Significance: DOT1L, the only H3K79 methyltransferase in human cells, forms a complex with AF10/AF17 and ENL/AF9, is dysregulated in mixed-lineage leukemia (MLLr), and is believed to regulate transcriptional elongation without direct evidence. Here, functional genomic, proteomic, and biochemical studies aimed at an understanding of the role of DOT1L in erythroleukemic cells show no major role in elongation but, surprisingly, reveal that DOT1L evidently has a role in transcriptional initiation by recruiting initiation factor TFIID and enhancing H2B mono-ubiquitination by restricting deubiquitination by the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. The results thus implicate the DOT1L complex in transcriptional initiation and raise the intriguing possibility that MLLr leukemic fusion proteins may also promote transcriptional initiation through modified DOT1L complexes.

Haematopoietic stem cells (HSCs) possess unique physiological adaptations to sustain blood cell production and cope with stress responses throughout life. To maintain these adaptations, HSCs rely on maintaining a tightly controlled protein translation rate. However, the mechanism of how HSCs regulate protein translation remains to be fully elucidated. In this study, we investigate the role of transfer RNA (tRNA) m¹A58 ‘writer’ proteins TRMT6 and TRMT61A in regulating HSCs function. Trmt6 deletion promoted HSC proliferation through aberrant activation of mTORC1 signaling. TRMT6-deficient HSCs exhibited an impaired self-renewal ability in competitive transplantation assay. Mechanistically, single cell RNA-seq analysis reveals that the mTORC1 signaling pathway is highly upregulated in HSC-enriched cell populations after Trmt6 deletion. m¹A-tRNA-seq and Western blot analysis suggest that TRMT6 promotes methylation modification of specific tRNA and expression of TSC1, fine-tuning mTORC1 signaling levels. Furthermore, Pharmacological inhibition of the mTORC1 pathway rescued functional defect in TRMT6-deficient HSCs. To our knowledge, this study is the first to elucidate a mechanism by which TRMT6-TRMT61A complex-mediated tRNA-m¹A58 modification regulates HSC homeostasis.

TOX4 is one of the regulatory factors of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.

HMG protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that Tox4 conditional knockout in mice reduces thymic cellularity, partially blocks T cell development, and decreases ratio of CD8 to CD4 through decreasing proliferation and increasing apoptosis of CD8 cells. In addition, single-cell RNA-seq discovered that Tox4 loss also impairs proliferation of the fast-proliferating double positive (DP) blast population within DP cells in part due to downregulation of genes critical for proliferation, notably Cdk1. Moreover, genes with high and low expression level are more dependent on Tox4 than genes with medium expression level. Mechanistically, Tox4 may facilitate transcriptional reinitiation and restrict elongation in a dephosphorylation-dependent manner, a mechanism that is conserved between mouse and human. These results provide insights into the role of TOX4 in development and establish it as an evolutionarily conserved regulator of transcriptional elongation and reinitiation.

Phase separation, a biophysical process that segregates subcellular environments into condensates, has been recognized for its role in transcriptional regulation. However, the extent of its influence on cellular senescence processes remains to be fully elucidated. We established that MRG15 depletion leads to cellular senescence in human mesenchymal stem cells (hMSCs). MRG15 can form phase-separated liquid condensates via its intrinsically disordered region (IDR). IDR deletion and replacement assays revealed that MRG15 condensation is crucial to hMSC senescence. According to the epigenomic and transcriptomic analysis, MRG15 depletion impacts pathways integral to the cell cycle and the senescence process, as evidenced by the diminished binding and the modified expression of key genes, including p53, CDKN1A, LMNB1, CCNB1, NPM1, MYC, and HMGB2. Our findings establish a link between phase separation and senescence regulation and present a promising new therapeutic target for the alleviation of age-related diseases and the potential extension of lifespan.

The human immune system executes immune defense against foreign antigens, immune surveillance against mutant cells, and immune homeostasis to maintain bodily physiological equilibrium. Transfer RNA (tRNA) is an important part of gene expression regulation. Recent studies have found that aberrant tRNA modifications can disturb the function of the immune system by altering translation efficiency, including regulating immune recognition and signal transduction, as well as influencing apoptosis and autophagy. tRNA modifications are also closely related to the development and occurrence of immune-related disorders such as infections, tumors, and autoimmune diseases. By integrating current research on immune-related tRNA modifications, this review systematically summarizes the mechanisms of tRNA modifications involved in three major immune functions and their relationship with related diseases, encompassing the direct regulation of immune cells and the indirect immune effects mediated through tissue or tumor cells. In addition, some tRNA modifications can also modulate immune responses by affecting tRNA cleavage and the consequent generation of tRNA-derived small RNAs (tsRNAs). Accordingly, this review outlines promising intervention targets and unresolved research bottlenecks, facilitating follow-up investigations into tRNA modification-dependent immune regulation and translational therapeutic research.