DNA Regulatory Elements Research Articles

Common as well as unique methylation-sensitive DNA regulatory elements in three mammalian SLC9C1 genes

The SLC9C1 gene (which encodes the NHE10 protein) is essential for male fertility in both mice and humans, however the epigenetic mechanisms regulating its testis/sperm-specific gene expression have yet to be studied. Here we identify and characterize DNA regulatory elements of the SLC9C1 gene across three mammalian species: mouse, rat, and human. First, in silico analysis of these mammalian SLC9C1 genes identified a CpG island located upstream of the transcription start site in the same relative position in all three genes. Further analysis reveals that this CpG island behaves differently, with respect to gene regulatory activity, in the mouse SLC9C1 gene than it does in the rat and human SLC9C1 gene. The mouse SLC9C1 CpG island displays strong promoter activity by itself and seems to have a stronger gene regulatory effect than either the rat or human SLC9C1 CpG islands. While the function of the upstream SLC9C1 CpG island may be divergent across the three studied species, it appears that the promoters of these three mammalian SLC9C1 genes share similar DNA methylation-sensitive regulatory mechanisms. All three SLC9C1 promoter regions are differentially methylated in lung and testis, being more hypermethylated in lung relative to the testis, and DNA sequence alignments provide strong evidence of primary sequence conservation. Luciferase assays reveal that in vitro methylation of constructs containing different elements of the three SLC9C1 genes largely exhibit methylation-sensitive promoter activity (reduced promoter activity when methylated) in both HEK 293 and GC-1spg cells. In total, our data suggest that the DNA methylation-sensitive elements of the mouse, rat, and human SLC9C1 promoters are largely conserved, while the upstream SLC9C1 CpG island common to all three species seems to perform a different function in mouse than it does in rat and human. This work provides evidence that while homologous genes can all be regulated by DNA methylation-dependent epigenetic mechanisms, the location of the specific cis-regulatory elements responsible for this regulation can differ across species.

OR33-05 Glucocorticoids Instigate Transcriptional Waves To Maintain Temporally Organized Fuel Production During Fasting

Abstract Disclosure: D. Goldberg: None. I. Goldstein: None. Most animals are well equipped to endure prolonged fasting periods by converting energy stores into available biochemical fuel. This is achieved by the hepatic production of two fuel types supplying other tissues: glucose (originating from gluconeogenesis or glycogen breakdown) and ketone bodies (produced via ketogenesis). The adaptive response to fasting involves several hormones that together maintain homeostasis in the face of energy shortage. Glucocorticoids (GCs) are principal hormonal cues regulating the transcriptional response to fasting and were shown to augment both glucose and ketone production. We aimed to decipher how GCs control both the gluconeogenic and ketogenic programs. We profiled the transcriptional response and genome-wide enhancer dynamics of primary mouse hepatocytes to GC treatment. In addition to the induction of the expected fasting metabolism genes, we were surprised to find that GCs induced various genes encoding transcription factors (TFs). In fact, 13% of GC-induced genes were TFs, many of which were similarly induced in livers of fasted mice. Perturbation experiments and chromatin immunoprecipitation sequencing (ChIP-seq) showed that GCs induced these TFs via the glucocorticoid receptor (GR) which bound to DNA regulatory elements of the genes. We found that GCs induce two groups of TFs. The first group are TFs that maintain the early gluconeogenic fasting response. Indeed, knock-down of GC-induced TFs (C/EBPβ, MAFB or KLF15) reduced gluconeogenic gene expression. The second group are TFs which dictate a secondary transcriptional wave serving to produce fuel in prolonged fasting. Notably, GCs Initiate a TF cascade in which GR induces the expression of the Ppara gene, leading to increased PPARα protein. Upon subsequent ligand stimulation of PPARα, a ketogenic gene program is activated. We show that a dual treatment of GCs with a PPARα agonist leads to synergistic induction of ketogenic genes and that this induction is dependent on protein synthesis. We have analyzed the transcriptomic result of this TF cascade and found that dozens of ketogenic genes are synergistically induced. Genome-wide analysis of enhancer dynamics revealed numerous enhancers activated by the GR-PPARα cascade. These enhancers are proximal to ketogenic genes, are enriched for the PPARα binding motif and show increased PPARα binding as profiled by ChIP-seq. Taken together, our work reveals a GC-induced program whereby the expression of TFs increases during fasting, serving to rapidly support gluconeogenic capacity in short-term fasting and initiate a delayed ketogenic program brought about in prolonged fasting. These two regulatory modules may dictate the well-known temporal organization of the fasting response whereby gluconeogenesis predominates in early fasting but is gradually replaced by ketogenesis as fasting persists. Presentation: Sunday, June 18, 2023

Insight into the regulatory mechanism of dynamic chromatin 3D interactions during cardiomyocyte differentiation in human

Cardiogenesis is an extremely complicated process involved with DNA regulatory elements, and trans factors regulate gene expression pattern spatiotemporally. Enhancers, as the well-known DNA elements, activate target gene expression by transcription factors (TFs) occupied to organize dynamic three-dimensional (3D) interactions, which when affected or interrupted might cause heart defects or diseases. In this study, we integrated transcriptome, 3D genome, and regulatome to reorganize the global 3D genome in cardiomyogenesis, showing a gradually decreased trend of both chromatin interactions and topological associating domains (TADs) during cardiomyocyte differentiation. And almost all of the chromatin interactions occurred within the same or between adjacent TADs involved with enhancers, indicating that dynamical rewiring of enhancer-related chromatin interactions in the continuous expansive TADs is closely correlated to cardiogenesis. Moreover, we found stage-specific interactions activate stage-specific expression to be involved within corresponding biological functions, and the stage-specific combined regulations of enhancers and binding TFs form connected networks to control stage-specific expression and biological processes, which promote cardiomyocyte differentiation. Finally, we identified markers based on regulatory networks, which might drive cardiac development. This study demonstrates the power of enhancer interactome combined with active TFs to reveal insights into transcriptional regulatory networks during cardiomyogenesis.

Functional characterization of the Komagataella phaffii 1033 gene promoter and transcriptional terminator.

The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step. The transcriptional terminator is another regulatory element in the expression cassette that can modify the expression levels of the heterologous gene. In this work, we identified and functionally characterized the promoter (P1033) and transcriptional terminator (T1033) of a constitutive gene (i.e., the 1033 gene) with a weak non-methanol-dependent transcriptional activity. We constructed two K. phaffii strains with two combinations of the regulatory DNA elements from the 1033 and AOX1 genes (i.e., P1033-TAOX1 and P1033-T1033 pairs) and evaluated the impact of the regulatory element combinations on the transcript levels of the heterologous gene and endogenous 1033 and GAPDH genes in cells grown in glucose or glycerol, and on the extracellular product/biomass yield. The results indicate that the P1033 has a 2-3% transcriptional activity of the GAP promoter and it is tunable by cell growth and the carbon source. The combinations of the regulatory elements rendered different transcriptional activity of the heterologous and endogenous genes that were dependent on the carbon source. The promoter-terminator pair and the carbon source affected the heterologous gene translation and/or protein secretion pathway. Moreover, low heterologous gene-transcript levels along with glycerol cultures increased translation and/or protein secretion.

Prediction of prokaryotic and eukaryotic promoters based on information-theoretic features

Promoters are DNA regulatory elements located near the transcription start site and are responsible for regulating the transcription of genes. DNA fragments arranged in a certain order form specific functional regions with different information contents. Information theory is the science that studies the extraction, measurement and transmission of information. The genetic information contained in DNA follows the general laws of information storage. Therefore, method in information theory can be used for the analysis of promoters carrying genetic information.In this study, we introduced the concept of information theory to the study of promoter prediction. We used 107 features extracted based on information theory methods and a backpropagation neural network to build a classifier. Then, the trained classifier was applied to predict the promoters of 6 organisms. The average AUCs of the 6 organisms obtained by using hold-out validation and ten-fold cross-validation were 0.885 and 0.886, respectively. The results verified the effectiveness of information-theoretic features in promoter prediction. Considering the possible redundancy in the feature set, we performed feature selection and obtained key feature subsets related to promoter characteristics. The results indicate the potential utility of information-theoretic features in promoter prediction.

TETRASPANIN 8-1 from Phaseolus vulgaris plays a key role during mutualistic interactions.

Arbuscular mycorrhizal (AM) fungi and rhizobia form two of the most important plant-microbe associations for the assimilation of phosphorus (P) and nitrogen (N). Symbiont-derived signals are able to coordinate the infection process by triggering multiple responses in the plant root, such as calcium influxes and oscillations, increased reactive oxygen species (ROS), cytoskeletal rearrangements and altered gene expression. An examination was made of the role of tetraspanins, which are transmembrane proteins that self-organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking, and ROS generation. In plant cells, tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, plasmodesmata and hormonal regulation. Some plant tetraspanins, such as Arabidopsis thaliana TETRASPANIN 8 and TETRASPANIN 9 (AtTET8 and AtTET9) are associated with exosomes during inter-kingdom communication. In this study, a homolog of AtTET8, PvTET8-1, in common bean (Phaseolus vulgaris L. var. Negro Jamapa) was examined in roots during interactions with Rhizobium tropici and Rhizophagus irregularis. The promoter of PvTET8-1 contained several cis-acting regulatory DNA elements potentially related to mutualistic interactions, and PvTET8-1 was transcriptionally activated during AM fungal and rhizobial associations. Silencing it decreased the size and number of nodules, nitrogen fixation, and mycorrhizal arbuscule formation, whereas overexpressing it increased the size and number of nodules, and mycorrhizal arbuscule formation but decreased nitrogen fixation. PvTET8-1 appears to be an important element in both of these mutualistic interactions, perhaps through its interaction with NADPH oxidase and the generation of ROS during the infection processes.

Proximity-dependent recruitment of Polycomb repressive complexes by the lncRNA Airn

During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between the Airn lncRNA and chromatin correlated with underlying intensity of PRC recruitment and PRC-directed modifications. Deletion of CpG islands that contact the Airn locus altered long-distance repression and PRC activity in a manner that correlated with changes in chromatin architecture. Our data imply that the extent to which Airn expression recruits PRCs to chromatin is controlled by DNA regulatory elements that modulate proximity of the Airn lncRNA product to its target DNA.

The Cytomegalovirus M35 Protein Directly Binds to the Interferon-β Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

#3257 EFFECTS OF CKD ASSOCIATED GENETIC VARIATION ON TRANSCRIPTOMIC REGULATION OF RENAL CELLS

Abstract Background and Aims Chronic kidney disease (CKD) is an universal health problem characterized by a decline of renal function. This progressive disease leads to kidney failure and CKD patients have a high risk of cardiovascular diseases, including endothelial dysfunction in the heart. Genetic variability in the general population has a strong impact on the risk of CKD development. Genome wide association studies (GWAS) already identified more than 250 single nucleotide polymorphisms (SNPs) associated with renal function. These SNPs are studied in relation with their location in the genome in protein coding regions, however, genetic variability in the genome could also affect the function of DNA regulatory elements (DREs). These non-protein coding regions regulate transcriptional expression of genes by transcription factor binding. These elements are ubiquitously present in the genome and can regulate promotor activity from a long distance (>100 kb from the start site) via 3D chromatin folding. DRE activity is cell type dependent and also changes in different conditions (e.g. disease). Therefore, genetic variability e.g. SNPs located in DREs and their regulation of gene transcription could play a key role in the development and progression of CKD. Previous studies identified more than 30 DREs localized SNPs in renal cells which could be important in CKD development. We focus on SNP rs881858, a CKD associated SNP region without a protein coding sequence. Moreover, vascular endothelial growth factor (VEGF) is identified as target gene of this SNP which could therefore impact renal cell-endothelial cell interaction. Six possible active DREs were identified in this rs881858 DNA region. We hypothesize that the CKD associated SNP rs881858 induce DRE mediated transcriptional changes that contribute to the development and progression of CKD. We aim to study the effect of the DREs in DNA region rs881858 on transcriptional gene regulation in renal cells. Method All variations of the genetic variability in 6 identified DREs in DNA region rs881858 were introduced by site-directed mutagenesis. These variations including the reference variant, were cloned into a luciferase reporter plasmid with a minimal promoter and subsequently transfected in human embryonal kidney (HEK) cells. Luciferase activity was measured using spectrophotometry and represent DRE regulation of transcription. A decline in luciferase activity of one of the variants in a DRE imply a reduced activity of that specific variant compared to the reference variant. To assess the impact of the DREs in the genome, a CRISPR/Cas9 construct was made to knockout each individual DRE on the risk locus rs881858. Results We considered a DRE functional if (almost) all variants within this DRE demonstrate a change in luciferase activity. Based on this, we identified 3 DRE DNA regions in which SNPs gave a decline in luciferase activity. CRISPR/Cas9 mediated knockout of DRE in the HEK cells was validated using PCR and qPCR analysis of SNP rs881858 associated genes is planned to confirm the DRE mediated transcriptomic regulation of these genes. Conclusion These primary results shows that CKD associated SNP rs881858 has a negative impact on DRE function compared to the reference variant. Next we will study the effect of DRE mediated gene transcription in the DRE knockout lines using qPCR. Furthermore, CRISPR/Cas9 mediated DRE-knockout will be performed in an iPSC derived renal organoid model. Since this in vitro model consists of both renal and vascular cell types, the interaction between these cell types can be evaluated in a background of epigenetic variability. This study provides new knowledge in the effect of genetic variant in the development of CKD. Results could lead to identification of novel pathways and therefore personalized strategies for intervention. This method is a stepping stone for studying other known genetic variants in other diseases, including renal and cardiovascular diseases.

Hypoxia controls expression of kidney-pathogenic MUC1 variants.

The interplay between genetic and environmental factors influences the course of chronic kidney disease (CKD). In this context, genetic alterations in the kidney disease gene MUC1 (Mucin1) predispose to the development of CKD. These variations comprise the polymorphism rs4072037, which alters splicing of MUC1 mRNA, the length of a region with variable number of tandem repeats (VNTR), and rare autosomal-dominant inherited dominant-negative mutations in or 5' to the VNTR that causes autosomal dominant tubulointerstitial kidney disease (ADTKD-MUC1). As hypoxia plays a pivotal role in states of acute and chronic kidney injury, we explored the effects of hypoxia-inducible transcription factors (HIF) on the expression of MUC1 and its pathogenic variants in isolated primary human renal tubular cells. We defined a HIF-binding DNA regulatory element in the promoter-proximal region of MUC1 from which hypoxia or treatment with HIF stabilizers, which were recently approved for an anti-anemic therapy in CKD patients, increased levels of wild-type MUC1 and the disease-associated variants. Thus, application of these compounds might exert unfavorable effects in patients carrying MUC1 risk variants.

A Novel Small NPC1 Promoter Enhances AAV-Mediated Gene Therapy in Mouse Models of Niemann-Pick Type C1 Disease.

Niemann-Pick disease type C1 (NP-C) is a prematurely lethal genetic lysosomal storage disorder with neurological and visceral pathology resulting from mutations in the NPC1 gene encoding the lysosomal transmembrane protein NPC1. There is currently no cure for NP-C, and the only disease modifying treatment, miglustat, slows disease progression but does not significantly attenuate neurological symptoms. AAV-mediated gene therapy is an attractive option for NP-C, but due to the large size of the human NPC1 gene, there may be packaging and truncation issues during vector manufacturing. One option is to reduce the size of DNA regulatory elements that are essential for gene expression, such as the promoter sequence. Here, we describe a novel small truncated endogenous NPC1 promoter that leads to high gene expression both in vitro and in vivo and compare its efficacy to other commonly used promoters. Following neonatal intracerebroventricular (ICV) injection into the CNS, this novel promoter provided optimal therapeutic efficacy compared to all other promoters including increased survival, improved behavioural phenotypes, and attenuated neuropathology in mouse models of NP-C. Taken together, we propose that this novel promoter can be extremely efficient in designing an optimised AAV9 vector for gene therapy for NP-C.

The transcription factors Creb1 and CEBPB regulate Sox9 promoter activity in TM4 Sertoli cells

In Sertoli cells, the Sox9 gene is essential for testicular development and normal spermatogenesis. SOX9 is critical for postnatal Sertoli cells differentiation and proliferation in the testis. However, the molecular mechanisms that specifically regulate its expression are not entirely understood. Sox9 expression is regulated by CREB1 and CEBPB in other biological contexts such as during chondrogenesis and in rat thyroid follicular cells. We hypothesized that Sox9 promoter activity is regulated by CREB1 and CEBPB in Sertoli cells. Our results show that Sox9 expression is dependent on the activation of these transcription factors by the cAMP/PKA signaling pathway in TM4 Sertoli cells. Chromatin immunoprecipitation and promoter/reporter luciferase assays with 5′ promoter deletions and site-directed mutagenesis demonstrated that CREB1 is being recruited to a DNA regulatory element at -141 bp of the Sox9 promoter region. Such regulation is dependent on the cAMP/PKA signaling pathway, resulting in phosphorylation of CREB1. Activation of Sox9 expression by CEBPB may involve its recruitment to the proximal promoter region by protein–protein interaction with CREB1. Thus, we have shown that the Sox9 promoter is being regulated by the transcription factors CREB1 and CEBPB in TM4 Sertoli cells and involve their recruitment to the proximal promoter region.

Hypoxia induces polycystin-1 expression in the renal epithelium.

Mutations in polycystin-1 which is encoded by the PKD1 gene are the main causes for the development of autosomal dominant polycystic kidney disease. However, only little is known about the physiological function of polycystin-1 and even less about the regulation of its expression. Here, we show that expression of PKD1 is induced by hypoxia and compounds that stabilize the hypoxia-inducible transcription factor (HIF) 1α in primary human tubular epithelial cells. Knockdown of HIF subunits confirms HIF-1α-dependent regulation of polycystin-1 expression. Furthermore, HIF ChIP-seq reveals that HIF interacts with a regulatory DNA element within the PKD1 gene in renal tubule-derived cells. HIF-dependent expression of polycystin-1 can also be demonstrated in vivo in kidneys of mice treated with substances that stabilize HIF. Polycystin-1 and HIF-1α have been shown to promote epithelial branching during kidney development. In line with these findings, we show that expression of polycystin-1 within mouse embryonic ureteric bud branches is regulated by HIF. Our finding links expression of one of the main regulators of accurate renal development with the hypoxia signalling pathway and provides additional insight into the pathophysiology of polycystic kidney disease.

Hypoxia hits APOL1 in the kidney

Individuals of African ancestry carrying two pathogenic variants of apolipoprotein 1 (APOL1) have a substantially increased risk for developing chronic kidney disease. The course of APOL1 nephropathy is extremely heterogeneous and shaped by systemic factors such as a response to interferon. However, additional environmental factors operating in this second-hit model have been less well defined. Here, we reveal that stabilization of hypoxia-inducible transcription factors (HIF) by hypoxia or HIF prolyl hydroxylase inhibitors activates transcription of APOL1 in podocytes and tubular cells. An active regulatory DNA-element upstream of APOL1 that interacted with HIF was identified. This enhancer was accessible preferentially in kidney cells. Importantly, upregulation of APOL1 by HIF was additive to the effects of interferon. Furthermore, HIF stimulated expression of APOL1 in tubular cells derived from the urine of an individual carrying a risk variant for kidney disease. Thus, hypoxic insults may serve as important modulators of APOL1 nephropathy.

Predicting Corynebacterium glutamicum promoters based on novel feature descriptor and feature selection technique.

The promoter is an important noncoding DNA regulatory element, which combines with RNA polymerase to activate the expression of downstream genes. In industry, artificial arginine is mainly synthesized by Corynebacterium glutamicum. Replication of specific promoter regions can increase arginine production. Therefore, it is necessary to accurately locate the promoter in C. glutamicum. In the wet experiment, promoter identification depends on sigma factors and DNA splicing technology, this is a laborious job. To quickly and conveniently identify the promoters in C. glutamicum, we have developed a method based on novel feature representation and feature selection to complete this task, describing the DNA sequences through statistical parameters of multiple physicochemical properties, filtering redundant features by combining analysis of variance and hierarchical clustering, the prediction accuracy of the which is as high as 91.6%, the sensitivity of 91.9% can effectively identify promoters, and the specificity of 91.2% can accurately identify non-promoters. In addition, our model can correctly identify 181 promoters and 174 non-promoters among 400 independent samples, which proves that the developed prediction model has excellent robustness.

Abstract P6-11-01: Scaffold attachment factor B1 modulates cholesterol pathways in triple-negative breast cancer

Abstract Historically known as a tumor suppressor by estrogen receptor (ER) co-repression in breast cancer (BC), the matrix binding protein Scaffold Attachment Factor B1 (SAFB) binds scaffold or matrix attachment region DNA elements (S/MAR DNA) in eukaryotic DNA. SAFB1 plays a role in cellular stress response, DNA repair, differentiation, and apoptosis. SAFB loss in ER-independent BC and pancreatic cancer (PC) patients resulted in poor survival rates, hinting at the role of SAFB1 as a tumor suppressor. To understand the tumor suppressive mechanism of SAFB1, we performed shRNA-mediated knockdown (KD) of SAFB in PC cell lines and triple-negative breast cancer (TNBC) cells, an aggressive subgroup of BC. Analysis of onco-properties showed an increase in clonogenicity potential and cell proliferation in SAFB KDs in both PDACs and TNBCs cells. Further, RNA-Seq analysis of SAFB KDs in the TNBC cell line revealed activation of the mevalonate (MVA) pathway and the resulting cholesterol biosynthesis as the key metabolic change. Both pancreatic ductal adenocarcinoma (PDACs) and TNBC exhibited higher levels of MVA pathway gene expression upon loss of SAFB. Sterol regulatory element binding proteins (SREBP) 1 and 2 dictate cholesterol biosynthesis, and SREBP2 promotes tumor properties via the MVA pathway. Molecular and metabolic analysis of SAFB KD TNBC showed an increase in lipid droplets and SREBP2 maturation. Using Chromatin Immunoprecipitation and quantitative real-time PCR (ChIP-qPCR) in TNBC we demonstrate the direct binding of SAFB to the SREBP2 promoter region. In addition, byproducts of the MVA pathway have been shown to activate YAP/TAZ-dependent tissue homeostasis and tumorigenesis. We also observed increased YAP1 mRNA levels and decreased YAP1 (Ser127) phosphorylation with SAFB loss in TNBC, explaining the aggressive tumorigenicity gained with SAFB loss. Our study thus far suggests SAFB has overt control over the SREBP2-MVA-YAP1 pathway, and loss of SAFB results in an enhanced tumor phenotype. Citation Format: Meron Ghidey, Divya Murthy, Sukjin Yang, Songyeon Ahn, Jun Hyoung Park, Benny Kaipparettu. Scaffold attachment factor B1 modulates cholesterol pathways in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-01.

Demystifying the Druggability of the MYC Family of Oncogenes.

The MYC family of oncogenes (MYC, MYCN, and MYCL) encodes a basic helix-loop-helix leucine zipper (bHLHLZ) transcriptional regulator that is responsible for moving the cell through the restriction point. Through the HLHZIP domain, MYC heterodimerizes with the bHLHLZ protein MAX, which enables this MYC-MAX complex to bind to E-box regulatory DNA elements thereby controlling transcription of a large group of genes and their proteins. Translationally, MYC is one of the foremost oncogenic targets, and deregulation of expression of the MYC family gene/proteins occurs in over half of all human tumors and is recognized as a hallmark of cancer initiation and maintenance. Additionally, unexpected roles for this oncoprotein have been found in cancers that nominally have a non-MYC etiology. Although MYC is rarely mutated, its gain of function in cancer results from overexpression or from amplification. Moreover, MYC is a pleiotropic transcription factor possessing broad pathogenic prominence making it a coveted cancer target. A widely held notion within the biomedical research community is that the reliable modulation of MYC represents a tremendous therapeutic opportunity given its role in directly potentiating oncogenesis. However, the MYC-MAX heterodimer interaction contains a large surface area with a lack of well-defined binding sites creating the perception that targeting of MYC-MAX is forbidding. Here, we discuss the biochemistry behind MYC and MYC-MAX as it relates to cancer progression associated with these transcription factors. We also discuss the notion that MYC should no longer be regarded as undruggable, providing examples that a therapeutic window is achievable despite global MYC inhibition.

Mechanisms of chromate carcinogenesis by chromatin alterations.

In a dynamic environment, organisms must constantly mount an adaptive response to new environmental conditions in order to survive. Novel patterns of gene expression, driven by attendant changes in chromatin architecture, aid in adaptation and survival. Critical mechanisms in the control of gene transcription govern new spatiotemporal chromatin-chromatin interactions that make regulatory DNA elements accessible to the transcription factors that control the response. Consequently, agents that disrupt chromatin structure are likely to have a direct impact on the transcriptional programs of cells and organisms and to drive alterations in fundamental physiological processes. In this regard, hexavalent chromium (Cr(VI)) is of special interest because it interacts directly with cellular proteins, DNA, and other macromolecules, and is likely to upset cell functions that may cause generalized damage to the organism. Here, we will highlight chromium-mediated mechanisms that disrupt chromatin architecture and discuss how these mechanisms are integral to its carcinogenic properties. Emerging evidence indicates that Cr(VI) targets euchromatin, particularly in genomic locations flanking the binding sites of the essential transcription factors CTCF and AP1, and, in so doing, they disrupt nucleosomal architecture. Ultimately, the ensuing changes, if occurring in critical regulatory domains, may establish a new chromatin state, either toxic or adaptive, that will be governed by the corresponding gene transcription changes in key biological processes associated with that state.

CRISPR Activator Approaches to Study Endogenous Androglobin Gene Regulation.

Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.

Systematic identification and characterization of repressive domains in Drosophila transcription factors

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA‐binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co‐repressors (CoRs) they recruit have not been determined. Here, we develop a high‐throughput, next‐generation sequencing‐based screening method, repressive‐domain (RD)‐seq, to systematically identify RDs in complex DNA‐fragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription‐related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, we show that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance our understanding of repressors, their sequences, and the functional impact of sequence‐altering mutations and should provide a valuable resource for further studies.