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Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

The regulation of the beta-type globin genes has served as a powerful model for developmental transcriptional control, with humans expressing embryonic (HBE), two fetal (HBG1/2) and two adult (HBB and HBD) versions at the corresponding developmental stages. Reactivation of HBG in adult erythroid cells is of benefit to patients with sickle cell disease and β-thalassemia. While past efforts focused on the identification of repressors of the HBG genes, including BCL11A, ZBTB7A, and NFI-A/X, to date no transcriptional activators have been identified with selectivity for any of the globin genes. Using a CRISPR-Cas9 screen in the HUDEP2 erythroid cell line, we recently identified BRG1/SMARCA4 as a potential activator of HBG production. BRG1 is a core ATPase component of the multi-subunit BAF (BRG1/BRM-associated factor) complex; different subtypes of this large chromatin remodeler family are specific to particular cell types or developmental stages. BRG1 was previously implicated in globin gene regulation but its molecular composition and whether it has selectivity for any of the genes in the cluster is unknown. Notably, we found that loss of BRG1 in both HUDEP2 and primary erythroid cells impairs transcription of HBG to a much greater extent than that of HBB. Here we functionally dissected BAF subunits to decipher composition of the relevant BAF complex and the mechanism for this unexpected HBG gene selectivity.

Although increasing levels of fetal hemoglobin (HbF) in sickle cell disease (SCD) significantly reduces mortality, effective pharmacologic induction of HbF has remained elusive. To identify potentially druggable molecules involved in HbF control, we carried out a domain-focused CRISPR-Cas9-based genetic screen in HUDEP2 cells targeting all 218 serine/threonine protein phosphatases with 6 independent sgRNAs each. A single phosphatase, PPP6C, emerged as a potential HbF repressor. PPP6C is the catalytic subunit of protein phosphatase 6 (PP6), a serine/threonine phosphatase complex that has been implicated in cell cycle regulation, autophagy, and innate immunity mechanisms in other cell types, but its role in erythropoiesis and HbF regulation has not previously been described.

Large granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 TCRαβ T-LGL and 12 TCRγδ T-LGL). 76 mutations were observed in three or more patients in the cohort, and out of those, STAT3, KMT2D PIK3R1, TTN, EYS, and SULF1mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole exome cohort.Hotspot mutations in STAT3, PIK3R1, and FASwere detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2Dand TET2were observed. Moreover, STAT3mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2Dand SETD1B(p< 0.01, p< 0.05 respectively). STAT3was mutated in 50.5% of the patients. Most common Y640F STAT3mutation was associated with lower ANC values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3coiled-coil domain were characterized. STAT3mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferongamma signaling anddecreased PI3K-Akt signaling for STAT3mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.