Analysis of optimized DNase-seq reveals intrinsic bias in transcription factor footprint identification

DNase-seq is a powerful technique for identifying cis-regulatory elements across the genome. We studied the key experimental parameters to optimize the performance of DNase-seq. We found that sequencing short 50-100bp fragments that accumulate in long inter-nucleosome linker regions is more efficient for identifying transcription factor binding sites than using longer fragments. We also assessed the potential of DNase-seq to predict transcription factor occupancy through the generation of nucleotide-resolution transcription factor footprints. In modeling the sequence-specific DNaseI cutting bias we found a surprisingly strong effect that varied over more than two orders of magnitude. This confounds DNaseI footprint analysis to the extent that the nucleotide resolution cleavage patterns at most transcription factor binding sites are derived from intrinsic DNaseI cleavage bias rather than from specific protein-DNA interactions. In contrast, quantitative comparison of DNaseI hypersensitivity between states can predict transcription factor occupancy associated with particular biological perturbations

Digital Quantification of Gene Expression in Sequential Breast Cancer Biopsies Reveals Activation of an Immune Response

Advancements in molecular biology have unveiled multiple breast cancer promoting pathways and potential therapeutic targets. Large randomized clinical trials remain the ultimate means of validating therapeutic efficacy, but they require large cohorts of patients and are lengthy and costly. A useful approach is to conduct a window of opportunity study in which patients are exposed to a drug pre-surgically during the interval between the core needle biopsy and the definitive surgery. These are non-therapeutic studies and the end point is not clinical or pathological response but rather evaluation of molecular changes in the tumor specimens that can predict response. However, since the end points of the non-therapeutic studies are biologic, it is critical to first define the biologic changes that occur in the absence of treatment. In this study, we compared the molecular profiles of breast cancer tumors at the time of the diagnostic biopsy versus the definitive surgery in the absence of any intervention using the Nanostring nCounter platform. We found that while the majority of the transcripts did not vary between the two biopsies, there was evidence of activation of immune related genes in response to the first biopsy and further investigations of the immune changes after a biopsy in early breast cancer seem warranted

Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state–specific “regulatory transposable elements.” Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements.</p

Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state–specific “regulatory transposable elements.” Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements.</p

Nucleosome dynamics define transcriptional enhancers

Chromatin plays a central role in eukaryotic gene regulation. We have performed genome-wide mapping of epigenetically-marked nucleosomes to determine their position both near transcription start sites and at distal regulatory elements including enhancers. In prostate cancer cells where androgen receptor (AR) binds primarily to enhancers, we found that androgen treatment dismisses a central nucleosome present over AR binding sites that is flanked by a pair of marked nucleosomes. A novel quantitative model built on the behavior of such nucleosome pairs correctly identified regions bound by the regulators of the immediate androgen response including AR and FoxA1. More importantly this model also correctly predicted novel binding sites for other transcription factors present following prolonged androgen stimulation including Oct1 and NKX3.1. Thus quantitative modeling of enhancer structure provides a powerful predictive method to infer the identity of transcription factors involved in cellular responses to specific stimuli

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified HNRNPM as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and back-splicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM dependent linear splicing events using splice-switching-antisense-oligonucleotides (SSOs) was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors

Biological basis of extensive pleiotropy between blood traits and cancer risk

Background: The immune system has a central role in preventing carcinogenesis. Alteration of systemic immune cell levels may increase cancer risk. However, the extent to which common genetic variation influences blood traits and cancer risk remains largely undetermined. Here, we identify pleiotropic variants and predict their underlying molecular and cellular alterations. Methods: Multivariate Cox regression was used to evaluate associations between blood traits and cancer diagnosis in cases in the UK Biobank. Shared genetic variants were identified from the summary statistics of the genome-wide association studies of 27 blood traits and 27 cancer types and subtypes, applying the conditional/conjunctional false-discovery rate approach. Analysis of genomic positions, expression quantitative trait loci, enhancers, regulatory marks, functionally defined gene sets, and bulk- and single-cell expression profiles predicted the biological impact of pleiotropic variants. Plasma small RNAs were sequenced to assess association with cancer diagnosis. Results: The study identified 4093 common genetic variants, involving 1248 gene loci, that contributed to blood-cancer pleiotropism. Genomic hotspots of pleiotropism include chromosomal regions 5p15-TERT and 6p21-HLA. Genes whose products are involved in regulating telomere length are found to be enriched in pleiotropic variants. Pleiotropic gene candidates are frequently linked to transcriptional programs that regulate hematopoiesis and define progenitor cell states of immune system development. Perturbation of the myeloid lineage is indicated by pleiotropic associations with defined master regulators and cell alterations. Eosinophil count is inversely associated with cancer risk. A high frequency of pleiotropic associations is also centered on the regulation of small noncoding Y-RNAs. Predicted pleiotropic Y-RNAs show specific regulatory marks and are overabundant in the normal tissue and blood of cancer patients. Analysis of plasma small RNAs in women who developed breast cancer indicates there is an overabundance of Y-RNA preceding neoplasm diagnosis. Conclusions: This study reveals extensive pleiotropism between blood traits and cancer risk. Pleiotropism is linked to factors and processes involved in hematopoietic development and immune system function, including components of the major histocompatibility complexes, and regulators of telomere length and myeloid lineage. Deregulation of Y-RNAs is also associated with pleiotropism. Overexpression of these elements might indicate increased cancer risk

Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance

Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations

K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression.

peer reviewedT-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8+ T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo