Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites.

Many different transcription factors (TFs) regulate gene expression in a combinatorial fashion, often by binding in close proximity to each other on composite cis-regulatory DNA elements. Here, we investigated how ETS TFs bind with the AP1 TFs JUN-FOS at composite DNA-binding sites. DNA-binding ability with JUN-FOS correlated with the phenotype of ETS proteins in prostate cancer. We found that the oncogenic ETS-related gene (ERG) and ETS variant (ETV) 1/4/5 subfamilies co-occupy ETS-AP1 sites with JUN-FOS in vitro, whereas JUN-FOS robustly inhibited DNA binding by the tumor suppressors ETS homologous factor (EHF) and SAM pointed domain-containing ETS TF (SPDEF). EHF bound ETS-AP1 DNA with tighter affinity than ERG in the absence of JUN-FOS, possibly enabling EHF to compete with ERG and JUN-FOS for binding to ETS-AP1 sites. Genome-wide mapping of EHF- and ERG-binding sites in prostate epithelial cells revealed that EHF is preferentially excluded from closely spaced ETS-AP1 DNA sequences. Structural modeling and mutational analyses indicated that adjacent positively charged surfaces from EHF and JUN-FOS use electrostatic repulsion to disfavor simultaneous DNA binding. Conservation of positive residues on the JUN-FOS interface identified E74-like ETS TF 1 (ELF1) as an additional ETS TF exhibiting anticooperative DNA binding with JUN-FOS, and we found that ELF1 is frequently down-regulated in prostate cancer. In summary, divergent electrostatic features of ETS TFs at their JUN-FOS interface enable distinct binding events at ETS-AP1 DNA sites, which may drive specific targeting of ETS TFs to facilitate distinct transcriptional programs.

ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain.

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. High-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-motifs, which bind JUN and FOS transcription factor families, were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor.

The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNA-recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.

Steric mechanism of auto-inhibitory regulation of specific and non-specific DNA binding by the ETS transcriptional repressor ETV6.

DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus (5')GGAA(3') recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing (5')GGAA(3')versus(5')GGAT(3'). These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

Autoinhibition of ETV6 (TEL) DNA binding: appended helices sterically block the ETS domain.

ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS domain. The C-terminal inhibitory domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and (15)N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors.