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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.

Transposable elements (TEs) are abundant components of constitutive heterochromatin of the most diverse evolutionarily distant organisms. TEs enrichment in constitutive heterochromatin was originally described in the model organism Drosophila melanogaster, but it is now considered as a general feature of this peculiar portion of the genomes. The phenomenon of TE enrichment in constitutive heterochromatin has been proposed to be the consequence of a progressive accumulation of transposable elements caused by both reduced recombination and lack of functional genes in constitutive heterochromatin. However, this view does not take into account classical genetics studies and most recent evidence derived by genomic analyses of heterochromatin in Drosophila and other species. In particular, the lack of functional genes does not seem to be any more a general feature of heterochromatin. Sequencing and annotation of Drosophila melanogaster constitutive heterochromatin have shown that this peculiar genomic compartment contains hundreds of transcriptionally active genes, generally larger in size than that of euchromatic ones. Together, these genes occupy a significant fraction of the genomic territory of heterochromatin. Moreover, transposable elements have been suggested to drive the formation of heterochromatin by recruiting HP1 and repressive chromatin marks. In addition, there are several pieces of evidence that transposable elements accumulation in the heterochromatin might be important for centromere and telomere structure. Thus, there may be more complexity to the relationship between transposable elements and constitutive heterochromatin, in that different forces could drive the dynamic of this phenomenon. Among those forces, preferential transposition may be an important factor. In this article, we present an overview of experimental findings showing cases of transposon enrichment into the heterochromatin and their positive evolutionary interactions with an impact to host genomes.

Background ATP-dependent chromatin remodeling complexes are multi-protein machines highly conserved across eukaryotic genomes. They control sliding and displacing of the nucleosomes, modulating histone-DNA interactions and making nucleosomal DNA more accessible to specific binding proteins during replication, transcription, and DNA repair, which are processes involved in cell division. The SRCAP and p400/Tip60 chromatin remodeling complexes in humans and the related Drosophila Tip60 complex belong to the evolutionary conserved INO80 family, whose main function is promoting the exchange of canonical histone H2A with the histone variant H2A in different eukaryotic species. Some subunits of these complexes were additionally shown to relocate to the mitotic apparatus and proposed to play direct roles in cell division in human cells. However, whether this phenomenon reflects a more general function of remodeling complex components and its evolutionary conservation remains unexplored. Results We have combined cell biology, reverse genetics, and biochemical approaches to study the subcellular distribution of a number of subunits belonging to the SRCAP and p400/Tip60 complexes and assess their involvement during cell division progression in HeLa cells. Interestingly, beyond their canonical chromatin localization, the subunits under investigation accumulate at different sites of the mitotic apparatus (centrosomes, spindle, and midbody), with their depletion yielding an array of aberrant outcomes of mitosis and cytokinesis, thus causing genomic instability. Importantly, this behavior was conserved by the Drosophila melanogaster orthologs tested, despite the evolutionary divergence between fly and humans has been estimated at approximately 780 million years ago. Conclusions Overall, our results support the existence of evolutionarily conserved diverse roles of chromatin remodeling complexes, whereby subunits of the SRCAP and p400/Tip60 complexes relocate from the interphase chromatin to the mitotic apparatus, playing moonlighting functions required for proper execution of cell division.

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.

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.

The TIP60 complex is an evolutionarily conserved, multifunctional chromatin remodeling complex involved in critical cellular processes, including DNA repair, transcription regulation, and cell cycle control. Although its molecular organization and functions have been extensively studied, a comparative synthesis of its context-specific roles across evolutionarily distant species and pathological conditions is important to fully grasp its biological and clinical significance. In this review, we provide an integrative overview of the TIP60 complex, emphasizing its composition and conserved functions in Homo sapiens and Drosophila melanogaster , with comparative insights from plant systems. We explore how TIP60 complex dysregulation contributes to the molecular pathology of cancer and neurodevelopmental disorders, highlighting recent mechanistic insights. We also examine the emerging interplay between TIP60 complex subunits and long non-coding RNAs, which are increasingly recognized as pivotal regulators of genome accessibility and transcriptional programs. Finally, in this intriguing scenario, we highlight the non-canonical functions of the TIP60 complex in mitosis and cytokinesis, underscoring its moonlighting roles in maintaining genomic and cellular integrity, beyond its established contribution to epigenetic regulation. By connecting these diverse aspects, our review aims to provide an integrated perspective on the TIP60 complex and its expanding functional landscape in health and disease.

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.

Genome sequences for most metazoans and plants are incomplete because of the presence of repeated DNA in the heterochromatin. The heterochromatic regions of Drosophila melanogaster contain 20 million bases (Mb) of sequence amenable to mapping, sequence assembly, and finishing. We describe the generation of 15 Mb of finished or improved heterochromatic sequence with the use of available clone resources and assembly methods. We also constructed a bacterial artificial chromosome-based physical map that spans 13 Mb of the pericentromeric heterochromatin and a cytogenetic map that positions 11 Mb in specific chromosomal locations. We have approached a complete assembly and mapping of the nonsatellite component of Drosophila heterochromatin. The strategy we describe is also applicable to generating substantially more information about heterochromatin in other species, including humans.

ATP-dependent chromatin remodeling complexes are involved in nucleosome sliding and eviction and/or the incorporation of histone variants into chromatin to facilitate several cellular and biological processes, including DNA transcription, replication and repair. The DOM/TIP60 chromatin remodeling complex of Drosophila melanogaster contains 18 subunits, including the DOMINO (DOM), an ATPase that catalyzes the exchange of the canonical H2A with its variant (H2A.V), and TIP60, a lysine-acetyltransferase that acetylates H4, H2A and H2A.V histones. In recent decades, experimental evidence has shown that ATP-dependent chromatin remodeling factors, in addition to their role in chromatin organization, have a functional relevance in cell division. In particular, emerging studies suggested the direct roles of ATP-dependent chromatin remodeling complex subunits in controlling mitosis and cytokinesis in both humans and D. melanogaster. However, little is known about their possible involvement during meiosis. The results of this work show that the knockdown of 12 of DOM/TIP60 complex subunits generates cell division defects that, in turn, cause total/partial sterility in Drosophila males, providing new insights into the functions of chromatin remodelers in cell division control during gametogenesis.

The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, we have identified and mapped 53 orthologs of D. melanogaster heterochromatic genes in the genomes of two evolutionarily distant species, D. pseudoobscura and D. virilis. Our results show that the orthologs of the D. melanogaster heterochromatic genes are clustered at three main genomic regions in D. virilis and D. pseudoobscura. In D. virilis, the clusters lie in the middle of euchromatin, while those in D. pseudoobscura are located in the proximal portion of the chromosome arms. Some orthologs map to the corresponding Muller C element in D. pseudoobscura and D. virilis, while others localize on the Muller B element, suggesting that chromosomal rearrangements that have been instrumental in the fusion of two separate elements involved the progenitors of genes currently located in D. melanogaster heterochromatin. These results demonstrate an evolutionary repositioning of gene clusters from ancestral locations in euchromatin to the pericentromeric heterochromatin of descendent D. melanogaster chromosomes. Remarkably, in both D. virilis and D. pseudoobscura the gene clusters show a conserved association with the HP1a protein, one of the most highly evolutionarily conserved epigenetic marks. In light of these results, we suggest a new scenario whereby ancestral HP1-like proteins (and possibly other epigenetic marks) may have contributed to the evolutionary repositioning of gene clusters into heterochromatin.