Targeted protein inactivation (TPI) is an elegant approach to investigate protein function and its role in the cellular landscape, overcoming limitations of genetic perturbation strategies. These systems act in a reversible manner and reduce off-target effects exceeding the limitations of CRISPR/Cas9 and RNA interference, respectively. Several TPI have been developed and wisely improved, including compartment delocalization tools and protein degradation systems. However, unlike chemical tools such as PROTACs (PROteolysis TArgeting Chimeras), which work in a wild-type genomic background, TPI technologies require adding an aminoacidic signal sequence (tag) to the protein of interest (POI). On the other hand, the design and optimization of PROTACs are very laborious and time-consuming. In this review, we focus on anchor-away, deGradFP, auxin-inducible degron (AID) and dTAG technologies and discuss their recent applications and advances. Finally, we propose nano-grad, a novel nanobody-based protein degradation tool, which specifically proteolyzes endogenous tag-free target protein.

The human Cranio Facial Development Protein 1 (Cfdp1) gene maps to chromosome 16q22.2-q22.3 and encodes the CFDP1 protein, which belongs to the evolutionarily conserved Bucentaur (BCNT) family. Craniofacial malformations are developmental disorders of particular biomedical and clinical interest, because they represent the main cause of infant mortality and disability in humans, thus it is important to understand the cellular functions and mechanism of action of the CFDP1 protein. We have carried out a multi-disciplinary study, combining cell biology, reverse genetics and biochemistry, to provide the first in vivo characterization of CFDP1 protein functions in human cells. We show that CFDP1 binds to chromatin and interacts with subunits of the SRCAP chromatin remodeling complex. An RNAi-mediated depletion of CFDP1 in HeLa cells affects chromosome organization, SMC2 condensin recruitment and cell cycle progression. Our findings provide new insight into the chromatin functions and mechanisms of the CFDP1 protein and contribute to our understanding of the link between epigenetic regulation and the onset of human complex developmental disorders.

Background: A variety of human genetic diseases is known to be caused by mutations in genes encoding chromatin factors and epigenetic regulators, such as DNA or histone modifying enzymes and members of ATP-dependent chromatin remodeling complexes. Floating-Harbor syndrome is a rare genetic disease affecting human development caused by dominant truncating mutations in the SRCAP gene, which encodes the ATPase SRCAP, the core catalytic subunit of the homonymous chromatin-remodeling complex. The main function of the SRCAP complex is to promote the exchange of histone H2A with the H2A.Z variant. According to the canonical role played by the SRCAP protein in epigenetic regulation, the Floating-Harbor syndrome is thought to be a consequence of chromatin perturbations. However, additional potential physiological functions of SRCAP have not been sufficiently explored. Results: We combined cell biology, reverse genetics, and biochemical approaches to study the subcellular localization of the SRCAP protein and assess its involvement in cell cycle progression in HeLa cells. Surprisingly, we found that SRCAP associates with components of the mitotic apparatus (centrosomes, spindle, midbody), interacts with a plethora of cytokinesis regulators, and positively regulates their recruitment to the midbody. Remarkably, SRCAP depletion perturbs both mitosis and cytokinesis. Similarly, DOM-A, the functional SRCAP orthologue in Drosophila melanogaster, is found at centrosomes and the midbody in Drosophila cells, and its depletion similarly affects both mitosis and cytokinesis. Conclusions: Our findings provide first evidence suggesting that SRCAP plays previously undetected and evolutionarily conserved roles in cell division, independent of its functions in chromatin regulation. SRCAP may participate in two different steps of cell division: by ensuring proper chromosome segregation during mitosis and midbody function during cytokinesis. Moreover, our findings emphasize a surprising scenario whereby alterations in cell division produced by SRCAP mutations may contribute to the onset of Floating-Harbor syndrome.

Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.

The Drosophila Yeti gene (CG40218) was originally identified by recessive lethal mutation and subsequently mapped to the deep pericentromeric heterochromatin of chromosome 2. Functional studies have shown that Yeti encodes a 241 amino acid protein called YETI belonging to the evolutionarily conserved family of Bucentaur (BCNT) proteins and exhibiting a widespread distribution in animals and plants. Later studies have demonstrated that YETI protein: (i) is able to bind both subunits of the microtubule-based motor kinesin-I; (ii) is required for proper chromosome organization in both mitosis and meiosis divisions; and more recently (iii) is a new subunit of dTip60 chromatin remodeling complex. To date, other functions of YETI counterparts in chicken (CENtromere Protein 29, CENP-29), mouse (Cranio Protein 27, CP27), zebrafish and human (CranioFacial Development Protein 1, CFDP1) have been reported in literature, but the fully understanding of the multifaceted molecular function of this protein family remains still unclear. In this review we comprehensively highlight recent work and provide a more extensive hypothesis suggesting a broader range of YETI protein functions in different cellular processes.

Reporter genes inserted via P-element integration into different locations of the Drosophila melanogaster genome have been routinely used to monitor the functional state of chromatin domains. It is commonly thought that P-element-derived reporter genes are subjected to position effect variegation (PEV) when transposed into constitutive heterochromatin because they acquire heterochromatin-like epigenetic modifications that promote silencing. However, sequencing and annotation of the D. melanogaster genome have shown that constitutive heterochromatin is a genetically and molecularly heterogeneous compartment. In fact, in addition to repetitive DNAs, it harbors hundreds of functional genes, together accounting for a significant fraction of its entire genomic territory. Notably, most of these genes are actively transcribed in different developmental stages and tissues, irrespective of their location in heterochromatin. An open question in the genetic and molecular studies on PEV in D. melanogaster is whether functional heterochromatin domains, i.e., heterochromatin harboring active genes, are able to silence reporter genes therein transposed or, on the contrary, can drive their expression. In this work, we provide experimental evidence showing that strong silencing of the Pw⁺ reporters is induced even when they are integrated within or near actively transcribed loci in the pericentric regions of chromosome 2. Interestingly, some Pw⁺ reporters were found insensitive to the action of a known PEV suppressor. Two of them are inserted within Yeti, a gene expressed in the deep heterochromatin of chromosome 2 which carries active chromatin marks. The difference sensitivity to suppressors-exhibited Pw⁺ reporters supports the view that different epigenetic regulators or mechanisms control different regions of heterochromatin. Together, our results suggest that there may be more complexity regarding the molecular mechanisms underlying PEV.