Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST). RESULTS: Using CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity. CONCLUSIONS: Our results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Collaborators or competitors: the communication between RNA polymerase II and the nucleosome during eukaryotic transcription.

Decades of scientific research have been devoted to unraveling the intricacies of eukaryotic transcription since the groundbreaking discovery of eukaryotic RNA polymerases in the late 1960s. RNA polymerase II, the polymerase responsible for mRNA synthesis, has always attracted the most attention. Despite its structural resemblance to its bacterial counterpart, eukaryotic RNA polymerase II faces a unique challenge in progressing transcription due to the presence of nucleosomes that package DNA in the nuclei. In this review, we delve into the impact of RNA polymerase II and histone signaling on the progression of eukaryotic transcription. We explore the pivotal points of interactions that bridge the RNA polymerase II and histone signaling systems. Finally, we present an analysis of recent cryo-electron microscopy structures, which captured RNA polymerase II-nucleosome complexes at different stages of the transcription cycle. The combination of the signaling crosstalk and the direct visualization of RNA polymerase II-nucleosome complexes provides a deeper understanding of the communication between these two major players in eukaryotic transcription.

Variation of C-terminal domain governs RNA polymerase II genomic locations and alternative splicing in eukaryotic transcription.

The C-terminal domain of RPB1 (CTD) orchestrates transcription by recruiting regulators to RNA Pol II upon phosphorylation. Recent insights highlight the pivotal role of CTD in driving condensate formation on gene loci. Yet, the molecular mechanism behind how CTD-mediated recruitment of transcriptional regulators influences condensates formation remains unclear. Our study unveils that phosphorylation reversibly dissolves phase separation induced by the unphosphorylated CTD. Phosphorylated CTD, upon specific association with transcription regulatory proteins, forms distinct condensates from unphosphorylated CTD. Function studies demonstrate CTD variants with diverse condensation properties in vitro exhibit difference in promoter binding and mRNA co-processing in cells. Notably, varying CTD lengths lead to alternative splicing outcomes impacting cellular growth, linking the evolution of CTD variation/length with the complexity of splicing from yeast to human. These findings provide compelling evidence for a model wherein post-translational modification enables the transition of functionally specialized condensates, highlighting a co-evolution link between CTD condensation and splicing.

Histone acetyltransferase HAF2 associates with PDC to control H3K14ac and H3K23ac in ethylene response.

Ethylene plays its essential roles in plant development, growth, and defense responses by controlling the transcriptional reprogramming, in which EIN2-C-directed regulation of histone acetylation is the first key-step for chromatin to perceive ethylene signaling. However, the histone acetyltransferase in this process remains unknown. Here, we identified histone acetyltransferase HAF2, and mutations in HAF2 confer plants with ethylene insensitivity. Furthermore, we found that HAF2 interacts with EIN2-C in response to ethylene. Biochemical assays demonstrated that the bromodomain of HAF2 binds to H3K14ac and H3K23ac peptides with a distinct affinity for H3K14ac; the HAT domain possesses acetyltransferase catalytic activity for H3K14 and H3K23 acetylation, with a preference for H3K14. ChIP-seq results provide additional evidence supporting the role of HAF2 in regulating H3K14ac and H3K23ac levels in response to ethylene. Finally, our findings revealed that HAF2 co-functions with pyruvate dehydrogenase complex (PDC) to regulate H3K14ac and H3K23ac in response to ethylene in an EIN2 dependent manner. Overall, this research reveals that HAF2 as a histone acetyltransferase that forms a complex with EIN2-C and PDC, collectively governing histone acetylation of H3H14ac and H3K23ac, preferentially for H3K14 in response to ethylene.

Distinctive interactomes of RNA polymerase II phosphorylation during different stages of transcription.

During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription.

Introduction of Asymmetry in the Fused 4-Oxalocrotonate Tautomerases.

Members of the 4-oxalocrotonate tautomerase (4-OT) subgroup in the tautomerase superfamily (TSF) are constructed from a single ß-α-ß unit and form homo- or heterohexamers, whereas those of the other four subgroups are composed of two consecutively joined ß-α-ß units and form trimers. A subset of sequences, double the length of the short 4-OTs, is found in the 4-OT subgroup. These "fused" 4-OTs form a separate subgroup that connects to the short 4-OTs in a sequence similarity network (SSN). The fused gene can be a template for the other four subgroups, resulting in the diversification of activity. Analysis of the SSN shows that multiple nodes in the fused 4-OTs connect to five linker nodes, which in turn connect to the short 4-OTs. Some fused 4-OTs are symmetric trimers and others are asymmetric trimers. The origin of this asymmetry was investigated by subjecting the sequences in three linker nodes and a closely associated fourth node to kinetic, mutagenic, and structural analyses. The results show that each sequence corresponds to the α- or ß-subunit of a heterohexamer that functions as a 4-OT. Mutagenesis indicates that the key residues in both are αPro1 and ßArg-11, like that of a typical 4-OT. Crystallographic analysis shows that both heterohexamers are asymmetric, where one heterodimer is flipped 180° relative to the other two heterodimers. The fusion of two subunits (α and ß) of one asymmetric heterohexamer generates an asymmetric trimer with 4-OT activity. Hence, asymmetry can be introduced at the heterohexamer level and then retained in the fused trimers.