Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions.

Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.

CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality.

CHD3 and CHD4 (Chromodomain Helicase DNA binding protein), two highly similar representatives of the Mi-2 subfamily of SF2 helicases, are coexpressed in many cell lines and tissues and have been reported to act as the motor subunit of the NuRD complex (nucleosome remodeling and deacetylase activities). Besides CHD proteins, NuRD contains several repressors like HDAC1/2, MTA2/3 and MBD2/3, arguing for a role as a transcriptional repressor. However, the subunit composition varies among cell- and tissue types and physiological conditions. In particular, it is unclear if CHD3 and CHD4 coexist in the same NuRD complex or whether they form distinct NuRD complexes with specific functions. We mapped the CHD composition of NuRD complexes in mammalian cells and discovered that they are isoform-specific, containing either the monomeric CHD3 or CHD4 ATPase. Both types of complexes exhibit similar intranuclear mobility, interact with HP1 and rapidly accumulate at UV-induced DNA repair sites. But, CHD3 and CHD4 exhibit distinct nuclear localization patterns in unperturbed cells, revealing a subset of specific target genes. Furthermore, CHD3 and CHD4 differ in their nucleosome remodeling and positioning behaviour in vitro. The proteins form distinct CHD3- and CHD4-NuRD complexes that do not only repress, but can just as well activate gene transcription of overlapping and specific target genes.

Assessing HDAC Function in the Regulation of Signal Transducer and Activator of Transcription 5 (STAT5) Activity Using Chromatin Immunoprecipitation (ChIP).

Transcriptional activation by STAT5 is repressed by deacetylase inhibitors. Investigating the role of deacetylases (HDACs) in STAT5-mediated transcription implies the analysis of molecular events taking place at the chromatin level. We describe here two alternative methods of chromatin immunoprecipitation that allow the characterization of chromatin modifications ensuing STAT5 activation and its inhibition by deacetylase inhibitors, in particular changes in histone acetylation, in histone occupancy, and in the association/dissociation of transcription factors and other chromatin-associated factors.

Inhibition of interleukin-3- and interferon- α-induced JAK/STAT signaling by the synthetic α-X-2',3,4,4'-tetramethoxychalcones α-Br-TMC and α-CF3-TMC.

The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2',3,4,4'-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.

Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer.

BACKGROUND: c-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1-E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter. RESULTS: We now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression. CONCLUSIONS: Our data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6.

Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function.

Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function.

The natural chemopreventive agent sulforaphane inhibits STAT5 activity.

Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.

The synthetic α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) inhibits the JAK/STAT signaling pathway.

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.