Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis

ABSTRACT The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

Inactivation of the CIC-DUX4 oncogene through P300/CBP inhibition, a therapeutic approach for CIC-DUX4 sarcoma

CIC-DUX4 sarcoma (CDS) is a highly aggressive and metastatic small round type of predominantly pediatric sarcoma driven by a fusion oncoprotein comprising the transcriptional repressor Capicua (CIC) fused to the C-terminal transcriptional activation domain of DUX4. CDS rapidly develops resistance to chemotherapy, thus novel specific therapies are greatly needed. We demonstrate that CIC-DUX4 requires P300/CBP to induce histone H3 acetylation, activate its targets, and drive oncogenesis. We describe the synthetic route to a selective and highly potent P300/CBP inhibitor named iP300w and related stereoisomers, and find that iP300w efficiently suppresses CIC-DUX4 transcriptional activity and reverses CIC-DUX4 induced acetylation. iP300w is active at 100-fold lower concentrations than related stereoisomers or A-485. At low doses, iP300w shows specificity to CDS cancer cell lines, rapidly inducing cell cycle arrest and preventing growth of established CDS xenograft tumors when delivered in vivo. The effectiveness of iP300w to inactivate CIC-DUX4 highlights a promising therapeutic opportunity for CDS.

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Promoting sustainable proliferation of NK cells by up-regulating IL2 and IL12 through CRISPR-CAS9Promoting sustainable proliferation of NK cells by up-regulating IL2 and IL12 through CRISPR-CAS9

NK cells are an important part of human immunity and are often used in clinical antitumor adoptive cell immunotherapy.However, the small number and low activity of NK cells in the tumor microenvironment limit its clinical efficacy.Studies have found that cytokines IL2 and IL12 play key roles in the proliferation of NK cells and the improvement of the activity of killing tumor cells, the toxic and side effects, however, are relatively large when the systemic application reaches the effective concentration. To study the effect of autocrine secretion of IL2 and IL12 on proliferation and anti-tumor activity of NK92 cell lines by editing NK cells' genes, and to construct NK92 cells with sustainable proliferation. Lentivirus was transfected into NK92 cells through the synergistic effect of crispr/cas9 localization gene editing technology and VP64 transcriptional activation domain to construct NK92-IL2-IL12 cell lines. The expression levels of IL2 and IL12 in the cell lines were detected by qPCR and ELISA.NK92 cell lines were inoculated into mouse tumor and the proliferation and anti-tumor effect of NK cells in tumor were observed. The vector plasmids IL2 and IL12 were successfully transferred into NK92 cells, which activated the up-regulated expression of IL2/IL12 in NK92, significantly increased the activity and lethality of NK92 cells, and sustained the proliferation of NK92 cells in mouse tumor.IL2 and IL12 can significantly promote the proliferation and killing activity of NK92 cell lines through autocrine, which have stronger anti-tumor activity than the wild-type ones, with continuous proliferation in the tumor.

Structural basis of phosphorylation kinetics in the transcriptional activation domain (TAD) of c-Jun and insight of two new phosphorylation sites Ser58 and Thr62

Protein phosphorylation is one of the most important posttranslational modifications observed on biomolecules. Nearly one-third of the cell cycle protein undergoes phosphorylation at some stage of the lifespan. Multi-site phosphorylation is well known in biological systems, including those in transcription factors. Multisite phosphorylation on transcription factors brings about their activation and/or inactivation. c-Jun is one of such transcription factors, whose function is dependent upon the state of phosphorylation. N-terminal phosphorylation required for c-Jun activity, while C-terminal one suppresses its activity. c-Jun contains a transcriptional activation domain (TAD) at N-terminus. It is known that four residues viz., Ser63, Ser73, Thr91 and Thr93 get phosphorylated which is required for its functional dimerization. However, there is no evidence if there exists any phosphorylation kinetics in c-Jun. In this paper, for the first time, it has been demonstrated that there exist phosphorylation kinetics within TAD. NMR based analysis suggested that Ser63 follows the fast kinetic while, Thr91 slow and Ser73 and Thr93 fall in the intermediate category. The four sites follow the following trend in their kinetics Ser63 > Ser73 > Thr93 > Thr91. Similar phosphorylation kinetics was also observed inside the C3H 10T0.5 fibroblast. NMR-based experiments also suggested the phosphorylation of two additional sites at Ser58 and Thr62. However, a detailed significance of these phosphorylation kinetic, as well as newly identified sites, is yet to be discovered.

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

SUMMARYGene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.