Mechanically enhanced biogenesis of gut spheroids with instability-driven morphomechanics.

Region-specific gut spheroids are precursors for gastrointestinal and pulmonary organoids that hold great promise for fundamental studies and translations. However, efficient production of gut spheroids remains challenging due to a lack of control and mechanistic understanding of gut spheroid morphogenesis. Here, we report an efficient biomaterial system, termed micropatterned gut spheroid generator (µGSG), to generate gut spheroids from human pluripotent stem cells through mechanically enhanced tissue morphogenesis. We show that µGSG enhances the biogenesis of gut spheroids independent of micropattern shape and size; instead, mechanically enforced cell multilayering and crowding is demonstrated as a general, geometry-insensitive mechanism that is necessary and sufficient for promoting spheroid formation. Combining experimental findings and an active-phase-field morphomechanics theory, our study further reveals an instability-driven mechanism and a mechanosensitive phase diagram governing spheroid pearling and fission in µGSG. This work unveils mechanobiological paradigms based on tissue architecture and surface tension for controlling tissue morphogenesis and advancing organoid technology.

Stimuli-Responsive PROTACs for Controlled Protein Degradation.

Proteolysis Targeting Chimeras (PROTACs) represent a promising therapeutic modality to address undruggable and resistant issues in drug discovery. However, potential on-target toxicity remains clinically challenging. We developed a generalized caging strategy to synthesize a series of stimuli-responsive PROTACs (sr-PROTACs) with diverse molecular blocks bearing robust and cleavable linkers, presenting "turn on" features in manipulating protein degradation. By leveraging pathological cues, such as elevated ROS, phosphatase, H2 S, or hypoxia, and external triggers, such as ultraviolet light, X-Ray, or bioorthogonal reagents, we achieved site-specific activation and traceless release of original PROTACs through de-caging and subsequent self-immolative cleavage, realizing selective uptake and controlled protein degradation in vitro. An in vivo study revealed that two sr-PROTACs with phosphate- and fluorine-containing cages exhibited high solubility and long plasma exposure, which were specifically activated by tumor overexpressing phosphatase or low dosage of X-Ray irradiation in situ, leading to efficient protein degradation and potent tumor remission. With more reactive biomarkers to be screened from clinical practice, our caging library could provide a general tool to design activatable PROTACs, prodrugs, antibody-drug conjugates, and smart biomaterials for personalized treatment, tissue engineering or regenerative medicine.

HMCES modulates the transcriptional regulation of nodal/activin and BMP signaling in mESCs.

Despite the fundamental roles of TGF-ß family signaling in cell fate determination in all metazoans, the mechanism by which these signals are spatially and temporally interpreted remains elusive. The cell-context-dependent function of TGF-ß signaling largely relies on transcriptional regulation by SMAD proteins. Here, we discover that the DNA repair-related protein, HMCES, contributes to early development by maintaining nodal/activin- or BMP-signaling-regulated transcriptional network. HMCES binds with R-SMAD proteins, co-localizing at active histone marks. However, HMCES chromatin occupancy is independent on nodal/activin or BMP signaling. Mechanistically, HMCES competitively binds chromatin to limit binding by R-SMAD proteins, thereby forcing their dissociation and resulting in repression of their regulatory effects. In Xenopus laevis embryo, hmces KD causes dramatic development defects with abnormal left-right axis asymmetry along with increasing expression of lefty1. These findings reveal HMCES transcriptional regulatory function in the context of TGF-ß family signaling.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

Cellular responses to transforming growth factor ß (TGF-ß) depend on cell context. Here, we explored how TGF-ß/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

Crosstalk between TGF-ß signaling and epigenome.

The transforming growth factor beta (TGF-ß) family of ligands plays major roles in embryonic development, tissue homeostasis, adult immunity, and wound repair. Dysregulation of TGF-ß signaling pathway leads to severe diseases. Its key components have been revealed over the past two decades. This family of cytokines acts by activating receptor activated SMAD (R-SMAD) transcription factors, which in turn modulate the expression of specific sets of target genes. Cells of a multicellular organism have the same genetic information, yet they show structural and functional differences owing to differential expression of their genes. Studies have demonstrated that epigenetic regulation, an integral part of the TGF-ß signaling, enables cells to sense and respond to TGF-ß signaling in a cell context-dependent manner. R-SMAD, as the central transcription factor of TGF-ß signaling, can recruit various epigenetic regulators to shape the transcriptome. In this review, we focus on epigenetic regulatory mechanisms in the TGF-ß signaling during mammalian development and diseases and discuss the central role of the interaction between R-SMAD and various epigenetic regulators in this epigenetic regulation. The crosstalk between TGF-ß signaling and the epigenome could serve as a versatile fine-tuning mechanism for transcriptional regulation during embryonic development and progression of diseases, particularly cancer.

Role of TRIM33 in Wnt signaling during mesendoderm differentiation.

Tripartite motif 33 (TRIM33), a member of the transcription intermediate factor 1 (TIF1) family of transcription cofactors, mediates transforming growth factor-beta (TGF-ß) signaling through its PHD-Bromo cassette in mesendoderm differentiation during early mouse embryonic development. However, the role of the TRIM33 RING domain in embryonic differentiation is less clear. Here, we report that TRIM33 mediates Wnt signaling by directly regulating the expression of a specific subset of Wnt target genes, and this action is independent of its RING domain. We show that TRIM33 interacts with ß-catenin, a central player in Wnt signaling in mouse embryonic stem cells (mESCs). In contrast to previous reports in cancer cell lines, the RING domain does not appear to function as the E3 ligase for ß-catenin, since neither knockout nor overexpression of TRIM33 had an effect on ß-catenin protein levels in mESCs. Furthermore, we show that although TRIM33 seems to be dispensable for Wnt signaling through a reporter assay, loss of TRIM33 significantly impairs the expression of a subset of Wnt target genes, including Mixl1, in a Wnt signaling-dependent manner. Together, our results indicate that TRIM33 regulates Wnt signaling independent of the E3 ligase activity of its RING domain for ß-catenin in mESCs.

The NF-κB-modulated microRNAs miR-195 and miR-497 inhibit myoblast proliferation by targeting Igf1r, Insr and cyclin genes.

MicroRNAs (miRNAs) play important roles in the development of skeletal muscle. In our previous study, expression of miR-195 and miR-497 were shown to be upregulated during muscle development in pigs. In this study, we investigated the roles of these two miRNAs in myogenesis and analyzed their transcriptional regulation. Our results showed that miR-195 and miR-497 were upregulated during muscle development and myoblast differentiation. Moreover, miR-195 and miR-497 inhibited proliferation but not differentiation in C2C12 cells. Further investigation revealed that Igf1r, Insr, Ccnd2 and Ccne1 were directly targeted by miR-195 and miR-497 in myoblasts. In addition, we confirmed that miR-195 and miR-497, which shared the similar expression profiling, were negatively regulated by nuclear factor κB (NF-κB) in both myoblasts and skeletal muscle tissue. Our data illustrate that the signaling pathway NF-κB-miR-195/497-Igf1r/Insr-Ccnd2/Ccne1 plays important roles in myogenesis. Our study provides novel evidence for the roles of miR-195 and miR-497 in muscle development.