scholarly journals High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ludovica Vanzan ◽  
Hadrien Soldati ◽  
Victor Ythier ◽  
Santosh Anand ◽  
Simon M. G. Braun ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Binding Sites ◽  
High Throughput Screening ◽  
Methylation Status ◽  
Chromatin Accessibility ◽  
Dna Demethylation ◽  
Active Dna Demethylation ◽  
Nucleosomal Dna ◽  
Select Group

AbstractBinding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.

scholarly journals High throughput screening identifies SOX2 as a Super Pioneer Factor that inhibits DNA methylation maintenance at its binding sites

2020 ◽  
Author(s):  
Ludovica Vanzan ◽  
Hadrien Soldati ◽  
Victor Ythier ◽  
Santosh Anand ◽  
Nicole Francis ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Binding Sites ◽  
High Throughput Screening ◽  
Dna Demethylation ◽  
Active Dna Demethylation ◽  
Nucleosomal Dna ◽  
Select Group ◽  
Pioneer Factor ◽  
Set Up

AbstractAccess of mammalian transcription factors (TFs) to regulatory regions, an essential event for transcription regulation, is hindered by chromatin compaction involving nucleosome wrapping, repressive histone modifications and DNA methylation. Moreover, methylation of TF binding sites (TBSs) affects TF binding affinity to these sites. Remarkably, a special class of TFs called pioneer transcription factors (PFs) can access nucleosomal DNA, leading to nucleosome remodelling and chromatin opening. However, whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown.Here, we set up a highly parallelized approach to investigate PF ability to bind methylated DNA and induce demethylation. Our results indicate that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs that have a strong pioneering activity; while most PFs do not induce changes in DNA methylation at their binding sites, we identified PFs that can protect DNA from methylation and PFs that can induce DNA demethylation at methylated binding sites. We called the latter “super pioneer transcription factors” (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Importantly, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation by inhibition of the maintenance methyltransferase DNMT1 during replication. This important finding suggests a novel mechanism allowing TFs to interfere with the epigenetic memory during DNA replication.

scholarly journals The epigenetic pioneer EGR2 initiates DNA demethylation in differentiating monocytes at both stable and transient binding sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Karina Mendes ◽  
Sandra Schmidhofer ◽  
Julia Minderjahn ◽  
Dagmar Glatz ◽  
Claudia Kiesewetter ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Binding Sites ◽  
Chromatin Accessibility ◽  
Dna Demethylation ◽  
Blood Monocytes ◽  
Early Growth Response ◽  
Gm Csf ◽  
Cell Functions ◽  
Active Dna Demethylation ◽  
Interferon Regulatory Factor 4

AbstractThe differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), is accompanied by the active turnover of DNA methylation, but the extent, consequences and mechanisms of DNA methylation changes remain unclear. Here, we profile and compare epigenetic landscapes during IL-4/GM-CSF-driven MO differentiation across the genome and detect several thousand regions that are actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We further identify TF that are globally associated with DNA demethylation processes. While interferon regulatory factor 4 (IRF4) is found to control hallmark dendritic cell functions with less impact on DNA methylation, early growth response 2 (EGR2) proves essential for MO differentiation as well as DNA methylation turnover at its binding sites. We also show that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites.

scholarly journals Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2

2019 ◽  
Vol 47 (17) ◽  
pp. 9069-9086 ◽  
Author(s):  
Filippo M Cernilogar ◽  
Stefan Hasenöder ◽  
Zeyang Wang ◽  
Katharina Scheibner ◽  
Ingo Burtscher ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Specific Binding ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Specific Binding Sites ◽  
Nucleosomal Dna ◽  
Cell Type Specific ◽  
Chromatin Opening ◽  
Selection Of

Abstract Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

scholarly journals Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2

2019 ◽  
Author(s):  
Filippo M. Cernilogar ◽  
Stefan Hasenöder ◽  
Zeyang Wang ◽  
Katharina Scheibner ◽  
Ingo Burtscher ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Specific Binding ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Specific Binding Sites ◽  
Nucleosomal Dna ◽  
Cell Type Specific ◽  
Chromatin Opening ◽  
Selection Of

AbstractPioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions can they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

scholarly journals Azacitidine Blocks GATA-1-Mediated Repression of the PU.1 Gene in Human Leukemic Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5220-5220
Author(s):  
Pavel Burda ◽  
Jarmila Vargova ◽  
Nikola Curik ◽  
John Strouboulis ◽  
Giorgio Lucio Papadopoulos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Regulatory Element ◽  
Methylation Status ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Leukemic Cells ◽  
Epigenetic Therapy ◽  
Myeloid Differentiation

Abstract Introduction: GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. Expression of PU.1 is controlled by several transcription factors including PU.1 itself by binding to the distal URE enhancer (upstream regulatory element) whose deletion leads to acute myeloid leukemia (AML) (Rosenbauer F et al. 2004). Co-expression of PU.1 and GATA-1 in AML-erythroleukemia (EL) blasts prevents efficient differentiation regulated by these transcription factors. Inhibition of transcriptional activity of PU.1 protein by GATA-1 has been reported (Nerlov C et al. 2000), however it is not known whether GATA-1 can inhibit PU.1 gene in human early erythroblasts directly. We have recently found that MDS/AML erythroblasts display repressive histone modifications and DNA methylation status of PU.1 gene that respond to 5-azacitidine (AZA) leading to inhibited blast cell proliferation and stimulated myeloid differentiation (Curik N et al. 2012). We hypothesize that l eukemia blockade during early erythroid differentiation includes direct GATA-1-mediated inhibition of the PU.1 gene. Results: We herein document the GATA-1 mediated repression of the PU.1 gene in human EL cell lines (OCI-M2 and K562) together with the recruitment of DNA methyl transferase I (DNMT1) to the URE known to guide most of the PU.1 gene transcription. Repression of the PU.1 gene involves both DNA methylation at the URE and methylation/deacetylation of the histone H3 lysine-K9 residue and methylation of H3K27 at additional DNA elements and the PU.1 promoter. Inhibition of GATA-1 by siRNA as well as the AZA treatment in AML-EL led to the significant DNA-demethylation of the URE thorough the mechanism of DNMT1 depletion leading to upregulation of the PU.1 expression. Conclusions: Our data indicate that GATA-1 binds to the PU.1 gene at the URE and initiate events leading to the PU.1 gene repression in human ELs. The mechanism includes repressive epigenetic remodeling of the URE that is important for the PU.1 downregulation and leukemogenesis and that is also simultaneously sensitive to the DNA demethylation treatment with AZA. The GATA-1-mediated inhibition likely contributes to the PU.1 downregulation during progenitor cell differentiation that could be employed during leukemogenesis. Importantly, we also observed important differences between murine and human ELs and found that repression of the PU.1 gene in human ELs can become reverted by the epigenetic therapy with AZA. Our work also suggests that hypomethylating therapy using DNA methylation inhibitors in MDS/AML may become potentially effective in MDS/EL patients. We think that during early erythroid differentiation the GATA-1 binds and represses the PU.1 gene, however this is not fully completed in EL and therefore the erythroid as well as myeloid differentiation are blocked. Grants: GACR P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/1. Disclosures Off Label Use: Azacitidine, DNA demethylation agens tested in vitro in AML/MDS treatment. Stopka:Celgene: Research Funding.

Dynamic Changes Of DNA Methylation and a Functional Role For TET2 DNA Dioxygenase In Human Erythroid Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3415-3415
Author(s):  
Jie Li ◽  
Papoin Julien ◽  
Chao An ◽  
Jingpin Hu ◽  
Ari Melnick ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Functional Role ◽  
Methylation Status ◽  
Erythroid Differentiation ◽  
Dna Demethylation ◽  
Parent Cell ◽  
Hematopoietic Stem ◽  
Mature Erythrocyte ◽  
Terminal Erythroid Differentiation

Abstract Erythropoiesis is a process by which multipotent hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. This process contains eight distinct differentiation stages including burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and mature erythrocyte. Unlike most cell types, an important feature of erythropoiesis is that following each of the three or four mitoses that occur during terminal erythroid differentiation, the daughter cells are distinctly different from the parent cell from which they are derived. Thus, erythropoiesis is a complex process that requires tight regulation. The most extensively studied regulators of erythroid differentiation include the EPO/EPOR system and two major transcription factors, GATA1 and KLF1. In contrast to the well-established roles of growth factors, cytokines and transcription factors in regulating erythropoiesis, the regulation of erythropoiesis by other mechanisms is much less understood. In the present study, we explore the changes in DNA methylation during human terminal erythroid differentiation and DNA methylation/demethylation in human erythropoiesis. The methylation status of DNA influences many biologic processes. It has been recently reported that global demethylation occurs during both murine and human erythropoiesis. However, the dynamics of DNA methylation changes, the underlying molecular mechanism(s), and the function of DNA demethylation in erythropoiesis are not clear. To address these issues, we performed next-generation bisulfite sequencing on highly purified human erythroblasts at distinct differentiation stages. We show that while there is a global hypomethylation as terminal erythropoiesis proceeds, stage-specific analysis revealed that a significant proportion of differential methylation includes gains of methylation. Moreover, genes that presented with DNA methylation changes could be categorized into 3 groups based on the dynamics of their methylation changes. As Ten-eleven-translocation proteins (TETs) have been implicated in DNA demethylation by converting 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), we attempted to explore the role of TETs in DNA demethylation and terminal erythroid differentiation. We show that 5hmc is progressively increased during human terminal erythroid differentiation. Importantly, knockdown of TET2 by shRNA in human CD34+ cells impaired the production of 5hmc as well as terminal erythroid differentiation. Our findings demonstrate the complexity of DNA methylation dynamics and identify a functional role for TET2 in human erythroid differentiation. These findings provide new and novel insights into the mechanistic understanding of normal and disordered erythropoiesis. As aberrant DNA methylation underlies many hematological diseases including the dyserythropoiesis of myelodysplastic syndromes, we suggest that these finding also provide novel insights into these diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA Methylation Repels Binding of HIF Transcription Factors to Maintain Tumour Immunotolerance

2020 ◽  
Author(s):  
Flora D’anna ◽  
Laurien Van Dyck ◽  
Jieyi Xiong ◽  
Hui Zhao ◽  
Rebecca V. Berrens ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Immune Checkpoint ◽  
Binding Sites ◽  
Dna Demethylation ◽  
Transcript Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Dna Hypomethylation ◽  
Cell Type Specific

AbstractBackgroundHypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia.ResultsHere, we report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modelling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid-down by the differential expression and binding of other transcription factors under normoxia control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumours with high immune checkpoint expression, but not in tumours with low immune checkpoint expression, where they would compromise tumour immunotolerance. In a low-immunogenic tumour model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumour growth.ConclusionsOur data elucidate the mechanism underlying cell-type specific responses to hypoxia, and suggest DNA methylation and hypoxia to underlie tumour immunotolerance.

scholarly journals Coordinate Regulation of TET2 and EBNA2 Controls the DNA Methylation State of Latent Epstein-Barr Virus

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Fang Lu ◽  
Andreas Wiedmer ◽  
Kayla A. Martin ◽  
Priyankara J. M. S. Wickramasinghe ◽  
Andrew V. Kossenkov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Dna Demethylation ◽  
Methylation State ◽  
Type Iii ◽  
Barr Virus ◽  
Active Dna Demethylation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylation-associated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state. IMPORTANCE Epstein-Barr virus (EBV) latency and carcinogenesis involve the selective epigenetic modification of viral and cellular genes. Here, we show that TET2, a cellular tumor suppressor involved in active DNA demethylation, plays a central role in regulating the DNA methylation state during EBV latency. TET2 is coordinately regulated and functionally interacts with the viral oncogene EBNA2. TET2 and EBNA2 function cooperatively to demethylate genes important for EBV-driven B-cell growth transformation.

Transcription Factor CTCF Inhibits Effects of 5-Azacitidine in MDS/AML Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3848-3848
Author(s):  
Martina Kapalova ◽  
Pavel Burda ◽  
Karin Vargova ◽  
Filipp Savvulidi ◽  
Tomas Zikmund ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factor ◽  
High Risk ◽  
Binding Sites ◽  
Conflicts Of Interest ◽  
Methylation Status ◽  
Significant Proportion ◽  
Dna Demethylation ◽  
Ctcf Binding

Abstract Abstract 3848 Introduction: 5-azacitidine (AZA) represents very promising albeit not fully efficient therapy for int-2 and high risk MDS patients. Molecules that interfere with AZA therapy are not known. In significant proportion of MDS patients, PU.1 gene is methylated at −17-kb-located upstream regulatory element (URE) where several key transcription factors regulate PU.1 expression. PU.1 represents major factor that controls normal myeloid differentiation. Methylated URE in MDS progenitors can be efficiently demethylated by AZA leading to restoration of cell differentiation capacity (Curik et al 2012). PU.1 gene contains several binding sites for transcription factor CTCF. CTCF represents very important modulator of gene expression, whose binding to DNA can be prevented by DNA methylation. We herein asked if CTCF regulates PU.1 and if so, whether its association with PU.1 gene coincides with DNA methylation status of MDS blasts. Methods: Human high risk MDS patient CD34+ progenitors and MDS-derived erytroleukaemia OCI-M2 and murine erythroleukaemia cell (MEL) lines were studied by RT-PCR, immunoblotting, and chromatin immunoprecipitation (ChIP) assays. Manipulation of gene expression was done by transfection of cDNA or siRNA. Results: We herein show that CTCF binding sites at PU.1 gene similarly to URE are severely methylated in CD34+ progenitors from high risk MDS patients and MDS-derived erytroleukaemia cell line, and as expected, AZA induced their rapid demethylation. Methylated CTCF binding sites are not occupied by CTCF. However upon AZA-mediated demethylation, CTCF is recruited to the binding sites at PU.1 gene as determined by ChIP. Our other data provided evidence that CTCF interacts with the ISWI ATPse SNF2H (SMARCA5). Indeed, the recruitment of CTCF at PU.1 gene in MDS/AML cells was coincident with recruitment of its interacting partner SMARCA5. In addition, SMARCA5 facilitates CTCF binding to the DNA as demonstrated at ICR locus (near H19 and Igf2 genes) upon siRNA-mediated downregulation of SMARCA5. To understand role of CTCF-SMARCA5 recruitment to the PU.1 gene and its effects on PU.1 expression we upregulated CTCF expression by transfecting an expression plasmid encoding CTCF cDNA and observed that upon increasing CTCF levels the PU.1 protein level was downregulated. Conversely, downregulation of SMARCA5 by siRNA caused upregulation of PU.1 levels. These data indicated that PU.1 is negatively regulated by CTCF and SMARCA5. Furthermore, inhibitory effects of CTCF and SMARCA5 on PU.1 expression were also demonstrated in presence of AZA in MDS cells following DNA demethylation of PU.1 gene. Conclusion: Our results indicate that CTCF and SMARCA5 are cooperating inhibitory factors to downregulate PU.1 and that AZA-mediated demethylation facilitates the CTCF-SMARCA5 binding to PU.1 gene in MDS patients. CTCF and SMARCA5 are novel factors that interfere with positive prodifferentiation effects of AZA. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012–264507, P301/12/P380, GAUK 251070 45410 and 251135 82210). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

1998 ◽  
Vol 18 (11) ◽  
pp. 6293-6304 ◽  
Author(s):  
Vesco Mutskov ◽  
Delphine Gerber ◽  
Dimitri Angelov ◽  
Juan Ausio ◽  
Jerry Workman ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Molecular Biology ◽  
Binding Sites ◽  
Cross Linking ◽  
Uv Laser ◽  
Histone Tail ◽  
Histone Tails ◽  
Nucleosomal Dna ◽  
Core Histones

ABSTRACT In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

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