Activity Of Multiple Transcription Factors Research Articles

Plant enhancers exhibit both cooperative and additive interactions among their functional elements.

Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unclear how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize three light-responsive plant enhancers-AB80, Cab-1, and rbcS-E9-derived from genes associated with photosynthesis. Saturation mutagenesis revealed mutations, many of which clustered in short regions, that strongly reduced enhancer activity in the light, in the dark or in both conditions. When tested in the light, these mutation-sensitive regions did not function on their own; rather, cooperative interactions with other such regions were required for full activity. Epistatic interactions occurred between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affected enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions acted independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

FOX transcription factors are common regulators of Wnt/β-catenin–dependent gene transcription

The Wnt/β-catenin pathway is a critical regulator of development and stem cell maintenance. Mounting evidence suggests that the outcome of Wnt signaling is determined by the collaborative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. However, the contribution of FOX transcription factors to Wnt signaling has not been investigated in a systematic manner. Here, we performed complementary screens of all 44 human FOX proteins to identify new Wnt pathway regulators. By combining β-catenin reporter assays with Wnt pathway-focused qPCR arrays and proximity proteomics of selected candidates, we determine that most FOX proteins are involved in the regulation of Wnt pathway activity. As proof-of-principle, we additionally characterize class D and I FOX transcription factors as physiologically relevant regulators of Wnt/β-catenin signaling. We conclude that FOX proteins are common regulators of Wnt/β-catenin-dependent gene transcription that may control Wnt pathway activity in a tissue-specific manner.

Effects of the Acetyltransferase p300 on Tumour Regulation from the Novel Perspective of Posttranslational Protein Modification.

p300 acts as a transcription coactivator and an acetyltransferase that plays an important role in tumourigenesis and progression. In previous studies, it has been confirmed that p300 is an important regulator in regulating the evolution of malignant tumours and it also has extensive functions. From the perspective of non-posttranslational modification, it has been proven that p300 can participate in regulating many pathophysiological processes, such as activating oncogene transcription, promoting tumour cell growth, inducing apoptosis, regulating immune function and affecting embryo development. In recent years, p300 has been found to act as an acetyltransferase that catalyses a variety of protein modification types, such as acetylation, propanylation, butyylation, 2-hydroxyisobutyration, and lactylation. Under the catalysis of this acetyltransferase, it plays its crucial tumourigenic driving role in many malignant tumours. Therefore, the function of p300 acetyltransferase has gradually become a research hotspot. From a posttranslational modification perspective, p300 is involved in the activation of multiple transcription factors and additional processes that promote malignant biological behaviours, such as tumour cell proliferation, migration, and invasion, as well as tumour cell apoptosis, drug resistance, and metabolism. Inhibitors of p300 have been developed and are expected to become novel anticancer drugs for several malignancies. We review the characteristics of the p300 protein and its functional role in tumour from the posttranslational modification perspective, as well as the current status of p300-related inhibitor research, with a view to gaining a comprehensive understanding of p300.

Nanoparticle-mediated CRISPR/dCas9a activation of multiple transcription factors to engineer insulin-producing cells.

Insulin may help to control blood glucose levels in diabetes; however, the long-term release of insulin is important for therapy. In this work, four guide RNAs (gRNA) for factors that promote specification and maturation of insulin-producing cells were synthesized: pancreatic and duodenal homeobox 1 (PDX1), protoendocrine factor (neurogenin 3, NGN3), NK6 homeobox 1 (NKX6.1), and musculoaponeurotic fibrosarcoma oncogene family A (MAFA). These gRNAs were used to form ribonucleoproteins (RNPs) with tracRNA and dCas9-VPR, and were then immobilized on magnetic peptide-imprinted chitosan nanoparticles, which enhanced transfection. The production and release of insulin from transfected cells were then measured using ELISA and staining with anti-insulin antibodies. The expression of the genes was evaluated using qRT-PCR; this was also used to investigate the cascade of additional transcriptional regulators. The magnitude and duration of insulin production were evaluated for single and repeated transfections (using different transfection schedules) to identify the most promising protocol.

HMGN2 represses gene transcription via interaction with transcription factors Lef-1 and Pitx2 during amelogenesis

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2–transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.

The BASP1 transcriptional corepressor modifies chromatin through lipid-dependent and lipid-independent mechanisms

SummaryThe transcriptional corepressor BASP1 requires N-terminal myristoylation for its activity and functions through interactions with nuclear lipids. Here we determine the role of BASP1 lipidation in histone modification and the modulation of chromatin accessibility. We find that the removal of the active histone modifications H3K9ac and H3K4me3 by BASP1 requires the N-terminal myristoylation of BASP1. In contrast, the placement of the repressive histone modification, H3K27me3, by BASP1 does not require BASP1 lipidation. RNA-seq and ATAC-seq analysis finds that BASP1 regulates the activity of multiple transcription factors and induces extensive changes in chromatin accessibility. We find that ∼50% of BASP1 target genes show lipidation-dependent chromatin compaction and transcriptional repression. Our results suggest that BASP1 elicits both lipid-dependent and lipid-independent functions in histone modification and transcriptional repression. In accordance with this, we find that the tumor suppressor activity of BASP1 is also partially dependent on its myristoylation.

HMGN2 regulates transcription factor activity through chromatin modifications and protein interactions, developmentally modulated by microRNA‐23

The chromatin‐associated high mobility group protein N2 (HMGN2) regulates transcription factor activity through both chromatin and protein interactions. Several homeodomain and HMG‐box domain transcription factors regulate development and differentiation and are modulated by protein interactions and epigenetic factors. We demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull‐down and co‐immunoprecipitation assays show HMGN2 directly interacts with Lef‐1 through its HMG‐box domain. Furthermore, electrophoretic mobility shift assays demonstrate that HMGN2 inhibits Lef‐1 DNA binding activity. Thus, HMGN2 inhibits the transcriptional activities of Lef‐1, Dlx2, FoxJ1 as well as Pitx2. Pitx2 and Lef‐1 activate the Hmgn2 promoter and HMGN2 expression inhibits their transcriptional activation. Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter demonstrating Hmgn2 association with open chromatin. Hmgn2 expression is developmentally regulated however, the post‐transcriptional mechanisms that regulate Hmgn2 expression remain unclear. We demonstrate that miR‐23a and miR‐23b directly target Hmgn2, promoting transcriptional activation of several gene promoters including the amelogenin promoter, regulated by Pitx2. Hmgn2 expression decreases correlating with increased miR‐23 expression in craniofacial tissues as the murine embryo develops. Analyses of Hmgn2 LacZ expression demonstrate that Hmgn2 expression is gradually decreased during embryonic development. Ablation of Hmgn2 in mice results in increased amelogenin expression due to increased Pitx2, Dlx2, Lef‐1 and FoxJ1 transcriptional activity. We demonstrate both post‐transcriptional regulation of Hmgn2 by miR‐23a/b and post‐translational gene expression by Hmgn2 protein interactions. HMGN2 appears to regulate tooth development through its interaction with multiple transcription factors.

Quantifying the regulatory role of individual transcription factors in Escherichia coli.

SUMMARYGene regulation often results from the action of multiple transcription factors (TFs) acting at a promoter, obscuring the individual regulatory effect of each TF on RNA polymerase (RNAP). Here we measure the fundamental regulatory interactions of TFs in E. coli by designing synthetic target genes that isolate individual TFs’ regulatory effects. Using a thermodynamic model, each TF’s regulatory interactions are decoupled from TF occupancy and interpreted as acting through (de)stabilization of RNAP and (de)acceleration of transcription initiation. We find that the contribution of each mechanism depends on TF identity and binding location; regulation immediately downstream of the promoter is insensitive to TF identity, but the same TFs regulate by distinct mechanisms upstream of the promoter. These two mechanisms are uncoupled and can act coherently, to reinforce the observed regulatory role (activation/repression), or incoherently, wherein the TF regulates two distinct steps with opposing effects.

Transcription factor enrichment analysis (TFEA) quantifies the activity of multiple transcription factors from a single experiment

Detecting changes in the activity of a transcription factor (TF) in response to a perturbation provides insights into the underlying cellular process. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects positional motif enrichment associated with changes in transcription observed in response to a perturbation. TFEA detects positional motif enrichment within a list of ranked regions of interest (ROIs), typically sites of RNA polymerase initiation inferred from regulatory data such as nascent transcription. Therefore, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent transcription (eg. PRO-Seq), CAGE, histone ChIP-Seq, and accessibility data (e.g., ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Mediator Subunit, Med14, Regulates Pre- and Post-Fertilization Developmental Events in <i>Arabidopsis</i>

In flowering plants, transitions from vegetative-to-reproductive and reproductive-to-seed stages are tightly regulated by coordinated activity of multiple transcription factors and cofactors. However, the mechanism of communication and coordination between these transcription regulators during ovule and seed development is not fully understood. This study reveals that the Mediator subunit MED14 integrates transcriptional signals from different transcription factors during pre-and post-fertilization developmental events in Arabidopsis. This is also the first study to demonstrate the importance of parental contribution of any Mediator subunit. We found that the promoter of AtMED14 is active in the auxin maxima regions of meristematic zones. We were unable to obtain any homozygous line of MED14 knock-out. The heterozygous mutants of med14 show defects in embryo sac, fertilization, and seed development. The genetic and cytological analyses revealed that the defective maternal sporophytic reproductive part of the med14+/- mutant is responsible for the fertilization defects. Some of the med14 embryos are arrested at globular stage whereas few others have uncontrolled cell division forming globular mass of cells. The auxin signaling essential for the ovule, embryo and seed development is compromised in med14 mutants. Further, MED14 is also important for seed coat development and its mucilage content as it interacts with different transcription regulators like ANT, WUS, LUG, MYB5, KNAT7 and LUH to regulate the expression of enzymes like MUM2 and MUM4 that are required for the synthesis of seed mucilage and its release, respectively. Overall, this study establishes MED14 as an important convergence point of transcriptional signals from various transcription factors required for development of gynoecium and seed in Arabidopsis.

Quantifying the Regulatory Role of Individual Transcription Factors in Escherichia coli

Gene regulation often results from the action of multiple transcription factors (TFs) acting at a promoter, with a net regulation that depends on both the direct interactions of TFs with RNA polymerase (RNAP) and the indirect interactions with each other. Here we measure the fundamental regulatory interactions of TFs in E. coli by designing synthetic target genes that isolate the individual TFs regulatory effect. Using a thermodynamic model, the direct regulatory impact of the TF on RNAP is decoupled from TF occupancy and interpreted as acting through two mechanisms: (de)stabilization of RNAP and (de)acceleration of transcription initiation. We nd the contributions of each mechanism depends on TF identity and binding location; for the set of TFs pro led, regulation immediately downstream of the promoter is insensitive to TF identity, yet these same TFs regulate by distinct mechanisms upstream of the promoter. Strikingly, we observe two fundamental regulatory paradigms with these two mechanisms acting coherently, to reinforce the observed regulatory role (activation or repression), or incoherently, where the TF regulates two distinct steps with opposing effect. This insight provides critical information on the scope of TF-RNAP regulation allowing for a stronger approach to characterize the endogenous regulatory function of TFs.

Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.

Gone Caving: Roles of the Transcriptional Regulators YAP and TAZ in Skeletal Development.

The development of the skeleton is controlled by cellular decisions determined by the coordinated activation of multiple transcription factors. Recent evidence suggests that the transcriptional regulator proteins, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), could have important roles in directing the activity of these transcriptional programs. However, in vitro evidence for the roles of YAP and TAZ in skeletal cells has been hopelessly contradictory. The goals of this review are to provide a cross-sectional view on the state of the field and to synthesize the available data toward a unified perspective. YAP and TAZ are regulated by diverse upstream signals and interact downstream with multiple transcription factors involved in skeletal development, positioning YAP and TAZ as important signal integration nodes in an hourglass-shaped signaling pathway. Here, we provide a survey of putative transcriptional co-effectors for YAP and TAZ in skeletal cells. Synthesizing the in vitro data, we conclude that TAZ is consistently pro-osteogenic in function, while YAP can exhibit either pro- or anti-osteogenic activity depending on cell type and context. Synthesizing the in vivo data, we conclude that YAP and TAZ combinatorially promote developmental bone formation, bone matrix homeostasis, and endochondral fracture repair by regulating a variety of transcriptional programs depending on developmental stage. Here, we discuss the current understanding of the roles of the transcriptional regulators YAP and TAZ in skeletal development, and provide recommendations for continued study of molecular mechanisms, mechanotransduction, and therapeutic implications for skeletal disease.

Bookmarking by Non-pioneer Transcription Factors during Liver Development Establishes Competence for Future Gene Activation

SummaryTranscription factor binding to enhancer and promoter regions critical for homeostatic adult gene activation is established during development. To understand how cell-specific gene expression patterns are generated, we study the developmental timing of association of two prominent hepatic transcription factors with gene regulatory regions. Most individual binding events display extraordinarily high temporal variations during liver development. Early and persistent binding is necessary, but not sufficient, for gene activation. Stable gene expression patterns are the result of combinatorial activity of multiple transcription factors, which mark regulatory regions long before activation and promote progressive broadening of active chromatin domains. Both temporally stable and dynamic, short-lived binding events contribute to the developmental maturation of active promoter configurations. The results reveal a developmental bookmarking function of master regulators and illuminate remarkable parallels between the principles employed for gene activation during development, during evolution, and upon mitotic exit.

SIX1 Transcription Factor Enhances Human Erythropoiesis Via GATA1

Erythropoiesis is orchestrated by the coordinated action of multiple transcription factors. The master erythropoietic regulator GATA1 is itself modulated via interactions with multiple co-regulatory factors, such as FOG1, KLF1, and LMO2. Though the PAX-SIX-EYA-DACH network (PSEDN) of conserved transcription factors has been well characterized in the formation of eyes, kidney, branchial structures, and skeletal muscles, a role for PSEDN members in hematopoietic systems has only recently been recognized (Liu et al., Nature 2019 PMID:30894749). Here, we studied the PSEDN member SIX1 and discovered its ability to drive erythroid differentiation of human hematopoietic cells. Enforced overexpression (OE) of SIX1 in human TF1 erythroleukemia cells or primary CD34+ hematopoietic stem-progenitor cells (HSPCs) stimulated the generation of erythroid cells, as determined by increased numbers of cells expressing erythroid-selective surface markers (CD235ahiCD71hiCD34-) and hemoglobin (HBB). Conversely, SIX1 knockout in TF1 cells or primary HSPCs reduced erythroid cell generation in response to erythropoietin (EPO). SIX1 OE could also stimulate TF1 cell erythroid differentiation in the absence of EPO. Further analysis of SIX1 OE in TF1 cells revealed that SIX1 stimulated the expression of multiple functionally important erythroid molecules including ALAS2, SLC4a1, EPOR, SPTA1, KLF1 and ANK1. By gene set enrichment analysis (GSEA) of global RNA-seq data, SIX1 OE stimulated heme metabolism genes as well as many genes known to be regulated by GATA1, including FOG1-dependent and -independent genes. SIX1 OE reduced GATA2 and increased GATA1 protein and RNA expression, resembling GATA switching downstream of EPO signaling. To determine whether GATA1 was necessary for SIX1 to stimulate erythropoiesis, we generated GATA1 knockout cells using CRISPR/Cas9 technology. In contrast to control cells, SIX1 OE in GATA1 knockout cells failed to stimulate erythropoiesis, indicating that SIX1 stimulation of erythropoiesis requires GATA1. To gain further insight into the mechanism by which SIX1 stimulates erythropoiesis, the promiscuous biotin ligase, BirA (Choi-Rhee et al., Protein Sci. 2004 PMID:15459338), was fused in-frame to SIX1 to determine the SIX1 proximal interactome. Streptavidin-enrichment of biotinylated proteins in SIX1-BirA OE lysates revealed GATA1 and FOG1 as proximal interactors of SIX1-BirA, but not of BirA alone. When co-expressed in HEK293T cells GATA1 and SIX1 were found to co-immunoprecipitate, suggesting the two proteins can physically interact in a complex. We demonstrated the functional consequence of the SIX1 interaction with GATA1 using a GATA1-dependent luciferase reporter gene harboring three copies of GATA binding sites. Cells in which SIX1 and GATA1 were co-expressed exhibited significantly higher levels of luciferase expression compared to cells expressing only GATA1, suggesting SIX1 could stimulate GATA1-dependent transcription. Introduction of mutations in SIX known to cause Branchio-Oto-Renal (BOR) (Ruf et al., PNAS 2004 PMID:15141091) syndrome did not inhibit the ability of SIX to bind GATA1 nor its ability to drive erythropoiesis. Taken together our results suggest that SIX1 can stimulate erythropoiesis via multiple mechanisms, including increased GATA1 expression and function. Our findings provide the first demonstration of a role for the PSEDN in erythropoiesis and reveal unknown physical and functional interactions between two central developmental transcriptional networks (GATA:FOG network and PSEDN). Disclosures No relevant conflicts of interest to declare.

Ubiquitous expressed transcript promotes tumorigenesis by acting as a positive modulator of the polycomb repressive complex 2 in clear cell renal cell carcinoma

BackgroundThe ubiquitous expressed transcript (UXT) plays a key role in various tumors by regulating transcriptional activity of multiple transcription factors, including androgen receptor (AR). However, the role of UXT in clear cell renal cell carcinoma (ccRCC) is still unknown.MethodsYeast two-hybrid screening, GST pull-down and co-immunoprecipitation assays were performed to detect the interacting protein of UXT. Chromatin immunoprecipitation (ChIP) was performed to investigate the levels of histone H3 lysine 27 trimethylation at the HOXA9 promoters. CCK-8 assays, colony formation assays and Transwell assays were performed to detect the proliferation, colony formation, migration and invasion of renal cancer cells. Quantitative PCR analysis was performed to detect the expressions of UXT in human ccRCC samples.ResultsThe enhancer of zeste homolog 2 (EZH2) is a novel UXT interacting protein and UXT interacts with EZH2 in the nucleus. In addition, UXT interacts with the polycomb repressive complex 2 (PRC2) through directly binding to EZH2 and suppressor of zeste 12 homolog (SUZ12), but not to embryonic ectoderm development (EED). Moreover, the UXT activates EZH2 histone methyltransferase activity by facilitating EZH2 binding with SUZ12. We further provided striking evidences that knockdown of UXT inhibits proliferation, colony formation, migration and invasion of renal cancer cells, in an EZH2-dependent manner. Importantly, the upregulation of UXT expression was observed in clinical ccRCC samples, and the high expression level of UXT was associated with advanced stage, distant metastasis and poor overall survival in patients with ccRCC.ConclusionThe UXT is a novel regulator of the PRC2 and acts as a renal cancer oncogene that affects the progression and survival of ccRCC patients.

Influence of Confounding Factors on Radiation Dose Estimation Using In Vivo Validated Transcriptional Biomarkers.

For triage purposes following a nuclear accident, blood-based gene expression biomarkers can provide rapid dose estimates for a large number of individuals. Ionizing-radiation-responsive genes are regulated through the DNA damage-response pathway, which includes activation of multiple transcription factors. Modulators of this pathway could potentially affect the response of these biomarkers and consequently compromise accurate dose estimation calculations. In the present study, four potential confounding factors were selected: cancer condition, sex, simulated bacterial infection (lipopolysaccharide), and curcumin, an anti-inflammatory/antioxidant agent. Their potential influence on the transcriptional response to radiation of the genes CCNG1 and PHPT1, two biomarkers of radiation exposure ex vivo, was assessed. First, both CCNG1 and PHPT1 were detected in vivo in blood samples from radiotherapy patients and as such were validated as biomarkers of exposure. Importantly, their basal expression level was slightly but significantly affected in vivo by patients' cancer condition. Moreover, lipopolysaccharide stimulation of blood irradiated ex vivo led to a significant modification of CCNG1 and PHPT1 transcriptional response in a dose- and time-dependent manner with opposite regulatory effects. Curcumin also affected CCNG1 and PHPT1 transcriptional response counteracting some of the radiation induction. No differences were observed based on sex. Dose estimations calculated using linear regression were affected by lipopolysaccharide and curcumin. In conclusion, several confounding factors tested in this study can indeed modulate the transcriptional response of CCNG1 and PHPT1 and consequently can affect radiation exposure dose estimations but not to a level which should prevent the biomarkers' use for triage purposes.

Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells.

Krüppel-like factors (Klfs) have a pivotal role in maintaining self-renewal of mouse embryonic stem cells (mESCs). The functions of three Klf family members (Klf2, Klf4 and Klf5) have been identified, and are suggested to largely overlap. For further dissection of their functions, we applied an inducible knockout system for these Klf family members and assessed the effects of combinatorial loss of function. As a result, we confirmed that any one of Klf2, Klf4 and Klf5 was sufficient to support self-renewal, whereas the removal of all three compromised it. The activity of any single transcription factor, except for a Klf family member, was not sufficient to restore self-renewal of triple-knockout mESCs. However, some particular combinations of transcription factors were capable of the restoration. The triple-knockout mESCs were successfully captured at primed state. These data indicate that the pivotal function of a Klf family member is transduced into the activation of multiple transcription factors in a naïve-state-specific manner.

Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7

The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255–1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix.