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High expression ofMYCand its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between theMYClocus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding genePVT1are enriched inMYC-intact cases. To identify genomic drivers ofMYCactivation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in theMYClocus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements betweenMYCand immunoglobulin (Ig) loci. Rearrangements betweenMYCand non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within theBCL6super-enhancer (BCL6-SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrentMYC::BCL6-SE rearrangement. In contrast, GCB-DLBCL cell lines withoutMYCrearrangement were highly dependent on a previously uncharacterized 3’ enhancer within theMYClocus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that thePVT1promoter limitsMYCactivation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3’ rearrangements that removePVT1from its position inciswith the rearrangedMYCgene.Key pointsCRISPR-interference screens identify a conserved germinal center B cellMYCenhancer that is essential for GCB-DLBCL lackingMYCrearrangements.Functional profiling ofMYCpartner loci reveals principles ofMYCenhancer-hijacking activation by non-immunoglobulin rearrangements.

Background: A large proportion of genetic risk remains unexplained for structural heart disease involving the interventricular septum (IVS) including hypertrophic cardiomyopathy and ventricular septal defects. This study sought to develop a reproducible proxy of IVS structure from standard medical imaging, discover novel genetic determinants of IVS structure, and relate these loci to diseases of the IVS, hypertrophic cardiomyopathy, and ventricular septal defect. Methods: We estimated the cross-sectional area of the IVS from the 4-chamber view of cardiac magnetic resonance imaging in 32 219 individuals from the UK Biobank which was used as the basis of genome wide association studies and Mendelian randomization. Results: Measures of IVS cross-sectional area at diastole were a strong proxy for the 3-dimensional volume of the IVS (Pearson r =0.814, P =0.004), and correlated with anthropometric measures, blood pressure, and diagnostic codes related to cardiovascular physiology. Seven loci with clear genomic consequence and relevance to cardiovascular biology were uncovered by genome wide association studies, most notably a single nucleotide polymorphism in an intron of CDKN1A (rs2376620; β, 7.7 mm 2 [95% CI, 5.8–11.0]; P =6.0×10 − 10 ), and a common inversion incorporating KANSL1 predicted to disrupt local chromatin structure (β, 8.4 mm 2 [95% CI, 6.3–10.9]; P =4.2×10 − 14 ). Mendelian randomization suggested that inheritance of larger IVS cross-sectional area at diastole was strongly associated with hypertrophic cardiomyopathy risk (p IVW =4.6×10 − 10 ) while inheritance of smaller IVS cross-sectional area at diastole was associated with risk for ventricular septal defect (p IVW =0.007). Conclusions: Automated estimates of cross-sectional area of the IVS supports discovery of novel loci related to cardiac development and Mendelian disease. Inheritance of genetic liability for either small or large IVS, appears to confer risk for ventricular septal defect or hypertrophic cardiomyopathy, respectively. These data suggest that a proportion of risk for structural and congenital heart disease can be localized to the common genetic determinants of size and shape of cardiovascular anatomy.

The ENCODE Consortium’s efforts to annotate non-coding,cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes which play a major role in health and disease. Pooled, non-coding CRISPR screens are a promising approach for systematically investigating gene regulatory mechanisms. Here, the ENCODE Functional Characterization Centers report 109 screens comprising 346,970 individual perturbations across 13.3Mb of the genome, using a variety of methods, readouts, and statistical analyses. Across 332 functionally confirmed CRE-gene links, we identify principles for screening endogenous, non-coding elements for causal regulatory mechanisms. Nearly all CREs show strong evidence of open chromatin, and targeting accessibility peak summits is a critical component of our proposed sgRNA design rules. We provide experimental guidelines to accurately detect CREs with variable, often low, transcriptional effects. We discover a previously undescribed DNA strand-bias for CRISPRi in transcribed regions with implications for screen design and analysis. Benchmarking five screen analysis tools, we find CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity sgRNAs. Together, we provide an accessible data resource, predesigned sgRNAs targeting 3,275,697 ENCODE SCREEN candidate CREs, and screening guidelines to accelerate functional characterization of the non-coding genome.

SummaryCongenital heart defects, the most common birth disorders, are the clinical manifestation of anomalies in fetal heart development - a complex process involving dynamic spatiotemporal coordination among various precursor cell lineages. This complexity underlies the incomplete understanding of the genetic architecture of congenital heart diseases (CHDs). To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We identified similarities and differences of regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts. We interpreted deep learning models that predict cell-type resolved, base-resolution chromatin accessibility profiles from DNA sequence to decipher underlying TF motif lexicons and infer the regulatory impact of non-coding variants. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in CHD cases versus controls. We used CRISPR-based perturbations to validate an enhancer harboring a nominated regulatory CHD mutation, linking it to effects on the expression of a known CHD gene JARID2. Together, this work defines the cell-type resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements as a component of the genetic etiology of CHD.

Background: A large proportion of genetic risk remains unexplained for structural heart disease involving the interventricular septum (IVS) including hypertrophic cardiomyopathy (HCM) and ventricular septal defects (VSD). Methods: We performed machine learning to estimate the cross-sectional area of the interventricular septum (IVS.csad) from the 4-chamber view of cardiac MRI in 32,219 individuals from the UK Biobank. We related IVS.csad to relevant clinical phenotypes, and performed genome-wide association studies and Mendelian Randomization. Results: Automated measures of IVS.csad were highly accurate, and strongly correlated with anthropometric measures, blood pressure, and diagnostic codes related to cardiovascular physiology. A single nucleotide polymorphism in an intron of CDKN1A was associated with IVS.csad (rs2376620, Beta 8.4 mm2, 95% confidence intervals (CI) 5.8 to 11.0, p=2.0e-10), and a common inversion incorporating KANSL1 predicted to disrupt local chromatin structure was associated with an increase in IVS.csad (Beta 8.6 mm2, 95% CI 6.3-10.9, p=1.3e-13). Mendelian Randomization suggested that inheritance of larger IVS.csad was causal for HCM (Beta 2.45 log odds ratio (OR) HCM per increase in SD of IVS.csad, standard error (SE) 0.48, pIVW = 2.8e-7) while inheritance of smaller IVS.csad was causal for VSD (Beta -2.06 log odds ratio (OR) VSD per decrease in SD of IVS.csad, SE 0.75, pIVW = 0.006) Conclusion: Automated derivation of the cross sectional area of the IVS allowed discovery of loci related to cardiac development and Mendelian disease. Inheritance of genetic liability for either large or small interventricular septum, appears to confer risk for HCM or VSD respectively, suggesting that a proportion of risk for structural and congenital heart disease may localize to the common genetic determinants of cardiovascular anatomy.

RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous RNA complexes and purifies these complexes through hybrid capture with biotinylated antisense oligos. DNA loci that interact with the target RNA are identified using high-throughput DNA sequencing.