Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Jauch, Ralf; Ng, Calista Keow Leng; Saikatendu, Kumar Singh; Stevens, Raymond C.; Kolatkar, Prasanna R.

doi:10.1016/j.jmb.2007.11.091

Cited by 79 publications

(96 citation statements)

References 56 publications

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“…There might be some skewing, as two purines at the 5 0 of CCAAT, which are usually well represented in NF-Y sites [11,38] and required for efficient in vitro binding [38] are not enriched, unlike two/three nucleotides at the 3 0 end, CA(G), which are clearly enriched. NANOG is an homeodomain TF shown to recognize the motif TAAT(G/T)(G/T) [39], which is one nucleotide-underligned-different from the core NF-Y consensus ATTGG: interestingly, in one of the ChIP-on-Chip location analysis reported, the NANOG motif was almost identical to an inverted CCAAT box [2]. The subtle difference in the NF-Y logo at NANOG sites might suggest that the latter binds immediately flanking, at the 5 0 or 3 0 of NF-Y, selecting for additional sequences that are only present in a subset of CCAAT sites.…”

Section: Mechanisms Of Nf-y Activity In Stem Cellsmentioning

confidence: 99%

“…It is unclear how the interplay occurs mechanistically, whether the two proteins interact physically or indirectly via other polypeptides. The availability of NANOG [39] and NF-Y crystal structures with DNA [M. Nardini, N. Gnesutta, G. Donati, M. Bolognesi, R. Mantovani, manuscript in preparation] could enable us to expand on these findings in the future.…”

Section: Mechanisms Of Nf-y Activity In Stem Cellsmentioning

confidence: 99%

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The Short Isoform of NF‐YA Belongs to the Embryonic Stem Cell Transcription Factor Circuitry

et al. 2012

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Totipotency of embryonic stem cells (ESCs) is controlled at the transcriptional level by a handful of transcription factors (TFs) that promote stemness and prevent differentiation. One of the most enriched DNA elements in promoters and enhancers of genes specifically active in ESCs is the CCAAT box, which is recognized by NF-Y, a trimer with histone-like subunits-NF-YB/NF-YC-and the sequence-specific NF-YA. We show that the levels of the short NF-YA isoform-NF-YAs-is high in mouse ESCs (mESCs) and drops after differentiation; a dominant negative mutant affects expression of important stem cells genes, directly and indirectly. Protein transfections of TAT-NF-YAs stimulate growth and compensate for withdrawal of leukemia inhibitory factor (LIF) in cell cultures. Bioinformatic analysis identifies NF-Y sites as highly enriched in genomic loci of stem TFs in ESCs. Specifically, 30%-50% of NANOG peaks have NF-Y sites and indeed NF-Y-binding is required for NANOG association to DNA. These data indicate that NF-Y belongs to the restricted circle of TFs that govern mESCs, and, specifically, that NF-YAs is the active isoform in these cells.

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Section: Mechanisms Of Nf-y Activity In Stem Cellsmentioning

confidence: 99%

Section: Mechanisms Of Nf-y Activity In Stem Cellsmentioning

confidence: 99%

The Short Isoform of NF‐YA Belongs to the Embryonic Stem Cell Transcription Factor Circuitry

et al. 2012

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“…Comparison of the crystal structure of our hNANOG HD in complex with DNA with the crystal structure of the apo-mNANOG HD shows that DNA binding does not result in any significant conformational changes in NANOG HD. Both hNANOG HD and mNANOG HD are structurally similar to other HDs with and without bound DNA (26). For example, the RMSDs between hNANOG HD and octamer-binding transcription factor 1 or ventral nervous system defective HDs are 0.88 Å and 0.94 Å, respectively.…”

Section: Resultsmentioning

confidence: 88%

“…Overall, the structure matches the canonical HD-type fold consisting of three α-helices, with H1 and H2 being antiparallel. Helices H1 and H2 position the longest helix H3 in a manner that promotes its insertion into the major groove of the DNA (26). Helices H1 and H2 interact with the DNA minor/major groove, which is half of the duplex DNA, whereas H3 has some interactions with the DNA backbone and helix H1.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, NANOG HD is distinct from other HD protein families and the amino acid similarity to the closely related NK-2 gene family is less than 50% (25). Although a previous study describing the structure of mNANOG HD provided hints into residues that may be involved in NANOG-DNA interactions (26), the exact mapping of NANOG-DNA interactions leading to unique functions of NANOG in self-renewal and reprogramming of PSCs remained unknown.…”

Section: Significancementioning

confidence: 98%

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Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

Hayashi

Caboni

Das

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering. Youth) is a key transcription factor regulating pluripotency in mammalian early embryos and pluripotent stem cells. Cooperating with other master regulators of pluripotency, NANOG plays a central role in pluripotency (1-3) and forms autoregulatory loops to maintain ES cell (ESC) identity (4-7). NANOG was initially identified from its ability to confer mouse (m)ESC selfrenewal without dependence on leukemia inhibitory factor (LIF) when overexpressed in mESCs (8, 9). Disruption of the NANOG gene in mESCs compromises their pluripotency (8); however, mESCs can maintain their self-renewal without NANOG (10). NANOG expression marks fully reprogrammed induced pluripotent stem cells (iPSCs) during reprogramming mammalian somatic cells into a pluripotent state (11); however, NANOG is dispensable for generating iPSCs from somatic cells both exogenously (12) and endogenously (13,14). In contrast, NANOG robustly promotes reprogramming of epiblast stem cells (EpiSCs) (15,16) or human iPSCs (HiPSCs), which have primed state pluripotency with distinct gene expression patterns and cell signaling dependence, into ground-state pluripotency (17)(18)(19)(20).NANOG is composed of 305 amino acids, including a central homeodomain (HD). HDs are ∼60 amino acid DNA-bin...

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Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self‐renewal

et al. 2020

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The self-renewal efficiency of mouse embryonic stem cells (ESCs) is determined by the concentration of the transcription factor NANOG. While NANOG binds thousands of sites in chromatin, the regulatory systems that control DNA binding are poorly characterised. Here, we show that NANOG is phosphorylated by casein kinase I, and identify target residues. Phosphomimetic substitutions at phosphorylation sites within the homeodomain (S130 and S131) have site-specific functional effects. Phosphomimetic substitution of S130 abolishes DNA binding by NANOG and eliminates LIF-independent self-renewal. In contrast, phosphomimetic substitution of S131 enhances LIFindependent self-renewal, without influencing DNA binding. Modelling the DNA-homeodomain complex explains the disparate effects of these phosphomimetic substitutions. These results indicate how phosphorylation may influence NANOG homeodomain interactions that underpin ESC self-renewal.

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Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

Cited by 79 publications

References 56 publications

The Short Isoform of NF‐YA Belongs to the Embryonic Stem Cell Transcription Factor Circuitry

The Short Isoform of NF‐YA Belongs to the Embryonic Stem Cell Transcription Factor Circuitry

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

Phosphorylation of NANOG by casein kinase I regulates embryonic stem cell self‐renewal

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