Canalizing cell fate by transcriptional repression.

Precision in the establishment and maintenance of cellular identities is crucial for the development of multicellular organisms and requires tight regulation of gene expression. While extensive research has focused on understanding cell type-specific gene activation, the complex mechanisms underlying the transcriptional repression of alternative fates are not fully understood. Here, we provide an overview of the repressive mechanisms involved in cell fate regulation. We discuss the molecular machinery responsible for suppressing alternative fates and highlight the crucial role of sequence-specific transcription factors (TFs) in this process. Depletion of these TFs can result in unwanted gene expression and increased cellular plasticity. We suggest that these TFs recruit cell type-specific repressive complexes to their cis-regulatory elements, enabling them to modulate chromatin accessibility in a context-dependent manner. This modulation effectively suppresses master regulators of alternative fate programs and their downstream targets. The modularity and dynamic behavior of these repressive complexes enables a limited number of repressors to canalize and maintain major and minor cell fate decisions at different stages of development.

The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II.

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.

Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos.

The correct wiring of neuronal circuits is one of the most complex processes in development, since axons form highly specific connections out of a vast number of possibilities. Circuit structure is genetically determined in vertebrates and invertebrates, but the mechanisms guiding each axon to precisely innervate a unique pre-specified target cell are poorly understood. We investigated Drosophila embryonic motoneurons using single-cell genomics, imaging, and genetics. We show that a cell-specific combination of homeodomain transcription factors and downstream immunoglobulin domain proteins is expressed in individual cells and plays an important role in determining cell-specific connections between differentiated motoneurons and target muscles. We provide genetic evidence for a functional role of five homeodomain transcription factors and four immunoglobulins in the neuromuscular wiring. Knockdown and ectopic expression of these homeodomain transcription factors induces cell-specific synaptic wiring defects that are partly phenocopied by genetic modulations of their immunoglobulin targets. Taken together, our data suggest that homeodomain transcription factor and immunoglobulin molecule expression could be directly linked and function as a crucial determinant of neuronal circuit structure.

Role of a versatile peptide motif controlling Hox nuclear export and autophagy in the Drosophila fat body.

Hox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deletion forms of the Drosophila Hox protein Ultrabithorax (Ubx), we identified the presence of an unconventional nuclear export signal (NES) that overlaps with a highly conserved motif originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors that act in development and cancer. We show that this unconventional NES is involved in the interaction with the major exportin protein CRM1 (also known as Embargoed in flies) in vivo and in vitro We find that this interaction is tightly regulated in the Drosophila fat body to control the autophagy-repressive activity of Ubx during larval development. The role of the PBC interaction motif as part of an unconventional NES was also uncovered in other Drosophila and human Hox proteins, highlighting the evolutionary conservation of this novel function. Together, our results reveal the extreme molecular versatility of a unique short peptide motif for controlling the context-dependent activity of Hox proteins both at transcriptional and non-transcriptional levels.

Closing the gap: a roadmap to single-cell regulatory genomics.

Studying the spatiotemporal control of gene regulatory networks at the single-cell level is still a challenge, yet it is key to understanding the mechanisms driving cellular identity. In their recent study, Aerts and colleagues (González-Blas et al, 2020) develop a new strategy to spatially map and integrate single-cell transcriptome and epigenome profiles in the Drosophila eye-antennal disc and to deduce in each cell precise enhancer-to-gene activity relationships. This opens a new era in the transcriptional regulation field, as it allows extracting from each of the thousands of cells forming a tissue the critical features driving their identity, from enhancer sequences to transcription factors to gene regulatory networks.

Stk33 is required for spermatid differentiation and male fertility in mice.

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.

A global circadian repressor controls antiphasic expression of metabolic genes in Neurospora.

The white-collar complex (WCC), the core transcription factor of the circadian clock of Neurospora, activates morning-specific expression of the transcription repressor CSP1. Newly synthesized CSP1 exists in a transient complex with the corepressor RCM1/RCO1 and the ubiquitin ligase UBR1. CSP1 is rapidly hyperphosphorylated and degraded via UBR1 and its ubiquitin conjugase RAD6. Genes controlled by CSP1 are rhythmically expressed and peak in the evening (i.e., in antiphase to morning-specific genes directly controlled by WCC). Rhythmic expression of these second-tier genes depends crucially on phosphorylation and rapid turnover of CSP1, which ensures tight coupling of CSP1 abundance and function to the circadian activity of WCC. Negative feedback of CSP1 on its own transcription buffers the amplitude of CSP1-dependent oscillations against fluctuations of WCC activity. CSP1 predominantly regulates genes involved in metabolism. It controls ergosterol synthesis and fatty acid desaturases and thereby modulates the lipid composition of membranes.

The cis-regulatory code of Hox function in Drosophila.

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

Antagonistic regulation of apoptosis and differentiation by the Cut transcription factor represents a tumor-suppressing mechanism in Drosophila.

Apoptosis is essential to prevent oncogenic transformation by triggering self-destruction of harmful cells, including those unable to differentiate. However, the mechanisms linking impaired cell differentiation and apoptosis during development and disease are not well understood. Here we report that the Drosophila transcription factor Cut coordinately controls differentiation and repression of apoptosis via direct regulation of the pro-apoptotic gene reaper. We also demonstrate that this regulatory circuit acts in diverse cell lineages to remove uncommitted precursor cells in status nascendi and thereby interferes with their potential to develop into cancer cells. Consistent with the role of Cut homologues in controlling cell death in vertebrates, we find repression of apoptosis regulators by Cux1 in human cancer cells. Finally, we present evidence that suggests that other lineage-restricted specification factors employ a similar mechanism to put the brakes on the oncogenic process.

Analysis of Bub3 and Nup75 in the Drosophila male germline lineage.

Extensive communication at the stem cell-niche interface and asymmetric stem cell division is key for the homeostasis of the Drosophila male germline stem cell system. To improve our understanding of these processes, we analysed the function of the mitotic checkpoint complex (MCC) component Bub3 and the nucleoporin Nup75, a component of the nuclear pore complex realizing the transport of signalling effector molecules to the nucleus, in the Drosophila testis. By lineage-specific interference, we found that the two genes control germline development and maintenance. Bub3 is continuously required in the germline, as its loss results in the beginning in an over-proliferation of early germ cells and later on in loss of the germline. The absence of the germline lineage in such testes has dramatic cell non-autonomous consequences, as cells co-expressing markers of hub and somatic cyst cell fates accumulate and populate in extreme cases the whole testis. Our analysis of Nups showed that some of them are critical for lineage maintenance, as their depletion results in the loss of the affected lineage. In contrast, Nup75 plays a role in controlling proliferation of early germ cells but not differentiating spermatogonia and seems to be involved in keeping hub cells quiescent. In sum, our analysis shows that Bub3 and Nup75 are required for male germline development and maintenance.

Multifactorial regulation of a hox target gene.

Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior-posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper (rpr) by the Hox protein Deformed (Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements (HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo.

Specificity of the Hox member Deformed is determined by transcription factor levels and binding site affinities.

Hox proteins have similar binding specificities in vitro, yet they control different morphologies in vivo. This paradox has been partially solved with the identification of Hox low-affinity binding sites. However, anterior Hox proteins are more promiscuous than posterior Hox proteins, raising the question how anterior Hox proteins achieve specificity. We use the AP2x enhancer, which is activated in the maxillary head segment by the Hox TF Deformed (Dfd). This enhancer lacks canonical Dfd-Exd sites but contains several predicted low-affinity sites. Unexpectedly, these sites are strongly bound by Dfd-Exd complexes and their conversion into optimal Dfd-Exd sites results only in a modest increase in binding strength. These small variations in affinity change the sensitivity of the enhancer to different Dfd levels, resulting in perturbed AP-2 expression and maxillary morphogenesis. Thus, Hox-regulated morphogenesis seems to result from the co-evolution of Hox binding affinity and Hox dosage for precise target gene regulation.

Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture.

Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM, ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.

An Evolutionary Perspective on Hox Binding Site Preferences in Two Different Tissues.

Transcription factor (TF) networks define the precise development of multicellular organisms. While many studies focused on TFs expressed in specific cell types to elucidate their contribution to cell specification and differentiation, it is less understood how broadly expressed TFs perform their precise functions in the different cellular contexts. To uncover differences that could explain tissue-specific functions of such TFs, we analyzed here genomic chromatin interactions of the broadly expressed Drosophila Hox TF Ultrabithorax (Ubx) in the mesodermal and neuronal tissues using bioinformatics. Our investigations showed that Ubx preferentially interacts with multiple yet tissue-specific chromatin sites in putative regulatory regions of genes in both tissues. Importantly, we found the classical Hox/Ubx DNA binding motif to be enriched only among the neuronal Ubx chromatin interactions, whereas a novel Ubx-like motif with rather low predicted Hox affinities was identified among the regions bound by Ubx in the mesoderm. Finally, our analysis revealed that tissues-specific Ubx chromatin sites are also different with regards to the distribution of active and repressive histone marks. Based on our data, we propose that the tissue-related differences in Ubx binding behavior could be a result of the emergence of the mesoderm as a new germ layer in triploblastic animals, which might have required the Hox TFs to relax their binding specificity.

The Hox Transcription Factor Ubx Ensures Somatic Myogenesis by Suppressing the Mesodermal Master Regulator Twist.

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.

Hox dosage contributes to flight appendage morphology in Drosophila.

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.

Vectors for efficient and high-throughput construction of fluorescent drosophila reporters using the PhiC31 site-specific integration system.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements or enhancers. Here, we present a rapid and highly efficient system for the large-scale analysis of enhancer elements, site-specifically integrated into the Drosophila genome. This system, which is scalable for either small projects or high-throughput approaches, makes use of the Gateway cloning technology and the PhiC31 site-specific integration system, which allows the insertion of constructs at predetermined genomic locations. Thus, this system allows not only a fast and easy analysis of reporter gene expression in live animals, but also the simultaneous analysis of different regulatory outputs on a cellular resolution by recombining in the same animal distinct enhancer elements fused to different fluorescent proteins.

Shaping segments: Hox gene function in the genomic age.

Despite decades of research, morphogenesis along the various body axes remains one of the major mysteries in developmental biology. A milestone in the field was the realisation that a set of closely related regulators, called Hox genes, specifies the identity of body segments along the anterior-posterior (AP) axis in most animals. Hox genes have been highly conserved throughout metazoan evolution and code for homeodomain-containing transcription factors. Thus, they exert their function mainly through activation or repression of downstream genes. However, while much is known about Hox gene structure and molecular function, only a few target genes have been identified and studied in detail. Our knowledge of Hox downstream genes is therefore far from complete and consequently Hox-controlled morphogenesis is still poorly understood. Genome-wide approaches have facilitated the identification of large numbers of Hox downstream genes both in Drosophila and vertebrates, and represent a crucial step towards a comprehensive understanding of how Hox proteins drive morphological diversification. In this review, we focus on the role of Hox genes in shaping segmental morphologies along the AP axis in Drosophila, discuss some of the conclusions drawn from analyses of large target gene sets and highlight methods that could be used to gain a more thorough understanding of Hox molecular function. In addition, the mechanisms of Hox target gene regulation are considered with special emphasis on recent findings and their implications for Hox protein specificity in the context of the whole organism.