains (TADs) influences key oncogenic and tumor-suppressive loci by modifying the promoter-enhancer landscape, deregulating the transcriptional program and ultimately causing disease progression. To comprehensively characterize the chromatin landscape of clinically relevant loci in T-ALL, we sought to identify changes in intra-TAD activity and TAD disruptions on a genome-wide scale. We developed and implemented new computational approaches for Hi-C data analysis with a focus on Hi-C data integration with ChIP-Seq and RNA-Seq. We found significant correlations of TAD activity with CTCF occupancy, super-enhancer activity and gene expression of leukemia relevant loci. Furthermore, among the dozens of TAD disruptions we identified, we found a novel TAD “fusion” event around the MYC locus, which allows for chromatin interactions between a recently reported Notch-dependent MYC super-enhancer and promoter to drive overexpression of MYC. This particular TAD fusion is accompanied by a complete CTCF loss in the TAD boundary and increased MYC expression in T-ALL samples. Overall, this study sheds new light into how changes in chromatin architecture influence disease progression by restructuring oncogene and tumor suppressor landscapes. Citation Format: Palaniraja Thandapani, Andreas Kloetgen, Charalampos Lazaris, Xufeng Chen, Panagiotis Ntziachristos, Aristotelis Tsirigos, Iannis Aifantis. Dynamic 3d chromosomal landscapes in acute leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2998.

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

Palindromic AT-Rich Repeat (PATRR) - mediated translocations entail exchange across chromosomes at sites enriched in palindromic repeats of the nucleotides adenine (A) and thymine (T). Their precise embryologic origin and associated pathobiology with clear cell renal cell carcinoma (ccRCC) remain incompletely described. In the present study we document an individual with familial non-VHL ccRCC (7 primary renal tumors) who harbors a germline de novo PATRR mediated balanced translocation involving chromosomes 3 and 8 [t(3;8)] validated by spectral karyotyping (SKY). Using translocation specific PCR and DNA sequencing we determined the chromosome 3 breakpoint to be located in an AT-rich palindromic sequence in the third intron of the FHIT gene (chr3p14.2) and the chromosome 8 breakpoint in the first intron of the TRC8 gene (chr8q24.1). Genotyping analysis, using a high density custom exomechip array by Illumina, revealed a loss of the entire aberrant chromosome 8 carrying the 3p segment [der(8)] in all renal tumors tested. We also determined that the [46, XY, t(3;8), (p14.2, q24.1)] translocation was paternally derived by performing a genotypic assessment of the regions that differ between the parental alleles and then establishing which haplotypes are associated with the translocation. The somatic mutational landscape was assessed by Whole Exome sequencing of the renal tumors and the proband’s germline DNA. No germline or somatic deleterious mutations were detected in VHL gene suggesting that the ccRCC phenotype is not associated with Von Hippel-Lindau disease. Furthermore, we measured the transcriptomic profiles of the renal tumors and matched normal tissues employing barcoded probe hybridization technology (NanoString) and RNASeq to fully characterize differential gene expression and define gene ontology networks that are dysregulated. Most significantly, no difference in expression of the VHL gene was detected between tumors and normal counterparts. Aberrant regulation was detected in members of WNT signaling pathway, TGF-beta and MAPK pathways, in addition to NOTCH signaling pathway genes and members of the VEGF family. In summary, in this study we employed advanced genomic and transcriptomic techniques, to delineate the embryologic origin and deleterious biological consequences of the PATRR-mediated t(3;8) balanced translocation associated with clear cell renal cell carcinoma. Citation Format: Marilena Melas, Kevin J. McDonnell, Christopher K. Edlund, Sarah J. Tash, Duveen Y. Sturgeon, Chenxu Qu, Charalampos Lazaris, Peter J. Gruber, Thomas W. Glover, Beverly S. Emanuel, Stephen B. Gruber. Elucidating de novo PATRR-mediated t(3;8) balanced translocation and clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2017-491

​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate ​HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the ​HSF1 transcriptional program both in the presence of exogenous stress and in cancer.