A Congenital Anemia Reveals Distinct Targeting Mechanisms for Master Transcription Factor GATA1

Blood ◽  
2022 ◽  
Leif Ludwig ◽  
Caleb A Lareau ◽  
Erik L. Bao ◽  
Nan Liu ◽  
Taiju Utsugisawa ◽  

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

2020 ◽  
Vol 295 (26) ◽  
pp. 8725-8735
Stephanie L. Safgren ◽  
Rachel L. O. Olson ◽  
Anne M. Vrabel ◽  
Luciana L. Almada ◽  
David L. Marks ◽  

The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.

2016 ◽  
Vol 113 (16) ◽  
pp. 4434-4439 ◽  
Aoi Wakabayashi ◽  
Jacob C. Ulirsch ◽  
Leif S. Ludwig ◽  
Claudia Fiorini ◽  
Makiko Yasuda ◽  

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.

Development ◽  
2020 ◽  
Vol 147 (22) ◽  
pp. dev185116
Xiaolian Cai ◽  
Ziwen Zhou ◽  
Junji Zhu ◽  
Qian Liao ◽  
Dawei Zhang ◽  

ABSTRACTThe hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) are master regulators of the cellular response to O2. In addition to HIF1α and HIF2α, HIF3α is another identified member of the HIFα family. Even though the question of whether some HIF3α isoforms have transcriptional activity or repressive activity is still under debate, it is evident that the full length of HIF3α acts as a transcription factor. However, its function in hypoxia signaling is largely unknown. Here, we show that loss of hif3a in zebrafish reduced hypoxia tolerance. Further assays indicated that erythrocyte number was decreased because red blood cell maturation was impeded by hif3a disruption. We found that gata1 expression was downregulated in hif3a null zebrafish, as were several hematopoietic marker genes, including alas2, band3, hbae1, hbae3 and hbbe1. Hif3α recognized the hypoxia response element located in the promoter of gata1 and directly bound to the promoter to transactivate gata1 expression. Our results suggested that hif3a facilities hypoxia tolerance by modulating erythropoiesis via gata1 regulation.

2017 ◽  
Diego Borges-Rivera

Life continues to shock and amaze us, reminding us that truth is far stranger than fiction. http://Euplotid.io is a quantized, geometric model of the eukaryotic cell, an attempt at quantifying the incredible complexity that gives rise to a living cell by beginning from the smallest unit, a quanta. Starting from the very bottom we are able to build the pieces which when hierarchically and combinatorially combined produce the emergent complex behavior that even a single celled organism can show. Euplotid is composed of a set of quantized geometric 3D building blocks and constantly evolving dockerized bioinformatic pipelines enabling a user to build and interact with the local regulatory architecture of every gene starting from DNA-interactions, chromatin accessibility, and RNA-sequencing. Reads are quantified using the latest computational tools and the results are normalized, quality-checked, and stored. The local regulatory architecture of each gene is built using a Louvain based graph partitioning algorithm parameterized by the chromatin extrusion model and CTCF-CTCF interactions. Cis-Regulatory Elements are defined using chromatin accessibility peaks which are mapped to Transcriptional Start Sites based on inclusion within the same neighborhood. Deep Neural Networks are trained in order to provide a statistical model mimicking transcription factor binding, giving the ability to identify all Transcription Factors within a given chromatin accessibility peak. By in-silico mutating and re-applying the neural network we are able to gauge the impact of a transition mutation on the binding of any transcription factor. The annotated output can be visualized in a variety of 1D, 2D, 3D and 4D ways overlaid with existing bodies of knowledge such as GWAS results or PDB structures. Once a particular CRE of interest has been identified a Base Editor mediated transition mutation can then be performed in a relevant model for further study.

eLife ◽  
2018 ◽  
Vol 7 ◽  
Sarah Clark ◽  
Janette B Myers ◽  
Ashleigh King ◽  
Radovan Fiala ◽  
Jiri Novacek ◽  

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.

2020 ◽  
Vol 21 (5) ◽  
pp. 1664 ◽  
Emmanuelle Havis ◽  
Delphine Duprez

Although the transcription factor EGR1 is known as NGF1-A, TIS8, Krox24, zif/268, and ZENK, it still has many fewer names than biological functions. A broad range of signals induce Egr1 gene expression via numerous regulatory elements identified in the Egr1 promoter. EGR1 is also the target of multiple post-translational modifications, which modulate EGR1 transcriptional activity. Despite the myriad regulators of Egr1 transcription and translation, and the numerous biological functions identified for EGR1, the literature reveals a recurring theme of EGR1 transcriptional activity in connective tissues, regulating genes related to the extracellular matrix. Egr1 is expressed in different connective tissues, such as tendon (a dense connective tissue), cartilage and bone (supportive connective tissues), and adipose tissue (a loose connective tissue). Egr1 is involved in the development, homeostasis, and healing processes of these tissues, mainly via the regulation of extracellular matrix. In addition, Egr1 is often involved in the abnormal production of extracellular matrix in fibrotic conditions, and Egr1 deletion is seen as a target for therapeutic strategies to fight fibrotic conditions. This generic EGR1 function in matrix regulation has little-explored implications but is potentially important for tendon repair.

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 436-436 ◽  
Christopher J. Ott ◽  
Alexander J. Federation ◽  
Siddha Kasar ◽  
Josephine L. Klitgaard ◽  
Stacey M. Fernandes ◽  

Abstract Genome sequencing efforts of chronic lymphocytic leukemia have revealed mutations that disrupt protein-coding elements of the genome (Puente et al, 2011; Wang et al, 2011; Landau et al, 2013). Recently, comprehensive whole-genome sequencing efforts have begun to reveal the genetic aberrations that occur outside of protein-coding exons, many that may perturb gene regulatory sites (Puente et al, 2015). These include enhancer elements that make physical contact with gene promoters to regulate gene expression in a cell-type specific manner. While mutations certainly promote CLL leukemogenesis, epigenomic alterations may also play an important role in facilitating disease progression and maintenance by inducing the gene expression aberrations that have long been observed in CLL. Epigenomic alterations include chromatin structure changes that facilitate altered transcription and chromatin factor recruitment to regulatory elements. While comprehensive genome-wide DNA methylation studies have been performed on human cancers and normal cell counterparts including CLL, other comprehensive studies of cancer epigenomes have been lacking. We have completed an analysis of chromatin structures in a cohort of primary chronic lymphocytic leukemia (CLL) samples with comparisons to normal CD19+ B lymphocytes (n = 18 CLL samples, n = 5 normal B lymphocyte samples). We used chromatin accessibility assays (ATAC-seq) and genome-wide enhancer mapping (H3K27ac ChIP-seq) to comprehensively define the transcriptionally active chromatin landscape of CLL. We have discovered greater than 15,000 novel regulatory elements when compared to previously annotated regulatory elements. Moreover, sites within the loci of several hundred genes were found to have large regions of gained chromatin accessibility and H3K27 acetylation, revealing the appearance of aberrant enhancer activity. These gained enhancer elements correspond with increased gene expression and are found at gene loci such as LEF1, PLCG1, CTLA4, and ITGB1. We have also systematically identified the super-enhancers of CLL - large complex regulatory regions that possess unique tissue-specific regulatory capabilities. Many of these super-enhancers are found in normal B lymphocytes, yet the super-enhancer at the ITGB1 and LEF1 loci are CLL-specific and may be considered to facilitate leukemia-specific expression. We have found CLL-specific enhancers are also significantly associated with annotated CLL risk variants, and have identified enhancer-associated SNPs found within CLL-risk loci predicted to disrupt transcription factor binding sites. These include SNPs at the IRF8 and LEF1 locithat lead to the creation and destruction of SMAD4 and RXRA binding sites, respectively. Additionally, we have analyzed whole-genome sequencing data from a subset of our sample cohort. Mutational hotspots in the CXCR4 and BACH2 promoters occur within open, acetylated regions. Moreover, we discover recurrent mutations in enhancers of the ETS1 and ST6GAL1 locus that have not been previously annotated. Using a transcription factor network modeling approach, we used these global chromatin structure characteristics to determine networks that are highly active in CLL. We find that transcription factors such as NFATc1, E2F5, and NR3C2 are among the most interconnected transcription factors of the CLL genome, and their connectivity is significantly higher in CLL cells compared to normal B cells. In contrast, network profiling of CLL cells predicts loss of MXI1 connectivity, a negative regulator of the MYC oncogene. By treating cells with specific pharmacological inhibitors of NFAT family members including cyclosporin and FK506, we are able to reduce NFAT-mediated network connectivity, resulting in a selective loss of NFAT-bound enhancers. This leads to CLL cell death in vitro of both cell lines and primary CLL patient samples. Our results reveal the unique chromatin structure landscape of CLL for the first time, and identify the CLL-specific enhancer elements that confer the transcriptional dysregulation that has long been observed in this disease. Use of these chromatin structure analyses and enhancer landscapes has allowed us to construct the intrinsic transcription factor network of CLL, and determine a particular dependency on NFAT signaling for cell survival. Disclosures No relevant conflicts of interest to declare.

2021 ◽  
Vol 12 (11) ◽  
Ren-Dong Hu ◽  
Wen Zhang ◽  
Liang Li ◽  
Zu-Qi Zuo ◽  
Min Ma ◽  

AbstractActivation of adipose tissue macrophages (ATMs) contributes to chronic inflammation and insulin resistance in obesity. However, the transcriptional regulatory machinery involved in ATM activation during the development of obesity is not fully understood. Here, we profiled the chromatin accessibility of blood monocytes and ATMs from obese and lean mice using assay for transposase-accessible chromatin sequencing (ATAC-seq). We found that monocytes and ATMs from obese and lean mice exhibited distinct chromatin accessibility status. There are distinct regulatory elements that are specifically associated with monocyte or ATM activation in obesity. We also discovered several transcription factors that may regulate monocyte and ATM activation in obese mice, specifically a predicted transcription factor named ETS translocation variant 5 (ETV5). The expression of ETV5 was significantly decreased in ATMs from obese mice and its downregulation was mediated by palmitate stimulation. The decrease in ETV5 expression resulted in macrophage activation. Our results also indicate that ETV5 suppresses endoplasmic reticulum (ER) stress and Il6 expression in macrophages. Our work delineates the changes in chromatin accessibility in monocytes and ATMs during obesity, and identifies ETV5 as a critical transcription factor suppressing ATM activation, suggesting its potential use as a therapeutic target in obesity-related chronic inflammation.

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1860
Marcel da Câmara Ribeiro-Dantas ◽  
Danilo Oliveira Imparato ◽  
Matheus Gibeke Siqueira Dalmolin ◽  
Caroline Brunetto de Farias ◽  
André Tesainer Brunetto ◽  

Ewing Sarcoma (ES) is a rare malignant tumor occurring most frequently in adolescents and young adults. The ES hallmark is a chromosomal translocation between the chromosomes 11 and 22 that results in an aberrant transcription factor (TF) through the fusion of genes from the FET and ETS families, commonly EWSR1 and FLI1. The regulatory mechanisms behind the ES transcriptional alterations remain poorly understood. Here, we reconstruct the ES regulatory network using public available transcriptional data. Seven TFs were identified as potential MRs and clustered into two groups: one composed by PAX7 and RUNX3, and another composed by ARNT2, CREB3L1, GLI3, MEF2C, and PBX3. The MRs within each cluster act as reciprocal agonists regarding the regulation of shared genes, regulon activity, and implications in clinical outcome, while the clusters counteract each other. The regulons of all the seven MRs were differentially methylated. PAX7 and RUNX3 regulon activity were associated with good prognosis while ARNT2, CREB3L1, GLI3, and PBX3 were associated with bad prognosis. PAX7 and RUNX3 appear as highly expressed in ES biopsies and ES cell lines. This work contributes to the understanding of the ES regulome, identifying candidate MRs, analyzing their methilome and pointing to potential prognostic factors.

2019 ◽  
Vol 20 (1) ◽  
Priscillia Lhoumaud ◽  
Gunjan Sethia ◽  
Franco Izzo ◽  
Theodore Sakellaropoulos ◽  
Valentina Snetkova ◽  

AbstractActivation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

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