GATA Factor Mechanisms and Globin Gene Regulation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-18-sci-18
Author(s):  
Emery H. Bresnick ◽  
Shin-Il Kim ◽  
Scott J. Bultman ◽  
Sherry Lee ◽  
Meghan E. Boyer ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Activation Mechanism ◽  
Globin Genes ◽  
Gata Factor ◽  
Pol Ii ◽  
Hypomorphic Mutation ◽  
Gata Factors ◽  
Promoter Complex ◽  
Full Complement ◽  
The Relationship

Abstract Key steps in hematopoiesis and the expression of genes encoding hemoglobin subunits are critically dependent upon specific members of the GATA factor family of transcription factors. Our recent efforts have focused on elucidating how GATA factors select functional sites in chromatin and how they function combinatorially with additional regulatory factors. GATA motifs are often arranged in close proximity to E-boxes, and such composite elements commonly mediate GATA factor- and Scl/TAL1-dependent transcriptional responses. Only a small fraction of these composite elements in chromatin are occupied by GATA factors and Scl/TAL1, and a specific epigenetic signature distinguishes occupied versus unoccupied elements genome-wide. In the context of hemoglobin synthesis, we are using genetic and molecular approaches to dissect the multistep mechanism underlying the control of β-globin transcription. GATA-1-containing complexes assemble at the β-globin Locus Control Region (LCR) prior to the murine adult βmajor promoter. Though the LCR physically interacts with the βmajor promoter, this interaction is not required for the binding of several trans-acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems. promoter. Though the LCR physically interacts with the β promoter, this interaction is not required for the binding of several -acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems.

Dissecting a Multi-Step Mechanism of Beta-Globin Transcriptional Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1207-1207
Author(s):  
Emery H. Bresnick ◽  
Hogune Im ◽  
Kirby D. Johnson ◽  
Jeffrey A. Grass
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Chromatin Modification ◽  
Early Event ◽  
Globin Genes ◽  
Beta Globin ◽  
Gata Factor ◽  
Pol Ii ◽  
Gata Factors

Abstract Defining factors and signals that establish and maintain the native nucleoprotein structure of endogenous chromatin domains represents a powerful approach for elucidating transcriptional mechanisms. In adult erythroid cells, the locus control region (LCR) and the adult beta-globin genes of the murine beta-globin locus are highly enriched in acetylated histones H3 and H4 (acH3, acH4) and H3 methylated at lysine 4 (H3-meK4). By contrast, the embryonic beta-globin genes reside in a broad region of reduced acetylation. Histone H3 methylated at lysine 79 (H3-meK79) is highly enriched at the adult beta-globin genes, but not at the LCR. To elucidate the molecular steps in beta-globin transcriptional activation, genetic complementation experiments were conducted in GATA-1-null G1E cells containing an estrogen receptor hormone binding domain-GATA-1 fusion protein (ER-GATA-1). Kinetic analysis of ER-GATA-1 occupancy of chromatin and establishment of the histone modification pattern by chromatin immunoprecipitation (ChIP) revealed that GATA-1 occupies multiple regions within the LCR prior to the beta-major promoter. Chromatin accessibility at the promoter was low until ER-GATA-1 assembled into regulatory complexes at the LCR. Subsequently, ER-GATA-1 accessed the beta-major promoter, induced acH3, RNA polymerase II (Pol II) recruitment, and elevated H3-meK79. Acquisition of transcriptional competence appears to require establishment of H3-meK4, which is GATA-1-independent. Blocking transcriptional elongation did not erase H3-meK79, indicating that maintenance of H3-meK79 does not require ongoing elongation. Analysis of N-terminal GATA-1 deletion mutants that retain Friend of GATA-1 (FOG-1) binding and DNA binding activities revealed that FOG-1 binding and DNA binding activities are insufficient for Pol II recruitment and chromatin modification at the promoter. These results support a model in which ER-GATA-1 binding to the LCR increases acH3 at the promoter as an early event in transcriptional activation, which is tightly coupled to ER-GATA-1 access to the promoter, increased promoter accessibility, and Pol II recruitment. Increased promoter accessibility, which likely permits ER-GATA-1 access to the promoter, precedes maximal induction of H3-meK79, a late event in activation. Given the dynamic regulation of H3-meK79 by GATA-1 and NF-E2 and the modulation of H3-meK79 levels during erythropoiesis, we propose that H3-meK79 is a crucial signal that controls the rate of beta-globin transcription. Studies are underway to test this hypothesis and to dissect mechanisms underlying the requirement of N-terminal sequences of GATA-1 for Pol II recruitment and chromatin modification. Furthermore, having identified individual steps in transcriptional activation of the endogenous beta-globin genes, we are testing whether inducers of human fetal hemoglobin affect these specific steps. GATA-1 has been reported by the Crispino group to be expressed as an N-terminally truncated species in megakaryoblastic leukemia. Defining how the N-terminus functions should therefore lead to a molecular understanding of this disorder. As the N-termini of GATA factors differ considerably, one might expect these divergent sequences to establish GATA factor-specific functions, and this prediction is being tested via detailed analysis of the activities of chimeric GATA factors.

scholarly journals Dissecting Molecular Steps in Chromatin Domain Activation during Hematopoietic Differentiation

2007 ◽  
Vol 27 (12) ◽  
pp. 4551-4565 ◽  
Author(s):  
Shin-Il Kim ◽  
Scott J. Bultman ◽  
Huie Jing ◽  
Gerd A. Blobel ◽  
Emery H. Bresnick
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Chromatin Domain ◽  
Loop Formation ◽  
Pol Ii ◽  
Gata Factors ◽  
Hypomorphic Allele ◽  
Promoter Complex ◽  
Complex Locus ◽  
Carboxy Terminal

ABSTRACT GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis with GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the β-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal locus control region (LCR), revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the carboxy-terminal domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation.

Manipulating Higher Order Chromatin Structure of the β-Globin Locus by Targeted Tethering of a “looping” Factor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 647-647
Author(s):  
Wulan Deng ◽  
Philip D Gregory ◽  
Andreas Reik ◽  
Gerd Blobel
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Conflicts Of Interest ◽  
Chromatin Loop ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Loop Formation ◽  
Pol Ii ◽  
Globin Promoter

Abstract Abstract 647 The mammalian β-globin locus is under the coordinated control of multiple transcription factors to ensure the correct expression of the globin genes during development. The distal β-globin locus control region (LCR) physically interacts with β-like globin promoters to form developmentally dynamic chromatin loops. The hematopoietic transcription factor GATA-1 and its associated cofactor Ldb1 bind to the LCR and the β-globin promoter and are essential for loop formation and β-globin expression. However, the molecular basis of chromatin looping and its cause-effect relationship with transcriptional activation are unclear. Here, we examined whether Ldb1 is an effector of GATA-1 during loop formation. Specifically, we tested whether artificial tethering of Ldb1 to the endogenous β-globin promoter and LCR can substitute for GATA-1 function. Ldb1 was fused to artificial zinc finger proteins (ZFP) designed to bind to the LCR and β-globin promoter. Ldb1-ZFPs were introduced pairwise into murine GATA-1 null erythroid cells in which the β-globin locus is relaxed and transcriptionally silent. In vivo binding of the Ldb1-ZFPs to their targets was verified by ChIP assay. Strikingly, expression of Ldb1-ZFPs but not Ldb1 or ZFPs alone led to substantial activation of β-globin transcription in the absence of GATA-1. Moreover, chromosome conformation capture experiments showed that Ldb1-ZFPs triggered physical association between the LCR and the β-globin promoter. Recruitment of RNA polymerase II (Pol II) and its phosphorylation at serine 5 are critical LCR-dependent regulatory steps in β-globin transcription. We found that Ldb1-ZFP expression facilitated Pol II recruitment at the β-globin promoter and serine 5 phosphorylation to the same level as GATA-1-expressing erythroid cells. This is consistent with an Ldb1-ZFP-induced LCR-β-globin promoter chromatin loop. In concert, these results indicate that Ldb1 is a critical effector for GATA-1 by mediating enhancer-promoter loops. In broader terms, our results suggest that chromatin loop formation can be sufficient for gene activation in the absence of an essential transcription factor. We are currently in the process of examining whether targeting of the LCR to embryonic and fetal globin genes can be used to activate them in adult cells. Targeted chromatin loop formation may provide a method to activate fetal or adult hemoglobin expression in individuals with β-thalassemia or sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation

2014 ◽  
Vol 25 (6) ◽  
pp. 904-915 ◽  
Author(s):  
Pierangela Sabbattini ◽  
Marcela Sjoberg ◽  
Svetlana Nikic ◽  
Alberto Frangini ◽  
Per-Henrik Holmqvist ◽  
...  
Keyword(s):  
Stem Cells ◽  
Rna Polymerase Ii ◽  
Embryonic Stem ◽  
Epigenetic Silencing ◽  
Aurora B ◽  
Developmentally Regulated ◽  
Pol Ii ◽  
Differentiated Cells ◽  
Silent Genes ◽  
The Relationship

Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

scholarly journals Transcription-dependent spreading of canonical yeast GATA factor across the body of highly expressed genes

2017 ◽  
Author(s):  
Aria Ronsmans ◽  
Maxime Wery ◽  
Camille Gautier ◽  
Marc Descrimes ◽  
Evelyne Dubois ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Cancer Progression ◽  
Transcription Initiation ◽  
Environmental Changes ◽  
The Body ◽  
Large Set ◽  
Gata Factor ◽  
Gata Factors ◽  
Gata Transcription Factors

AbstractGATA transcription factors are highly conserved among eukaryotes and play roles in transcription of genes implicated in cancer progression and hematopoiesis. However, although their consensus binding sites have been well definedin vitro, thein vivoselectivity for recognition by GATA factors remains poorly characterized. Using ChIP-Seq, we identified the Dal80 GATA factor targets in yeast. Our data reveal Dal80 binding to a large set of promoters, sometimes independently of GATA sites. Strikingly, Dal80 was also detected across the body of promoter-bound genes, correlating with high, Dal80-sensitive expression. Mechanistic single-gene experiments showed that Dal80 spreading across gene bodies is independent of intragenic GATA sites but requires transcription elongation. Consistently, Dal80 co-purified with the post-initiation form of RNA Polymerase II. Our work suggests that GATA factors could play dual, synergistic roles during transcription initiation and post-initiation steps, promoting efficient remodeling of the gene expression program in response to environmental changes.Author SummaryGATA transcription factors are highly conserved among eukaryotes and play key roles in cancer progression and hematopoiesis. In budding yeast, four GATA transcription factors are involved in the response to the quality of nitrogen supply. We have determined the whole genome binding profile of one of them, Dal80, and revealed that it also binds across the body or promoter-bound genes. Our observation that ORF binding correlated with elevated transcription levels and exquisite Dal80 sensitivity suggests that GATA factors could play other, unexpected roles at post-initiation stages in eukaryotes.

scholarly journals Promoter G-quadruplex folding precedes transcription and is controlled by chromatin

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jiazhen Shen ◽  
Dhaval Varshney ◽  
Angela Simeone ◽  
Xiaoyun Zhang ◽  
Santosh Adhikari ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Accessibility ◽  
Pol Ii ◽  
Chromatin Compaction ◽  
Transcriptional Inhibition ◽  
G Quadruplex ◽  
Active Transcription ◽  
Pre Treatment ◽  
Dna Secondary Structures ◽  
The Relationship

Abstract Background Four-stranded G-quadruplexes (G4s) are DNA secondary structures in the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of promoter G4s, transcription and chromatin state. Results Transcriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed by G4 ChIP-seq, does not depend on transcriptional activity. We then show that chromatin compaction can lead to loss of promoter G4s and is accompanied by a corresponding loss of RNA polymerase II (Pol II), thus establishing a link between G4 formation and chromatin accessibility. Furthermore, pre-treatment of cells with a G4-stabilising ligand mitigates the loss of Pol II at promoters induced by chromatin compaction. Conclusions Overall, our findings show that G4 folding is coupled to the establishment of accessible chromatin and does not require active transcription.

scholarly journals Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex

2021 ◽  
Vol 118 (3) ◽  
pp. e2019163118
Author(s):  
Yihu Xie ◽  
Christopher L. Lord ◽  
Bradley P. Clarke ◽  
Austin L. Ivey ◽  
Pate S. Hill ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Regulatory Mechanism ◽  
Critical Role ◽  
Activation Mechanism ◽  
Cyclin Dependent Kinase ◽  
Active Conformation ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Cdk Regulation ◽  
Ctd Kinase I

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

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