PIFiA: self-supervised approach for protein functional annotation from single-cell imaging data.

Fluorescence microscopy data describe protein localization patterns at single-cell resolution and have the potential to reveal whole-proteome functional information with remarkable precision. Yet, extracting biologically meaningful representations from cell micrographs remains a major challenge. Existing approaches often fail to learn robust and noise-invariant features or rely on supervised labels for accurate annotations. We developed PIFiA (Protein Image-based Functional Annotation), a self-supervised approach for protein functional annotation from single-cell imaging data. We imaged the global yeast ORF-GFP collection and applied PIFiA to generate protein feature profiles from single-cell images of fluorescently tagged proteins. We show that PIFiA outperforms existing approaches for molecular representation learning and describe a range of downstream analysis tasks to explore the information content of the feature profiles. Specifically, we cluster extracted features into a hierarchy of functional organization, study cell population heterogeneity, and develop techniques to distinguish multi-localizing proteins and identify functional modules. Finally, we confirm new PIFiA predictions using a colocalization assay, suggesting previously unappreciated biological roles for several proteins. Paired with a fully interactive website ( https://thecellvision.org/pifia/ ), PIFiA is a resource for the quantitative analysis of protein organization within the cell.

A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons.

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.

PIFiA: Self-supervised Approach for Protein Functional Annotation from Single-Cell Imaging Data.

Fluorescence microscopy data describe protein localization patterns at single-cell resolution and have the potential to reveal whole-proteome functional information with remarkable precision. Yet, extracting biologically meaningful representations from cell micrographs remains a major challenge. Existing approaches often fail to learn robust and noise-invariant features or rely on supervised labels for accurate annotations. We developed PIFiA, (Protein Image-based Functional Annotation), a self-supervised approach for protein functional annotation from single-cell imaging data. We imaged the global yeast ORF-GFP collection and applied PIFiA to generate protein feature profiles from single-cell images of fluorescently tagged proteins. We show that PIFiA outperforms existing approaches for molecular representation learning and describe a range of downstream analysis tasks to explore the information content of the feature profiles. Specifically, we cluster extracted features into a hierarchy of functional organization, study cell population heterogeneity, and develop techniques to distinguish multi-localizing proteins and identify functional modules. Finally, we confirm new PIFiA predictions using a colocalization assay, suggesting previously unappreciated biological roles for several proteins. Paired with a fully interactive website (https://thecellvision.org/pifia/), PIFiA is a resource for the quantitative analysis of protein organization within the cell.

Nonreplicative functions of the origin recognition complex.

Origin recognition complex (ORC), a heteromeric six-subunit complex, is the central component of the eukaryotic pre-replication complex. Recent data from yeast, frogs, flies and mammals present compelling evidence that ORC and its individual subunits have nonreplicative functions as well. The majority of these functions, such as heterochromatin formation, chromosome condensation, and segregation are dependent on ORC-DNA interactions. Furthermore, ORC is involved in the control of cell division via its participation in centrosome duplication and cytokinesis. Recent findings have also demonstrated a direct interaction between ORC and mRNPs and highlighted an essential role of ORC in mRNA nuclear export. Along with the growth of evolutionary complexity of organisms, ORC complex functions become more elaborate and new functions of the ORC sub-complexes and individual subunits have emerged.

Stability of the PHF10 subunit of PBAF signature module is regulated by phosphorylation: role of ß-TrCP.

The PBAF chromatin-remodeling complexes are multi-protein machines, regulating expression of genes involved in proliferation and differentiation. PHF10 is a subunit of the PBAF essential for its association with chromatin. Mammalian PHF10 is expressed as four ubiquitous isoforms, which are alternatively incorporated in the complex and differ by their influence on transcription of target genes. PHF10 have different domain structure and two of them (PHF10-S isoforms) lack C-terminal PHD domains, which enables their phosphorylation by CK-1. Here we have found that PBAF subunits have low turnover rate, except for PHF10 which has much lower half-life, and is degraded by ß-TrCP. The ß-TrCP knockdown stabilizes PBAF core subunits - BRG1 and BAF155 and specific subunits - PHF10, BAF200, BAF180 and BRD7. PHF10 isoforms contain two non-canonical ß-TrCP degrons and are degraded by ß-TrCP in a phospho-dependent manner. But phosphorylation of PHF10-S degrons by CK-1, contrary to previously described degrons, prevents their degradation. Targeted molecular docking demonstrated that phosphorylated forms of PHF10 bind to ß-TrCP with much lower affinity than non-phosphorylated ones, contrary to previously described degrons. This unorthodox mechanism proposes that phosphorylation of ß-TrCP degrons by CK-1 could not only degrade a set of proteins, but also stabilize a different set of targets.

ORC interacts with THSC/TREX-2 and its subunits promote Nxf1 association with mRNP and mRNA export in Drosophila.

The origin recognition complex (ORC) of eukaryotes associates with the replication origins and initiates the pre-replication complex assembly. In the literature, there are several reports of interaction of ORC with different RNAs. Here, we demonstrate for the first time a direct interaction of ORC with the THSC/TREX-2 mRNA nuclear export complex. The THSC/TREX-2 was purified from the Drosophila embryonic extract and found to bind with a fraction of the ORC. This interaction occurred via several subunits and was essential for Drosophila viability. Also, ORC was associated with mRNP, which was facilitated by TREX-2. ORC subunits interacted with the Nxf1 receptor mediating the bulk mRNA export. The knockdown of Orc5 led to a drop in the Nxf1 association with mRNP, while Orc3 knockdown increased the level of mRNP-bound Nxf1. The knockdown of Orc5, Orc3 and several other ORC subunits led to an accumulation of mRNA in the nucleus, suggesting that ORC participates in the regulation of the mRNP export.

Mammalian cells contain two functionally distinct PBAF complexes incorporating different isoforms of PHF10 signature subunit.

The PBAF subtype of the mammalian chromatin remodeling SWI/SNF complex has wide and diverse functions in transcription regulation and development, being both transcription activator and repressor. However, a mechanism accounting for such functional diversity remains unclear. Human PHF10/BAF45a subunit of the PBAF complex plays an important role in brain development but has not been studied sufficiently. We have shown that the PHF10 gene encodes 2 types of evolutionarily conserved, ubiquitously expressed isoforms that are incorporated into the PBAF complex in a mutually exclusive manner. One isoform contains C-terminal tandem PHD fingers, which in the other isoform are replaced by the consensus sequence for phosphorylation-dependent SUMO 1 conjugation (PDSM). PBAF complexes containing different PHF10 isoforms can bind to the promoters of the same genes but produce different effects on the recruitment of Pol II to the promoter and on the level of gene transcription. In addition, it is only the PBAF with PHD-containing isoform that activates proliferation. Our study demonstrates the existence of functionally different PBAF complexes in mammalian cell. It also provides an insight into the molecular structure and role of human PHF10/BAF45a and characterizes it as an essential PBAF subunit.