scholarly journals The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin

2020 ◽  
Vol 295 (14) ◽  
pp. 4748-4759 ◽  
Author(s):  
Hang Gyeong Chin ◽  
Pierre-Olivier Esteve ◽  
Cristian Ruse ◽  
Jiyoung Lee ◽  
Scott E. Schaus ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Organelle Transport ◽  
Histone H4 ◽  
Protein Protein Interactions ◽  
Molecule Inhibitor ◽  
Associated Proteins ◽  
Lysine Methyltransferase

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein–protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311. Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro. In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

scholarly journals The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Kyle T. Helzer ◽  
Mary Szatkowski Ozers ◽  
Mark B. Meyer ◽  
Nancy A. Benkusky ◽  
Natalia Solodin ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Protein Function ◽  
Estrogen Receptor Α ◽  
Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation

1994 ◽  
Vol 107 (8) ◽  
pp. 2055-2063 ◽  
Author(s):  
A.P. Wolffe
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Protein Interactions ◽  
Multigene Family ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Eukaryotic Genes ◽  
Rna Genes

Differential expression of the oocyte and somatic 5 S RNA genes during Xenopus development can be explained by changes in transcription factor and histone interactions with the two types of gene. Both factors and histones bind 5 S RNA genes with specificity. Protein-protein interactions determine the stability of potentially transcriptionally active or repressed nucleoprotein complexes. A decline in transcription factor abundance, differential binding of transcription factors to oocyte and somatic 5 S genes, and increased competition with the histones for association with DNA during early embryogenesis, can account for the developmental decision to selectively repress the oocyte genes, while retaining the somatic genes in the transcriptionally active state. The 5 S ribosomal genes of Xenopus are perhaps the simplest eukaryotic genes to show regulated expression during development. A large multigene family (oocyte 5 S DNA) is transcriptionally active in oocytes but is repressed in somatic cells, whereas a small multigene family (somatic 5 S DNA) is active in both cell types. A potential molecular mechanism to explain the developmental switch that turns off oocyte 5 S DNA transcription has been experimentally reconstructed in vitro and more recently tested in vivo. Central to this mechanism is the specific association of both transcription factors and histones with 5 S RNA genes. How the interplay of histones and transcription factors is thought to affect transcription, and how their respective contributions might change during development from an oocyte, to an embryo and eventually to a somatic cell is the focus of this review.

scholarly journals Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

2009 ◽  
Vol 29 (15) ◽  
pp. 4103-4115 ◽  
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Thomas E. Akie ◽  
Tyler B. Moran ◽  
Andrew J. Woo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fetal Liver ◽  
Gel Filtration ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

scholarly journals Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jeroen Overman ◽  
Frank Fontaine ◽  
Mehdi Moustaqil ◽  
Deepak Mittal ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Protein Interactions ◽  
Vascular Development ◽  
Great Promise ◽  
Vascular Density ◽  
Protein Protein Interactions ◽  
Clinical Model

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.

scholarly journals Transcription factor LSF facilitiates lysine methylation of α-tubulin by microtubule-associated SET8

2019 ◽  
Author(s):  
Hang Gyeong Chin ◽  
Pierre Olivier Estève ◽  
Cristian Ruse ◽  
Jiyoung Lee ◽  
Scott E. Schaus ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Organelle Transport ◽  
Lysine Methylation ◽  
Mitotic Progression ◽  
Methyl Transferase ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Molecule Inhibitor ◽  
Dual Functions

AbstractMicrotubules are critical for mitosis, cell motility, and protein and organelle transport, and are a validated target for anticancer drugs. However, tubulin regulation and recruitment in these cellular processes is less understood. Post-translational modifications of tubulin are proposed to regulate microtubule functions and dynamics. Although many such modifications have been investigated, tubulin methylations and enzymes responsible for methylation have only recently begun to be described. Here we report that N-lysine methyl transferase KMT5A (SET8/PR-Set7), which methylates histone H4K20, also methylates α-tubulin. Furthermore, the transcription factor LSF binds both tubulin and SET8, and enhances α-tubulin methylationin vitro, countered by FQI1, a specific small molecule inhibitor of LSF. Thus, the three proteins SET8, LSF, and tubulin, all essential for mitotic progression, interact with each other. Overall, these results point to dual functions for both SET8 and LSF not only in chromatin regulation, but also for cytoskeletal modification.

scholarly journals SUMOylation of the Corepressor N-CoR Modulates Its Capacity to Repress Transcription

2006 ◽  
Vol 17 (4) ◽  
pp. 1643-1651 ◽  
Author(s):  
Jens Tiefenbach ◽  
Natalia Novac ◽  
Miryam Ducasse ◽  
Maresa Eck ◽  
Frauke Melchior ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Hormone Receptors ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Lysine Residues

In the absence of ligands the corepressor N-CoR mediates transcriptional repression by some nuclear hormone receptors. Several protein–protein interactions of N-CoR are known, of which mainly complex formation with histone deacetylases (HDACs) leads to the repression of target genes. On the other hand, the role of posttranslational modifications in corepressor function is not well established. Here, we show that N-CoR is modified by Sumo-1. We found SUMO-E2–conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1 as novel N-CoR interaction partners. The SANT1 domain of N-CoR was found to mediate this interaction. We show that K152, K1117, and K1330 of N-CoR can be conjugated to SUMO and that mutation of all sites is necessary to fully block SUMOylation in vitro. Because these lysine residues are located within repression domains I and III, respectively, we investigated a possible correlation between the functions of the repression domains and SUMOylation. Coexpression of Ubc9 protein resulted in enhanced N-CoR–dependent transcriptional repression. Studies using SUMOylation-deficient N-CoR RDI mutants suggest that SUMO modification contributes to repression by N-CoR. Mutation of K152 to R in RD1, for example, not only significantly reduced repression of a reporter gene, but also abolished the effect of Ubc9 on transcriptional repression.

scholarly journals Progress in the Discovery of Small-Molecule Inhibitors of Bromodomain–Histone Interactions

2011 ◽  
Vol 16 (10) ◽  
pp. 1170-1185 ◽  
Author(s):  
Chun-wa Chung ◽  
Jason Witherington
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Ligand Selectivity ◽  
Small Molecule Ligands ◽  
Associated Proteins ◽  
In Vitro Systems

Bromodomains are structurally conserved protein modules present in a large number of chromatin-associated proteins and in many nuclear histone acetyltransferases. The bromodomain functions as an acetyl-lysine binding domain and has been shown to be pivotal in regulating protein–protein interactions in chromatin-mediated cellular gene transcription, cell proliferation, and viral transcriptional activation. Structural analyses of these modules in complex with acetyl-lysine peptide ligands provide insights into the molecular basis for recognition and ligand selectivity within this epigenetic reader family. However, there are significant challenges in configuring assays to identify inhibitors of these proteins. This review focuses on the progress made in developing methods to identify peptidic and small-molecule ligands using biophysical label-free and biochemical approaches. The advantage of each technique and the results reported are summarized, highlighting the potential applicably to other reader domains and the caveats in translation from simple in vitro systems to a biological context.

scholarly journals Phosphorylation of Enabled by the DrosophilaAbelson Tyrosine Kinase Regulates the In Vivo Function and Protein-Protein Interactions of Enabled

1998 ◽  
Vol 18 (1) ◽  
pp. 152-160 ◽  
Author(s):  
Allen R. Comer ◽  
Shawn M. Ahern-Djamali ◽  
Jyh-Lyh Juang ◽  
P. David Jackson ◽  
F. M. Hoffmann
Keyword(s):  
Tyrosine Kinase ◽  
Protein Interactions ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Tyrosine Residues ◽  
Abelson Tyrosine Kinase ◽  
Genetic Suppressors ◽  
Associated Proteins

ABSTRACT Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila enawere discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

scholarly journals The microtubule-binding protein Cep170 promotes the targeting of the kinesin-13 depolymerase Kif2b to the mitotic spindle

2012 ◽  
Vol 23 (24) ◽  
pp. 4786-4795 ◽  
Author(s):  
Julie P. I. Welburn ◽  
Iain M. Cheeseman
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Microtubule Depolymerization ◽  
Extrinsic Factors ◽  
Microtubule Binding ◽  
Unique Protein ◽  
C Terminus ◽  
Primary Determinant ◽  
Associated Proteins

Microtubule dynamics are essential throughout mitosis to ensure correct chromosome segregation. Microtubule depolymerization is controlled in part by microtubule depolymerases, including the kinesin-13 family of proteins. In humans, there are three closely related kinesin-13 isoforms (Kif2a, Kif2b, and Kif2c/MCAK), which are highly conserved in their primary sequences but display distinct localization and nonoverlapping functions. Here we demonstrate that the N-terminus is a primary determinant of kinesin-13 localization. However, we also find that differences in the C-terminus alter the properties of kinesin-13, in part by facilitating unique protein–protein interactions. We identify the spindle-localized proteins Cep170 and Cep170R (KIAA0284) as specifically associating with Kif2b. Cep170 binds to microtubules in vitro and provides Kif2b with a second microtubule-binding site to target it to the spindle. Thus the intrinsic properties of kinesin-13s and extrinsic factors such as their associated proteins result in the diversity and specificity within the kinesin-13 depolymerase family.

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