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Transcription factors (TFs) play a pivotal role in cell fate decision by coordinating gene expression programs. Although most TFs act at the DNA layer, few TFs bind RNA and modulate splicing. Yet, the mechanistic cues underlying TFs activity in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct outcomes in defined cellular contexts. Our results demonstrate a new RNA-binding ability of Ubx. We find that the N51 amino acid of the DNA-binding Homeodomain is non-essential for RNA interaction in vitro, but is required for RNA interaction in vivo and Ubx splicing activity. Moreover, mutation of the N51 amino acid weakens the interaction between Ubx and active RNA Polymerase II (Pol II). Our results reveal that Ubx regulates elongation-coupled splicing, which could be coordinated by a dynamic interplay with active Pol II on chromatin. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster. We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx. We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.

Early lineage-specific master regulators are essential for the specification of cell types. However, once cells are committed to a specific fate, it is critical to restrict the activity of such factors to enable differentiation. To date, it remains unclear how these factors are silenced. Using the Drosophila mesoderm as a model and a comparative genomic approach, we identify the Hox transcription factor Ultrabithorax (Ubx) to be critical for the repression of the master regulator Twist. Mesoderm-specific Ubx loss-of-function experiments using CRISPR-Cas9 and overexpression studies demonstrate that Ubx majorly impacts twist transcription. A mechanistic analysis reveals that Ubx requires the NK-homeodomain protein Tinman to bind to the twist promoter. Furthermore, we find these factor interactions to be critical for silencing by recruiting the Polycomb DNA binding protein Pleiohomeotic. Altogether, our data reveal that Ubx is a critical player in mediating the silencing of Twist, which is crucial for coordinated muscle differentiation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcription factors (TFs) control cell fates by precisely orchestrating gene expression. However, how individual TFs promote transcriptional diversity remains unclear. Here, we use the Hox TF Ultrabithorax (Ubx) as a model to explore how a single TF specifies multiple cell types. Using proximity-dependent Biotin IDentification in Drosophila, we identify Ubx interactomes in three embryonic tissues. We find that Ubx interacts with largely non-overlapping sets of proteins with few having tissue-specific RNA expression. Instead most interactors are active in many cell types, controlling gene expression from chromatin regulation to the initiation of translation. Genetic interaction assays in vivo confirm that they act strictly lineage- and process-specific. Thus, functional specificity of Ubx seems to play out at several regulatory levels and to result from the controlled restriction of the interaction potential by the cellular environment. Thereby, it challenges long-standing assumptions such as differential RNA expression as determinant for protein complexes.

During development cells become restricted in their differentiation potential by repressing alternative cell fates, and the Polycomb complex plays a crucial role in this process. However, how alternative fate genes are lineage-specifically silenced is unclear. We studied Ultrabithorax (Ubx), a multi-lineage transcription factor of the Hox class, in two tissue lineages using sorted nuclei and interfered with Ubx in mesodermal cells. We find that depletion of Ubx leads to the de-repression of genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by retaining the Polycomb Group protein Pleiohomeotic at Ubx targeted genomic regions, thereby stabilizing repressive chromatin marks in a lineage-dependent manner. Our study demonstrates that Ubx stabilizes lineage choice by suppressing the multipotency encoded in the genome via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it could set a block to transdifferentiation in adult cells., Competing Interests: KD, JC, VD, JF, NT, OE, MP, IL No competing interests declared, (© 2019, Domsch et al.)

Apoptosis is a cellular suicide program, which is on the one hand used to remove superfluous cells thereby promoting tissue or organ morphogenesis. On the other hand, the programmed killing of cells is also critical when potentially harmful cells emerge in a developing or adult organism thereby endangering survival. Due to its critical role apoptosis is tightly controlled, however so far, its regulation on the transcriptional level is less studied and understood. Hox genes, a highly conserved gene family encoding homeodomain transcription factors, have crucial roles in development. One of their prominent functions is to shape animal body plans by eliciting different developmental programs along the anterior-posterior axis. To this end, Hox proteins transcriptionally regulate numerous processes in a coordinated manner, including cell-type specification, differentiation, motility, proliferation as well as apoptosis. In this review, we will focus on how Hox proteins control organismal morphology and function by regulating the apoptotic machinery. We will first focus on well-established paradigms of Hox-apoptosis interactions and summarize how Hox transcription factors control morphological outputs and differentially shape tissues along the anterior-posterior axis by fine-tuning apoptosis in a healthy organism. We will then discuss the consequences when this interaction is disturbed and will conclude with some ideas and concepts emerging from these studies., (© 2015 Elsevier Inc. All rights reserved.)