Dual-role transcription factors stabilize intermediate expression levels.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

PhaSepDB in 2022: annotating phase separation-related proteins with droplet states, co-phase separation partners and other experimental information.

Phase separation (PS) proteins form droplets to regulate myriad membraneless organelles (MLOs) and cellular pathways such as transcription, signaling transduction and protein degeneration. PS droplets are usually liquid-like and can convert to hydrogel/solid-like under certain conditions. The PS behavior of proteins is regulated by co-PS partners and mutations, modifications, oligomerizations, repeat regions and alternative splicing of the proteins. With growing interest in PS condensates and associated proteins, we established PhaSepDB 1.0, which provided experimentally verified PS proteins and MLO-related proteins. The past few years witnessed a surge in PS-related research works; thus, we kept updating PhaSepDB. The current PhaSepDB contains 1419 PS entries, 770 low-throughput MLO-related entries and 7303 high-throughput MLO-related entries. We provided more detailed annotations of PS proteins, including PS verification experiments, regions used in experiments, phase diagrams of different experimental conditions, droplet states, co-PS partners and PS regulatory information. We believe that researchers can go further in studying PS proteins with the updated PhaSepDB (http://db.phasep.pro/).

Proteome-scale analysis of phase-separated proteins in immunofluorescence images.

Phase separation is an important mechanism that mediates the spatial distribution of proteins in different cellular compartments. While phase-separated proteins share certain sequence characteristics, including intrinsically disordered regions (IDRs) and prion-like domains, such characteristics are insufficient for making accurate predictions; thus, a proteome-wide understanding of phase separation is currently lacking. Here, we define phase-separated proteomes based on the systematic analysis of immunofluorescence images of 12 073 proteins in the Human Protein Atlas. The analysis of these proteins reveals that phase-separated candidate proteins exhibit higher IDR contents, higher mean net charge and lower hydropathy and prefer to bind to RNA. Kinases and transcription factors are also enriched among these candidate proteins. Strikingly, both phase-separated kinases and phase-separated transcription factors display significantly reduced substrate specificity. Our work provides the first global view of the phase-separated proteome and suggests that the spatial proximity resulting from phase separation reduces the requirement for motif specificity and expands the repertoire of substrates. The source code and data are available at https://github.com/cheneyyu/deepphase.

PhaSepDB: a database of liquid-liquid phase separation related proteins.

It's widely appreciated that liquid-liquid phase separation (LLPS) underlies the formation of membraneless organelles, which function to concentrate proteins and nucleic acids. In the past few decades, major efforts have been devoted to identify the phase separation associated proteins and elucidate their functions. To better utilize the knowledge dispersed in published literature, we developed PhaSepDB (http://db.phasep.pro/), a manually curated database of phase separation associated proteins. Currently, PhaSepDB includes 2914 non-redundant proteins localized in different organelles curated from published literature and database. PhaSepDB provides protein summary, publication reference and sequence features of phase separation associated proteins. The sequence features which reflect the LLPS behavior are also available for other human protein candidates. The online database provides a convenient interface for the research community to easily browse, search and download phase separation associated proteins. As a centralized resource, we believe PhaSepDB will facilitate the future study of phase separation.

Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index.

BACKGROUND: Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent. RESULTS: Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment. CONCLUSIONS: Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

OTUD5 cooperates with TRIM25 in transcriptional regulation and tumor progression via deubiquitination activity.

Oncogenic processes exert their greatest effect by targeting regulators of cell proliferation. Studying the mechanism underlying growth augmentation is expected to improve clinical therapies. The ovarian tumor (OTU) subfamily deubiquitinases have been implicated in the regulation of critical cell-signaling cascades, but most OTUs functions remain to be investigated. Through an unbiased RNAi screen, knockdown of OTUD5 is shown to significantly accelerate cell growth. Further investigation reveals that OTUD5 depletion leads to the enhanced transcriptional activity of TRIM25 and the inhibited expression of PML by altering the ubiquitination level of TRIM25. Importantly, OTUD5 knockdown accelerates tumor growth in a nude mouse model. OTUD5 expression is markedly downregulated in tumor tissues. The reduced OTUD5 level is associated with an aggressive phenotype and a poor clinical outcome for cancers patients. Our findings reveal a mechanism whereby OTUD5 regulates gene transcription and suppresses tumorigenesis by deubiquitinating TRIM25, providing a potential target for oncotherapy.

p53 ß-hydroxybutyrylation attenuates p53 activity.

p53 is an essential tumor suppressor, whose activity is finely tuned by the posttranslational modifications. Previous research has reported that ß-hydroxybutyrate (BHB) induces ß-hydroxybutyrylation (Kbhb), which is a novel histone posttranslational modification. Here we report that p53 is modified by kbhb and that this modification occurs at lysines 120, 319, and 370 of p53. We demonstrate that the level of p53 kbhb is dramatically increased in cultured cells treated with BHB and in thymus tissues of fasted mice, and that CBP catalyze p53 kbhb. We show that p53 kbhb results in lower levels of p53 acetylation and reduced expression of the p53 downstream genes p21 and PUMA, as well as reduced cell growth arrest and apoptosis in cultured cells under p53-activating conditions. Similar results were observed in mouse thymus tissue under starvation conditions, which result in increased concentrations of serum BHB, and in response to genotoxic stress caused by γ-irradiation to activate p53. Our findings thus show that BHB-mediated p53 kbhb is a novel mechanism of p53 activity regulation, which may explain the link between ketone bodies and tumor, and which may provide promising therapeutic target for cancer treatment.