Altered Chromatin Occupancy of Master Regulators Underlies Evolutionary Divergence in the Transcriptional Landscape of Erythroid Differentiation

Erythropoiesis is one of the best understood examples of cellular differentiation. Morphologically, erythroid differentiation proceeds in a nearly identical fashion between humans and mice, but recent evidence has shown that networks of gene expression governing this process are divergent between species. We undertook a systematic comparative analysis of six histone modifications and four transcriptional master regulators in primary proerythroblasts and erythroid cell lines to better understand the underlying basis of these transcriptional differences. Our analyses suggest that while chromatin structure across orthologous promoters is strongly conserved, subtle differences are associated with transcriptional divergence between species. Many transcription factor (TF) occupancy sites were poorly conserved across species (∼25% for GATA1, TAL1, and NFE2) but were more conserved between proerythroblasts and cell lines derived from the same species. We found that certain cis-regulatory modules co-occupied by GATA1, TAL1, and KLF1 are under strict evolutionary constraint and localize to genes necessary for erythroid cell identity. More generally, we show that conserved TF occupancy sites are indicative of active regulatory regions and strong gene expression that is sustained during maturation. Our results suggest that evolutionary turnover of TF binding sites associates with changes in the underlying chromatin structure, driving transcriptional divergence. We provide examples of how this framework can be applied to understand epigenomic variation in specific regulatory regions, such as the β-globin gene locus. Our findings have important implications for understanding epigenomic changes that mediate variation in cellular differentiation across species, while also providing a valuable resource for studies of hematopoiesis

Functional characterization of T2D-associated SNP effects on baseline and ER stress-responsive β cell transcriptional activation.

Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) at \u3e250 loci in the human genome to type 2 diabetes (T2D) risk. For each locus, identifying the functional variant(s) among multiple SNPs in high linkage disequilibrium is critical to understand molecular mechanisms underlying T2D genetic risk. Using massively parallel reporter assays (MPRA), we test the cis-regulatory effects of SNPs associated with T2D and altered in vivo islet chromatin accessibility in MIN6 β cells under steady state and pathophysiologic endoplasmic reticulum (ER) stress conditions. We identify 1,982/6,621 (29.9%) SNP-containing elements that activate transcription in MIN6 and 879 SNP alleles that modulate MPRA activity. Multiple T2D-associated SNPs alter the activity of short interspersed nuclear element (SINE)-containing elements that are strongly induced by ER stress. We identify 220 functional variants at 104 T2D association signals, narrowing 54 signals to a single candidate SNP. Together, this study identifies elements driving β cell steady state and ER stress-responsive transcriptional activation, nominates causal T2D SNPs, and uncovers potential roles for repetitive elements in β cell transcriptional stress response and T2D genetics

Vimentin DNA methylation predicts survival in breast cancer

The Vimentin gene plays a pivotal role in epithelial-to-mesenchymal transition and is known to be overexpressed in the prognostically poor basal-like breast cancer subtype. Recent studies have reported Vimentin DNA methylation in association with poor clinical outcomes in other solid tumors, but not in breast cancer. We therefore quantified Vimentin DNA methylation using MALDI-TOF mass spectrometry in breast tumors and matched normal pairs in association with gene expression and survival in a hospital-based study of breast cancer patients. Gene expression data via qRT-PCR in cell lines and oligomicroarray data from breast tissues were correlated with percent methylation in the Vimentin promoter. A threshold of 20 percent average methylation compared with matched normal pairs was set for bivariate and multivariate tests of association between methylation and tumor subtype, tumor histopathology, and survival. Vimentin was differentially methylated in luminal breast cancer cell lines, and in luminal A, luminal B, and HER2-enriched breast tumor subtypes, but was rare in basal-like cell lines and tumors. Increased methylation was strongly correlated with decreased mRNA expression in cell lines, and had a moderate inverse correlation in breast tumors. Vimentin methylation predicted poor overall survival independent of race, subtype, stage, nodal status, or metastatic disease and holds promise as a new prognostic biomarker for breast cancer patients

Functionally informed fine-mapping and polygenic localization of complex trait heritability

Fine-mapping aims to identify causal variants impacting complex traits. We propose PolyFun, a computationally scalable framework to improve fine-mapping accuracy by leveraging functional annotations across the entire genome-not just genome-wide-significant loci-to specify prior probabilities for fine-mapping methods such as SuSiE or FINEMAP. In simulations, PolyFun + SuSiE and PolyFun + FINEMAP were well calibrated and identified >20% more variants with a posterior causal probability >0.95 than identified in their nonfunctionally informed counterparts. In analyses of 49 UK Biobank traits (average n = 318,000), PolyFun + SuSiE identified 3,025 fine-mapped variant-trait pairs with posterior causal probability >0.95, a >32% improvement versus SuSiE. We used posterior mean per-SNP heritabilities from PolyFun + SuSiE to perform polygenic localization, constructing minimal sets of common SNPs causally explaining 50% of common SNP heritability; these sets ranged in size from 28 (hair color) to 3,400 (height) to 2 million (number of children). In conclusion, PolyFun prioritizes variants for functional follow-up and provides insights into complex trait architectures. PolyFun is a computationally scalable framework for functionally informed fine-mapping that makes full use of genome-wide data. It prioritizes more variants than previous methods when applied to 49 complex traits from UK Biobank.Peer reviewe

BCL11A deletions result in fetal hemoglobin persistence and neurodevelopmental alterations

A transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA) normally occurs within a few months after birth. Increased production of HbF after this period of infancy ameliorates clinical symptoms of the major disorders of adult ß-hemoglobin: ß-thalassemia and sickle cell disease. The transcription factor BCL11A silences HbF and has been an attractive therapeutic target for increasing HbF levels; however, it is not clear to what extent BCL11A inhibits HbF production or mediates other developmental functions in humans. Here, we identified and characterized 3 patients with rare microdeletions of 2p15-p16.1 who presented with an autism spectrum disorder and developmental delay. Moreover, these patients all exhibited substantial persistence of HbF but otherwise retained apparently normal hematologic and immunologic function. Of the genes within 2p15-p16.1, only BCL11A was commonly deleted in all of the patients. Evaluation of gene expression data sets from developing and adult human brains revealed that BCL11A expression patterns are similar to other genes associated with neurodevelopmental disorders. Additionally, common SNPs within the second intron of BCL11A are strongly associated with schizophrenia. Together, the study of these rare patients and orthogonal genetic data demonstrates that BCL11A plays a central role in silencing HbF in humans and implicates BCL11A as an important factor for neurodevelopment

Whole-exome sequencing identifies an α-globin cluster triplication resulting in increased clinical severity of β-thalassemia

Whole-exome sequencing (WES) has been increasingly useful for the diagnosis of patients with rare causes of anemia, particularly when there is an atypical clinical presentation or targeted genotyping approaches are inconclusive. Here, we describe a 20-yr-old man with a lifelong moderate-to-severe anemia with accompanying splenomegaly who lacked a definitive diagnosis. After a thorough clinical workup and targeted genetic sequencing, we identified a paternally inherited β-globin mutation (HBB:c.93-21G>A, IVS-I-110:G>A), a known cause of β-thalassemia minor. As this mutation alone was inconsistent with the severity of the anemia, we performed WES. Although we could not identify any relevant pathogenic single-nucleotide variants (SNVs) or small indels, copy-number variant (CNV) analyses revealed a likely triplication of the entire α-globin cluster, which was subsequently confirmed by multiplex ligation-dependent probe amplification. Treatment and follow-up was redefined according to the diagnosis of β-thalassemia intermedia resulting from a single β-thalassemia mutation in combination with an α-globin cluster triplication. Thus, we describe a case where the typical WES-based analysis of SNVs and small indels was unrevealing, but WES-based CNV analysis resulted in a definitive diagnosis that informed clinical decision-making. More generally, this case illustrates the value of performing CNV analysis when WES is otherwise unable to elucidate a clear genetic diagnosis

eQTL Catalogue 2023: New datasets, X chromosome QTLs, and improved detection and visualisation of transcript-level QTLs

The eQTL Catalogue is an open database of uniformly processed human molecular quantitative trait loci (QTLs). We are continuously updating the resource to further increase its utility for interpreting genetic associations with complex traits. Over the past two years, we have increased the number of uniformly processed studies from 21 to 31 and added X chromosome QTLs for 19 compatible studies. We have also implemented Leafcutter to directly identify splice-junction usage QTLs in all RNA sequencing datasets. Finally, to improve the interpretability of transcript-level QTLs, we have developed static QTL coverage plots that visualise the association between the genotype and average RNA sequencing read coverage in the region for all 1.7 million fine mapped associations. To illustrate the utility of these updates to the eQTL Catalogue, we performed colocalisation analysis between vitamin D levels in the UK Biobank and all molecular QTLs in the eQTL Catalogue. Although most GWAS loci colocalised both with eQTLs and transcript-level QTLs, we found that visual inspection could sometimes be used to distinguish primary splicing QTLs from those that appear to be secondary consequences of large-effect gene expression QTLs. While these visually confirmed primary splicing QTLs explain just 6/53 of the colocalising signals, they are significantly less pleiotropic than eQTLs and identify a prioritised causal gene in 4/6 cases

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.Peer reviewe

Association of Epidemiologic Factors and Genetic Variants Influencing Hypothalamic-Pituitary-Adrenocortical Axis Function With Postconcussive Symptoms After Minor Motor Vehicle Collision

To determine the influence of epidemiologic factors and the influence of genetic variants affecting FKBP5, a protein known to modulate hypothalamic-pituitary-adrenocortical (HPA) axis function, on the severity of somatic symptoms commonly termed “post-concussive” six and twelve months after motor-vehicle collision (MVC)

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex