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Background: Hypertension or elevated blood pressure (BP) is a worldwide clinical challenge and the leading primary risk factor for kidney dysfunctions, heart failure, and cerebrovascular disease. The kidney is a central regulator of BP by maintaining sodium-water balance. Multiple genome-wide association studies revealed that BP is a heritable quantitative trait, modulated by several genetic, epigenetic, and environmental factors. The SNPs identified in genome-wide association studies predominantly (>95%) reside within noncoding genomic regions, making it difficult to understand how they regulate BP. Given the central role of the kidney in regulating BP, we hypothesized that chromatin-accessible regions in renal tissue would be enriched for BP-associated single nucleotide polymorphisms., Methods: We manually dissected 2 important kidney segments that maintain the sodium-water balance: proximal tubules and medullary thick ascending limbs from the human and rat kidneys. To delineate their chromatin and transcriptomic profiles, we performed the assay for transposase-accessible chromatin and RNA sequencing, respectively., Results: The chromatin accessibility maps revealed the shared and unique cis -regulatory elements that modulate the chromatin accessibility in proximal tubule and medullary thick ascending limbs of humans and rats. We developed a visualization tool to compare the cross-species epigenomic maps to identify potential regulatory targets for hypertension pathogenesis. We also identified a significant enrichment of BP-associated single nucleotide polymorphisms (1064 for human proximal tubule and 1172 for human medullary thick ascending limbs) within accessible chromatin regions of both segments, including rs1173771 and rs1421811 at the NPR3 locus and rs1800470 at the TGFb1 locus., Conclusions: Collectively, this study lays a foundation for interrogating how intergenic single nucleotide polymorphisms may regulate polygenic traits such as BP.

The Switch/Sugar non-fermenting (SWI/SNF) nucleosome remodeling complexes are essential for normal hematopoiesis. The Brg1/Brm associated factor (BAF) is a form of mammalian SWI/SNF that is distinguished by the presence of either ARID1A or ARID1B protein. In this study, we used hematopoietic specific Cre mouse models to assess the function of Arid1b in blood development. We found Arid1b loss did not affect steady state hematopoiesis or hematopoietic regeneration. Nonetheless, Arid1b null hematopoietic stem and progenitor cells have reduced ability to reconstitute hematopoietic system compared to wild type cells. Overall, our data indicate Arid1b is largely dispensable for normal hematopoiesis but impairs the regenerative ability of HSPCs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Arnold et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Essential hypertension, a multifaceted disorder, is a worldwide health problem. A complex network of genetic, epigenetic, physiological, and environmental components regulates blood pressure (BP), and any dysregulation of this network may result in hypertension. Growing evidence suggests a role for epigenetic factors in BP regulation. Any alterations in the expression or functions of these epigenetic regulators may dysregulate various determinants of BP, thereby promoting the development of hypertension. Histone posttranslational modifications are critical epigenetic regulators that have been implicated in hypertension. Several studies have demonstrated a clear association between the increased expression of some histone-modifying enzymes, especially HDACs (histone deacetylases), and hypertension. In addition, treatment with HDAC inhibitors lowers BP in hypertensive animal models, providing an excellent opportunity to design new drugs to treat hypertension. In this review, we discuss the potential contribution of different histone modifications to the regulation of BP., Competing Interests: Disclosures None.

Objective As cohesin mutations are rarely found in MLL-rearranged acute myeloid leukemias, we investigated the potential synthetic lethality between cohesin mutations and MLL-AF9 using murine hematopoietic stem and progenitor cells. Results Contrary to our hypothesis, a complete loss of Stag2 or haploinsufficiency of Smc3 were well tolerated in MLL-AF9-expressing hematopoietic stem and progenitor cells. Minimal effect of cohesin subunit loss on the in vitro self-renewal of MLL-AF9-expressing cells was observed. Despite the differing mutational landscapes of cohesin-mutated and MLL fusion AMLs, previous studies showed that cohesin and MLL fusion mutations similarly drive abnormal self-renewal through HOXA gene upregulation. The utilization of a similar mechanism suggests that little selective pressure exists for the acquisition of cohesin mutations in AMLs expressing MLL fusions, explaining their lack of co-occurrence. Our results emphasize the importance of using genetic models to test suspected synthetic lethality and suggest that a lack of co-occurrence may instead point to a common mechanism of action between two mutations.

Acute myeloid leukemia (AML) is driven by mutations that occur in numerous combinations. A better understanding of how mutations interact with one another to cause disease is critical to developing targeted therapies. Approximately 50% of patients that harbor a common mutation in NPM1 (NPM1cA) also have a mutation in the cohesin complex. As cohesin and Npm1 are known to regulate gene expression, we sought to determine how cohesin mutation alters the transcriptome in the context of NPM1cA. We utilized inducible Npm1 cAflox/+ and core cohesin subunit Smc3 flox/+ mice to examine AML development. While Npm1 cA/+ ;Smc3 Δ/+ mice developed AML with a similar latency and penetrance as Npm1 cA/+ mice, RNA-seq suggests that the Npm1 cA/+ ; Smc3 Δ/+ mutational combination uniquely alters the transcriptome. We found that the Rac1/2 nucleotide exchange factor Dock1 was specifically upregulated in Npm1 cA/+ ;Smc3 Δ/+ HSPCs. Knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis only in Npm1 cA/+ ;Smc3 Δ/+ AML. Higher Rac activity was also observed in Npm1 cA/+ ;Smc3 Δ/+ vs. Npm1 cA/+ AMLs. Importantly, the Dock1/Rac pathway is targetable in Npm1 cA/+ ;Smc3 Δ/+ AMLs. Our results suggest that Dock1/Rac represents a potential target for the treatment of patients harboring NPM1cA and cohesin mutations and supports the use of combinatorial genetics to identify novel precision oncology targets., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Survival outcomes for relapsed/refractory pediatric acute myeloid leukemia (R/R AML) remain dismal. Epigenetic changes can result in gene expression alterations which are thought to contribute to both leukemogenesis and chemotherapy resistance. We report results from a phase I trial with a dose expansion cohort investigating decitabine and vorinostat in combination with fludarabine, cytarabine, and G-CSF (FLAG) in pediatric patients with R/R AML [NCT02412475]. Thirty-seven patients enrolled with a median age at enrollment of 8.4 (range, 1-20) years. There were no dose limiting toxicities among the enrolled patients, including two patients with Down syndrome. The recommended phase 2 dose of decitabine in combination with vorinostat and FLAG was 10 mg/m 2 . The expanded cohort design allowed for an efficacy evaluation and the overall response rate among 35 evaluable patients was 54% (16 complete response (CR) and 3 complete response with incomplete hematologic recovery (CRi)). Ninety percent of responders achieved minimal residual disease (MRD) negativity (<0.1%) by centralized flow cytometry and 84% (n = 16) successfully proceeded to hematopoietic stem cell transplant. Two-year overall survival was 75.6% [95%CI: 47.3%, 90.1%] for MRD-negative patients vs. 17.9% [95%CI: 4.4%, 38.8%] for those with residual disease (p < .001). Twelve subjects (34%) had known epigenetic alterations with 8 (67%) achieving a CR, 7 (88%) of whom were MRD negative. Correlative pharmacodynamics demonstrated the biologic activity of decitabine and vorinostat and identified specific gene enrichment signatures in nonresponding patients. Overall, this therapy was well-tolerated, biologically active, and effective in pediatric patients with R/R AML, particularly those with epigenetic alterations., (© 2022 Wiley Periodicals LLC.)

Transcriptional enhancers have been defined by their ability to operate independent of distance and orientation in plasmid-based reporter assays of gene expression. At present, histone marks are used to identify and define enhancers but do not consider the endogenous role of an enhancer in the context of native chromatin. We employed a combination of genomic editing, single cell analyses, and sequencing approaches to investigate a Nanog-associated cis-regulatory element, which has been reported by others to be either an alternative promoter or a super-enhancer. We first demonstrate both distance and orientation independence in native chromatin, eliminating the issues raised with plasmid-based approaches. We next demonstrate that the dominant super-enhancer modulates Nanog globally and operates by recruiting and/or initiating RNA Polymerase II. Our studies have important implications to how transcriptional enhancers are defined and how they regulate gene expression., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Fbw7 is a tumor suppressor that regulates the degradation of oncogenic substrates such as c-Jun, c-Myc, Notch1 intracellular domain (ICD), and cyclin E by functioning as the substrate recognition protein in the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. Consequently, low expression or loss of FBXW7 in breast cancer has been hypothesized to result in the accumulation of oncogenic transcription factors that are master regulators of proliferation, apoptosis, and ultimately transformation. Despite this, the direct effect of Fbw7 loss on mammary gland morphology and tumorigenesis has not been examined. Here, we demonstrate that conditional deletion of Fbxw7 in murine mammary tissue initiates breast tumor development and also results in lactation and involution defects. Further, while Fbxw7 loss results in the overexpression of Notch1-ICD, c-Jun, cyclin E, and c-Myc, the downstream transcription factor pathways associated with c-Myc and cyclin E are the most dysregulated, including at the single-cell level. These pathways are dysregulated early after Fbxw7 loss, and their sustained loss results in tumorigenesis and reinforced c-Myc and cyclin E-E2F pathway disruption. We also find that loss of Fbxw7 is linked to the acquisition of Trp53 mutations, similar to the mutational spectrum observed in patients. Our results demonstrate that the loss of Fbxw7 promotes the acquisition of Trp53 mutations and that the two cooperate in breast tumor development. Targeting c-Myc, E2F, or p53 may therefore be a beneficial treatment strategy for FBXW7-altered breast cancer patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal as this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of polycomb repressive complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying that both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.

Background: The cohesin complex consists of multiple core subunits that play critical roles in mitosis and transcriptional regulation. The cohesin-associated protein Wapal plays a central role in off-loading cohesin to facilitate sister chromatid separation, but its role in regulating mammalian gene expression is not understood. We used embryonic stem cells as a model, given that the well-defined transcriptional regulatory circuits were established through master transcription factors and epigenetic pathways that regulate their ability to maintain a pluripotent state., Results: RNAi-mediated depletion of Wapal causes a loss of pluripotency, phenocopying loss of core cohesin subunits. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), we determine that Wapal occupies genomic sites distal to genes in combination with CTCF and core cohesin subunits such as Rad21. Interestingly, genomic sites occupied by Wapal appear enriched for cohesin, implying that Wapal does not off-load cohesin at regions it occupies. Wapal depletion induces derepression of Polycomb group (PcG) target genes without altering total levels of Polycomb-mediated histone modifications, implying that PcG enzymatic activity is preserved. By integrating ChIP-seq and gene expression changes data, we identify that Wapal binding is enriched at the promoters of PcG-silenced genes and is required for proper Polycomb repressive complex 2 (PRC2) recruitment. Lastly, we demonstrate that Wapal is required for the interaction of a distal cis-regulatory element (CRE) with the c-Fos promoter., Conclusions: Collectively, this work indicates that Wapal plays a critical role in silencing of PcG target genes through the interaction of distal CREs with promoters.

Sal-like protein 4 (SALL4) is a transcription factor that exists in two splice isoforms, SALL4a and SALL4b, and regulates transcription in embryonic stem cells, hematopoiesis, and acute myeloid leukemia. Constitutive overexpression of SALL4 in mice induces acute myeloid leukemia. Interestingly, a potential benefit of using SALL4 to facilitate ex vivo hematopoietic stem cell expansion has been proposed. However, distinct roles for how SALL4 contributes to normal versus malignant processes remain undefined. Here we show that SALL4b is the predominant isoform in murine hematopoietic stem cells and progenitors. Overexpression of either SALL4 isoform in hematopoietic stem cells or progenitors impairs hematopoietic colony formation and expansion in vitro. Lineage-negative bone marrow overexpressing SALL4b fails to engraft and reconstitute hematopoiesis when transplanted. We found that both SALL4a and SALL4b overexpression impair hematopoiesis, in part through dose-dependent repression of BMI1. Additionally, we have identified the following potential novel SALL4 target genes in hematopoiesis: ARID5B (SALL4a and SALL4b), EZH2, and KLF2 (SALL4a). Lastly, we found that SALL4 expression is variable in acute myeloid leukemia, ranging from no expression to levels comparable to embryonic stem cells. These results show that SALL4 isoforms contribute to only a subset of acute myeloid leukemia and that overexpression of SALL4 isoforms impairs hematopoiesis through repression of BMI1. Together these data demonstrate the sensitivity of hematopoiesis to appropriately balanced SALL4 expression, highlighting the importance of regulating this dynamic in potential therapeutic applications such as ex vivo stem cell expansion., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Enhancers are cis-acting elements capable of regulating transcription in a distance and orientation-independent manner. A subset of enhancers are occupied by RNA polymerase II (RNAP II) and transcribed to produce long non-coding RNAs termed eRNAs. We thoroughly investigated the association between eRNA productivity and various chromatin marks and transcriptional regulators in mouse embryonic stem cells (ESCs) through an integrative approach. We found that eRNA-producing enhancers exhibited elevated levels of the active mark H3K27Ac, decreased DNA methylation, and enrichment for the DNA hydroxylase Tet1. Many eRNA-producing enhancers have recently been characterized as "super-enhancers," suggesting an important role in the maintenance of pluripotency. Using experimental methods, we focally investigated a well-characterized enhancer linked to the Nanog locus and confirmed its exclusive eRNA productivity in ESCs. We further demonstrate that the binding of Sall4 and Tet family proteins were required for eRNA productivity at this locus. Collectively, we demonstrate that Tet1 binding and DNA hypomethylation are hallmarks of eRNA production.

A previous genetic analysis comparing the Dahl salt-sensitive (S) rat with the spontaneously hypertensive rat identified a major locus on chromosome 2 that influences proteinuria in the S rat. In the present study, blood pressure, proteinuria, and renal hemodynamics were evaluated in congenic strains with small segments of the protective spontaneously hypertensive rat genome on the S background. Proteinuria and renal function were significantly improved in the congenic strains compared with the S. The causative locus interval was narrowed to <375 kb on the basis of congenic strains, haplotype data, comparative mapping, and concordance with human genetic studies. Sequencing of the coding region of genes in this region identified 36 single nucleotide polymorphisms (13 nonsynonymous and 23 synonymous). Gene expression profiling indicated that only a few genes exhibited differential expression. Arhgef11, Pear1, and Sh2d2 were identified as important candidate genes that may be linked to kidney injury in the S rat. In particular, Arhgef11 plays an important role in the activation of the Rho-ROCK signaling pathway. Inhibition of this pathway using fasudil resulted in a significant reduction of proteinuria in treated S rats (compared with untreated S). However, no difference was observed between treated or untreated spontaneously hypertensive rat or congenic strains. The homologous region in humans was found to be associated with estimated glomerular filtration rate in the Candidate Gene Association Resource population. In summary, these findings demonstrate that allelic variants in Arhgef11, acting through the Rho-ROCK pathway, could influence kidney injury in the S as well as provide insight into human kidney disease.

The Dahl salt-sensitive (S) rat is a widely studied model of salt-sensitive hypertension and develops proteinuria, glomerulosclerosis, and renal interstitial fibrosis. An earlier genetic analysis using a population derived from the S and spontaneously hypertensive rat (SHR) identified eight genomic regions linked to renal injury in the S rat and one protective locus on chromosome 11. The "protective" locus in the S rat was replaced with the SHR genomic segment conferring "susceptibility" to kidney injury. The progression of kidney injury in the S.SHR(11) congenic strain was characterized in the present study. Groups of S and S.SHR(11) rats were followed for 12 wk on either a low-salt (0.3% NaCl) or high-salt (2% NaCl) diet. By week 12 (low-salt), S.SHR(11) demonstrated a significant decline in kidney function compared with the S. Blood pressure was significantly elevated in both strains on high salt. Despite similar blood pressure, the S.SHR(11) exhibited a more significant decline in kidney function compared with the S. The decline in S.SHR(11) kidney function was associated with more severe kidney injury including tubular loss, immune cell infiltration, and tubulointerstitial fibrosis compared with the S. Most prominently, the S.SHR(11) exhibited a high degree of medullary fibrosis and a significant increase in renal vascular medial hypertrophy. In summary, genetic modification of the S rat generated a model of accelerated renal disease that may provide a better system to study progression to renal failure as well as lead to the identification of genetic variants involved in kidney injury.

The nephron number at birth is a quantitative trait that correlates inversely with the risk of hypertension and chronic kidney disease later in life. During kidney development, the nephron number is controlled by multiple factors including genetic, epigenetic, and environmental modifiers. Premature birth, which represents more than 12% of annual live births in the United States, has been linked to low nephron number and the development of hypertension later in life. In this report, we describe the development of a mouse model of prematurity-induced reduction of nephron number. Premature mice, delivered 1 and 2 days early, have 17.4 ± 2.3% (n = 6) and 23.6 ± 2% (n = 10) fewer nephrons, respectively, when compared with full-term animals (12,252 ± 571 nephrons/kidney, n = 10). After 5 weeks of age, the mice delivered 2 days premature show lower real-time glomerular filtration rate (GFR, 283 ± 13 vs 389 ± 26 μL/min). The premature mice also develop hypertension (mean arterial pressure [MAP], 134 ± 18 vs 120 ± 14 mm Hg) and albuminuria (286 ± 83 vs 176 ± 59 μg albumin/mg creatinine). This mouse model provides a proof of concept that prematurity leads to reduced nephron number and hypertension, and this model will be useful in studying the pathophysiology of prematurity-induced nephron number reductions and hypertension., (Published by Mosby, Inc.)

Chronic kidney disease is a growing medical concern, with an estimated 25.6 million people in the United States exhibiting some degree of kidney injury and/or decline in kidney function. Animal models provide great insight into the study of the genetics of complex diseases. In particular, heterogeneous stock (HS) rats represent a unique genetic resource enabling rapid fine-mapping of complex traits. However, they have not been explored as a model to study renal phenotypes. To evaluate the usefulness of HS rats in the genetics of renal traits, a time course evaluation (weeks 8-40) was performed for several renal phenotypes. As expected, a large degree of variation was seen for most renal traits. By week 24, three (of 40) rats exhibited marked proteinuria that increased gradually until week 40 and ranged from 33.7 to 80.2 mg/24 h. Detailed histological analysis confirmed renal damage in these rats. In addition, several rats consistently exhibited significant hematuria (5/41). Interestingly, these rats were not the same rats that exhibited proteinuria, indicating that susceptibility to different types of kidney injury is likely segregating within the HS population. One HS rat exhibited unilateral renal agenesis (URA), which was accompanied by a significant degree of proteinuria and glomerular and tubulointerstitial injury. The parents of this HS rat were identified and bred further. Additional offspring of this pair were observed to exhibit URA at frequency between 40% and 60%. In summary, these novel data demonstrate that HS rats exhibit variation in proteinuria and other kidney-related traits, confirming that the model harbors susceptibility alleles for kidney injury and providing the basis for further genetic studies.