Preferential Binding of Human Full-Length XPA and the Minimal DNA Binding Domain (XPA-MF122) with the Mitomycin C−DNA Interstrand Cross-Link†

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

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Regulation of Cell Cycle Transcription Factor Swi4 through Auto-Inhibition of DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.6729 ◽

1999 ◽

Vol 19 (10) ◽

pp. 6729-6741 ◽

Cited By ~ 26

Author(s):

Kristin Baetz ◽

Brenda Andrews

Keyword(s):

Cell Cycle ◽

Transcription Factors ◽

Dna Binding ◽

Binding Domain ◽

Terminal Region ◽

Full Length ◽

Intramolecular Interactions ◽

Dna Binding Domain ◽

C Terminus ◽

Binding Factor

ABSTRACTInSaccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G1/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively. The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role. SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains. Swi4 binding to SCBs is restricted to the late M and G1phases, when Swi6 is localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle. This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system. We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6. However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA intrans. Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain. Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs. The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA.

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The TAL1 Short Isoform Generated By PRMT1-Mediated Alternative RNA Splicing Promotes Erythroid Differentiation

Blood ◽

10.1182/blood.v128.22.1249.1249 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1249-1249

Author(s):

Shuiling Jin ◽

Ngoc-Tung Tran ◽

Hairui Su ◽

Suming Huang ◽

Xinyang Zhao ◽

...

Keyword(s):

Dna Binding ◽

Rna Splicing ◽

Short Form ◽

Hematopoietic Cells ◽

Erythroid Differentiation ◽

Binding Domain ◽

Full Length ◽

Dna Binding Domain ◽

Transcriptional Regulatory ◽

Megakaryocyte Differentiation

Abstract The isoforms of key transcription factors in hematopoiesis such as TAL1, GATA1 and RUNX1 are generated through alternative RNA splicing regulated by the PRMT1-RBM15 axis (Zhang et al. 2015). The functions of short isoforms of GATA1 (GATA1s) and RUNX1 (RUNX1a) are well characterized, yet it is unknown how the short isoform of TAL1 (TAL1s) regulates hematopoiesis. In this presentation, we report that the short isoform of TAL1, i.e. TAL1s, is generated via alternative RNA splicing as detected by isoform specific real-time PCR reactions using RNA isolated from leukemia cell lines and primary human cord blood cells. RBM15, an RNA binding protein, which is involved in chromosome translocation to produce RBM15-MKL1 fusion protein in acute megakaryocytic leukemia, regulates the alternative RNA splicing of TAL1. RBM15 promotes the production of full-length TAL1 mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway favors the production of TAL1s. RBM15 directly binds to intronic regions on TAL1 pre-mRNA. Binding of RBM15 is responsible for recruiting SF3B1-associated RNA splicing complex. Given that PRMT1 senses the hypoxia status of hematopoietic cells, the changing of TAL1s/TAL1fl ratio by PRMT1 activity may be an adaptive response of hematopoietic cells to hypoxia status. The short form TAL1s still contains the helix-loop-helix DNA binding domain but not the N terminal regions upstream of the DNA binding domain. Thus, the TAL1s may act as a dominant negative mutant of the full-length TAL1fl to block TAL1fl-regulated transcription. We demonstrated that overexpression of TAL1s not the full-length TAL1promotes the erythroid differentiation of K562 cells. Although TAL1 gene is required for both erythroid and megakaryocyte differentiation at early stage of hematopoiesis, TAL1s does not promote megakaryocyte differentiation. Therefore, fine-tuning the TAL1 isoforms by the PRMT1-RBM15 axis determine the cell fate of a MEP progenitor cell. Using immunoprecipitation assays and mass spectrometry analysis, we identified proteins specifically associated with the N terminal region of TAL1. How unique TAL1s-associated transcriptional regulatory complex plays in erythroid differentiation will be discussed in the presentation in comparison with the Tal1fl-asociated protein complex. In summary, our findings stratify another new layer of regulation by PRMT1, which relays extracellular signals (such as hypoxia signal) to transcriptional regulatory program. Given that PRMT1 is often constitutively highly expressed in leukemia cells, how overproduction of short form TAL1 interferes with normal hematopoiesis may help to explain the molecular mechanisms of many hematological malignancies associated with dysregulation of TAL1 expression. Disclosures No relevant conflicts of interest to declare.

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Differences in DNA-sequence recognition between the DNA-binding domain fragment and the full-length molecule of the heat-shock transcription factor of schistosome

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/s0167-4781(01)00220-2 ◽

2001 ◽

Vol 1519 (3) ◽

pp. 230-234 ◽

Cited By ~ 2

Author(s):

Vinca Lardans ◽

Daniela Ram ◽

Frida Lantner ◽

Etty Ziv ◽

Israel Schechter

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Dna Binding ◽

Dna Sequence ◽

Binding Domain ◽

Heat Shock Transcription Factor ◽

Full Length ◽

Dna Binding Domain ◽

Sequence Recognition ◽

Dna Sequence Recognition

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Evolution of human, chicken, alligator, frog and zebrafish mineralocorticoid receptors: Allosteric influence on steroid specificity

10.1101/151233 ◽

2017 ◽

Cited By ~ 2

Author(s):

Yoshinao Katsu ◽

Kaori Oka ◽

Michael E. Baker

Keyword(s):

Dna Binding ◽

Ligand Binding ◽

Binding Domain ◽

Ligand Binding Domain ◽

Full Length ◽

Dna Binding Domain ◽

Mineralocorticoid Receptors ◽

Terminal Domain ◽

Terrestrial Vertebrate ◽

Hinge Domain

AbstractWe studied the response to aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, corticosterone, progesterone, 19-norprogesterone and spironolactone of human, chicken, alligator, frog and zebrafish full-length mineralocorticoid receptors (MRs) and truncated MRs, lacking the N-terminal domain (NTD) and DNA-binding domain (DBD), in which the hinge domain and ligand binding domain (LBD) were fused to a GAL4-DBD. Compared to full-length MRs, some vertebrate MRs required higher steroid concentrations to activate GAL4-DBD-MR-hinge/LBD constructs. For example, 11-deoxycortisol activated all full-length vertebrate MRs, but did not activate truncated terrestrial vertebrate MRs and was an agonist for truncated zebrafish MR. Progesterone, 19-norProgesterone and spironolactone did not activate full-length and truncated human, alligator and frog MRs. However, at 10 nM, these steroids activated full-length chicken and zebrafish MRs; at 100 nM, these steroids had little activity for truncated chicken MRs, while retaining activity for truncated zebrafish MRs, evidence that regulation of progestin activation of chicken MR resides in NTD/DBD and of zebrafish MR in hinge-LBD. Zebrafish and chicken MRs contain a serine corresponding to Ser810 in human MR, required for its antagonism by progesterone, suggesting novel regulation of progestin activation of chicken and zebrafish MRs. Progesterone may be a physiological activator of chicken and zebrafish MRs.

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The biophysical characterization of the interaction of XPA and the minimal DNA binding domain, XPA-MF122, with a mitomycin C-DNA interstrand crosslink.

10.1349/ddlp.2837 ◽

2001 ◽

Author(s):

David James Mustra

Keyword(s):

Dna Binding ◽

Mitomycin C ◽

Binding Domain ◽

Dna Binding Domain ◽

Biophysical Characterization ◽

Interstrand Crosslink

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Ability of scalloped deletion constructs to rescue sd mutant wing phenotypes in Drosophila melanogaster

Genome ◽

10.1139/g04-060 ◽

2004 ◽

Vol 47 (5) ◽

pp. 849-859 ◽

Cited By ~ 6

Author(s):

Leola Chow ◽

Joel Berube ◽

Alice Fromont ◽

John B Bell

Keyword(s):

Drosophila Melanogaster ◽

Dna Binding ◽

Nuclear Protein ◽

Binding Domain ◽

Dominant Negative ◽

Full Length ◽

Binding Motif ◽

Wing Development ◽

Dna Binding Domain

Scalloped (SD) and Vestigial (VG) proteins physically interact to form a selector complex that activates genes involved in wing development in Drosophila melanogaster. SD belongs to a conserved family of transcription factors containing the TEA/ATTS DNA-binding motif. VG is also a nuclear protein providing the activator function for the SD VG complex. The TEA DNA-binding domain and the VG interacting domain (VID) of SD have been previously identified and described. However, they, and possibly other functional domains of SD, have not been thoroughly characterized in vivo. Herein, transgenic constructs encoding various truncations of SD were used to assess their respective ability to rescue the mutant wing phenotype of two viable sd recessive mutations (sdETX4 and sd58d). The transgenic strains produced were also tested for the ability to induce further sd expression, an ability possessed by full length SD. The functional dissection of SD confirms that specific regions are necessary for wing development and provides further information as to how the SD VG complex functions to promote wing fate. Previous experiments have shown that expression of full length SD can cause a dominant negative wing phenotype. We show that expression of constructs that delete the SD DNA-binding domain can also cause a dominant negative phenotype in a background with either of the two tester sd strains. In contrast, SD constructs that delete the VID have no effect on the wing phenotype in either tester background. Finally, a significant portion of SD at the N-terminal end appears to be dispensable with respect to normal wing development, as this construct behaves the same as full length SD in our assays.Key words: Drosophila melanogaster, wing, scalloped, vestigial, nuclear protein.

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Ex3αERKO male infertility phenotype recapitulates the αERKO male phenotype

Journal of Endocrinology ◽

10.1677/joe-10-0290 ◽

2010 ◽

Vol 207 (3) ◽

pp. 281-288 ◽

Cited By ~ 23

Author(s):

Eugenia H Goulding ◽

Sylvia C Hewitt ◽

Noriko Nakamura ◽

Katherine Hamilton ◽

Kenneth S Korach ◽

...

Keyword(s):

Dna Binding ◽

Male Infertility ◽

Binding Domain ◽

Full Length ◽

Dna Binding Domain ◽

Male Mice ◽

Truncated Form ◽

Exon 2 ◽

Transcript Levels ◽

Reproductive Phenotype

Disruption of the Esr1 gene encoding estrogen receptor α (ERα) by insertion of a neomycin resistance gene (neo) into exon 2 (αERKO mice) was shown previously to cause infertility in male mice. While full-length ERα protein was not expressed in αERKO mice, alternative splicing resulted in the low-level expression of a truncated form lacking the N-terminus A/B domain and containing the DNA- and ligand-binding domains. Thus, it was unclear whether the reproductive phenotype in αERKO males was only due to the lack of full-length ERα or was affected by the presence of the variant ERα isoform. The present study examined male mice with deletion of exon 3 of Esr1 gene, lacking the DNA-binding domain, and null for ERα (Ex3αERKO). Dilation of some seminiferous tubules was apparent in male Ex3αERKO mice as early as postnatal day 10 and was pronounced in all tubules from day 20 onward. At 6 weeks of age, sperm numbers and sperm motility were lower in Ex3αERKO mice than in wild-type (WT) mice, and the rete testis and efferent ductules were dilated. Mating studies determined that adult Ex3αERKO males were infertile and failed to produce copulatory plugs. Serum testosterone levels and Hsd17b3 and Cyp17a1 transcript levels were significantly higher, but serum estradiol, progesterone, LH, and FSH levels and Cyp19a1 transcript levels were not significantly different from those in WT mice. These results confirm and extend those seen in other studies on male mice with deletion of exon 3 of Esr1 gene. In addition, the reproductive phenotype of male Ex3αERKO mice recapitulated the phenotype of αERKO mice, strongly suggesting that the αERKO male infertility was not due to the presence of the DNA-binding domain in the truncated form of ERα and that full-length ERα is essential for maintenance of male fertility.

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The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010188 ◽

2019 ◽

Vol 295 (3) ◽

pp. 743-756 ◽

Cited By ~ 6

Author(s):

Christopher J. Hosford ◽

Anthony Q. Bui ◽

Joshua S. Chappie

Keyword(s):

Dna Binding ◽

Mutational Analysis ◽

Binding Pocket ◽

Binding Mode ◽

Binding Domain ◽

Dna Binding Domain ◽

Modified Dna ◽

Structural Superposition ◽

Aromatic Cage ◽

Preferential Binding

McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. Here we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrB homologs and expand the biological roles of YTH domains.

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