Homeodomain transcription factors in vertebrates: working together to make a difference
Lead Research Organisation:
University of Manchester
Department Name: School of Medical Sciences
Abstract
How do different anatomical features develop with remarkable precision along the body axis of each animal species? We know that this process is determined by a group of proteins belonging to the Hox family. The Hox proteins read and execute instructions encoded in the genome. Each member of the Hox family appears in a well-defined territory of the developing embryo, and recognises specific instructions (most likely DNA sequences) contained in the genome. A puzzling finding is that the Hox specificity, which is central to developmental programs, is entirely lost in a test tube, where all Hox proteins recognise similar DNA sequences. Thus some other essential unknown biochemical factor must be present in the developing embryo. This project will identify Hox functional targets in the genome, and thus reveal the mechanisms that allow Hox proteins to control anatomical development. We will apply state-of-the-art experimental technologies and develop new computational methods to investigate systems where Hox proteins control development and their malfunctioning causes disease. Collectively, these results will identify the fundamental mechanisms that underlie anatomical development in vertebrates and will cast light on how their malfunctioning can result in diseases.
Technical Summary
The remarkable diversity of the anatomical features appearing along the body axis of all animals is achieved through differential regulation of gene expression programs. Hox transcription factors (TFs) are at the core of this process. However the highly specific gene expression programs directed by different Hox proteins in vivo stand in sharp contrast to their general lack of DNA-binding specificity in vitro. How do Hox proteins achieve their specificity? We will build on our recent discoveries to answer this baffling but fundamental question. Using vertebrate model systems, we will apply a combination of epigenomic, transcriptomic and TF binding profiling to study Hox TFs directly in the areas where they operate. We will develop new computational methods to jointly analyse multiple datasets and uncover TF combinatorial binding patterns. This approach will identify the functional binding of different Hox paralogs genome-wide and will define the mechanisms that determine Hox specificity. Collectively, these results will reveal fundamental mechanisms underlying development, disease and evolution in vertebrates and will provide predictive, testable models of TF functional binding.
Planned Impact
This project tackles a fundamental question in biology, namely how do animal species form their diverse anatomical structures?
Our results will be of interest and benefit to multiple parties. Understanding how Hox transcription factors (TFs) operate will clarify how the remarkable diversity of anatomical features along the body axis of animals is achieved, a fundamental goal in developmental biology. In addition Hox TFs are shared by diverse animal species, which display and an astonishing variety in the anatomical structures controlled by these TFs. Thus, defining how Hox TFs operate will also represents a fundamental step forward in our understanding of evolution. Clarifying the fundamental mechanisms that control development and organogenesis will eventually pave the way for the development of stem cell and regenerative medicine approaches targeting new methods of treatment of degenerative diseases, cancer and other pathologies. Hox TFs continue to be expressed into adulthood, where they function in various physiologic and pathologic processes. This project investigates the changes determined by Hox TFs that transform normal blood cells into leukaemic cells: our results will provide insights into the behavior of leukaemic cells and the disease causative mechanisms. This will translate into the possibility to develop new drugs. All of the above will have an important impact on society (the health and well-being of individuals and families). This project includes the development of a new method to facilitate predictions of TFs combinatorial binding. This will provide a valuable new research tool for the community and improve our understanding of gene expression and the functions of the non-coding genome. The interdisciplinary nature of this study will provide opportunities to train junior researchers in the use of next generation sequencing and downstream analyses. Acquiring these highly in-demand skills will eventually benefit the UK research base. To maximize the benefits of our research, we will increase its openness and accessibility and store all data in public online repositories, where they will be freely available to the scientific community. We will also engage with schools, science festivals, local communities and exhibitions, to increase public awareness in the field of genomics research. Translational genomic research is playing an increasingly important role in the diagnosis, monitoring, and treatment of diseases. These advances also raise profound ethical, legal, and social issues related to the use (and possible abuse) of genomic information. Promoting the public understanding of genomics is therefore not only important to support research, but also to promote the changes in society that must accompany scientific and technological developments.
Our results will be of interest and benefit to multiple parties. Understanding how Hox transcription factors (TFs) operate will clarify how the remarkable diversity of anatomical features along the body axis of animals is achieved, a fundamental goal in developmental biology. In addition Hox TFs are shared by diverse animal species, which display and an astonishing variety in the anatomical structures controlled by these TFs. Thus, defining how Hox TFs operate will also represents a fundamental step forward in our understanding of evolution. Clarifying the fundamental mechanisms that control development and organogenesis will eventually pave the way for the development of stem cell and regenerative medicine approaches targeting new methods of treatment of degenerative diseases, cancer and other pathologies. Hox TFs continue to be expressed into adulthood, where they function in various physiologic and pathologic processes. This project investigates the changes determined by Hox TFs that transform normal blood cells into leukaemic cells: our results will provide insights into the behavior of leukaemic cells and the disease causative mechanisms. This will translate into the possibility to develop new drugs. All of the above will have an important impact on society (the health and well-being of individuals and families). This project includes the development of a new method to facilitate predictions of TFs combinatorial binding. This will provide a valuable new research tool for the community and improve our understanding of gene expression and the functions of the non-coding genome. The interdisciplinary nature of this study will provide opportunities to train junior researchers in the use of next generation sequencing and downstream analyses. Acquiring these highly in-demand skills will eventually benefit the UK research base. To maximize the benefits of our research, we will increase its openness and accessibility and store all data in public online repositories, where they will be freely available to the scientific community. We will also engage with schools, science festivals, local communities and exhibitions, to increase public awareness in the field of genomics research. Translational genomic research is playing an increasingly important role in the diagnosis, monitoring, and treatment of diseases. These advances also raise profound ethical, legal, and social issues related to the use (and possible abuse) of genomic information. Promoting the public understanding of genomics is therefore not only important to support research, but also to promote the changes in society that must accompany scientific and technological developments.
Publications
Bobola N
(2017)
From DNA binding to transcriptional activation: Is the TALE complete?
in The Journal of cell biology
Bobola N
(2017)
Homeodomain proteins in action: similar DNA binding preferences, highly variable connectivity.
in Current opinion in genetics & development
Bridoux L
(2020)
HOX paralogs selectively convert binding of ubiquitous transcription factors into tissue-specific patterns of enhancer activation.
in PLoS genetics
Mallen J
(2021)
Molecular Characterization of HOXA2 and HOXA3 Binding Properties.
in Journal of developmental biology
Phuycharoen M
(2020)
Uncovering tissue-specific binding features from differential deep learning.
in Nucleic acids research
Phuycharoen M
(2019)
Uncovering tissue-specific binding features from differential deep learning
Stanney W
(2020)
Combinatorial action of NF-Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish.
in Developmental biology
Description | One of our major questions was if HOXA2 and HOXA3, which in vitro recognize the same DNA sequences, are capable to choose different region of the genome in vivo, as this could explain their ability to control diverse processes in developing mammalian embryos. When the award started, we only knew where HOXA2 binds in the genome, but had no idea about HOXA3. We raised an antibody against HOXA3 and successfully performed HOXA3 ChIP-seq to 'visualize' HOXA3 binding in the genome in the physiological tissues where HOXA3 is active. Analysis of these experiments has established that HOXA2 and HOXA3 bind different genomic regions in vivo, which can explain their specificity of function. We have found that HOXA2 and HOXA3 directly cooperate with TALE transcription factors (MEIS and PBX) . We identified three main determinants of HOX paralog-selective binding, resulting in high-confidence cooperative HOX-TALE binding at different genomic locations: 1) recognition of unique variants of the HOX-PBX motif; 2) differential affinity at shared HOX-PBX motifs and, 3) additional contribution of tissue-specific TFs. Based on our findings, we propose that HOX paralogs operate, alone and in concert with tissue-specific transcription factors, to switch on the function of TALE transcription factors at selected, paralog-specific enhancers. |
Exploitation Route | How transcription factors select their precise sets of target genes is a key biological question, as it can explain how cell behaviour and ultimately cell identity are controlled. Using HOX transcription factors as model, we have identified mechanisms that direct transcription factors to their precise sets of target genes. These mechanisms provide a framework to explain how transcription factors control cell fate and identity. We have generated large scale datasets, which we made publicly available via ArrayExpress. |
Sectors | Education Healthcare Pharmaceuticals and Medical Biotechnology Other |
URL | https://journals.plos.org/plosgenetics/article/authors?id=10.1371/journal.pgen.1009162 |
Description | Guided activation as a model for transcription factor networks determining cell fate |
Amount | £850,000 (GBP) |
Funding ID | BB/T007761/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2020 |
End | 02/2023 |
Description | Role of binding site-flanking DNA conformation in genome-wide recognition by development-associated transcription factors |
Amount | £13,600 (GBP) |
Organisation | UK-India Education and Research Initiative (UKIERI) |
Sector | Academic/University |
Country | United Kingdom |
Start | 06/2019 |
End | 03/2021 |
Title | HOXA3 antibody |
Description | Rabbit polyclonal antibody that recognizes the homeoprotein HOXA3 |
Type Of Material | Antibody |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Identification of HOXA3 occupancy genome wide by chromatin immunoprecipitation sequencing; data are publicly available in ArrayExpress |
Title | deep learning models to identify sequence features predicting differential TF binding |
Description | Transcription factors (TFs) can bind DNA in a cooperative manner, enabling a mutual increase in occupancy. Through this type of interaction, alternative binding sites can be preferentially bound in different tissues to regulate tissue-specific expression programmes. We investigated the application of convolutional neural network (CNN) models to the discovery of sequence features determining cooperative and differential TF binding across tissues. We analysed ChIP-seq data from MEIS, TFs which are broadly expressed across mouse branchial arches, and HOXA2, which is expressed in the second and more posterior branchial arches. By developing models predictive of MEIS differential binding in all three tissues, we were able to accurately predict HOXA2 co-binding sites. We evaluated transfer-like and multitask approaches to regularizing the high-dimensional classification task with a larger regression dataset, allowing for the creation of deeper and more accurate models. We tested the performance of perturbation and gradient-based attribution methods in identifying the HOXA2 sites from differential MEIS data. Our results show that deep regularized models significantly outperform shallow CNNs as well as k-mer methods in the discovery of tissue-specific sites bound in vivo. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | The method has been recently published (Biorxiv 2019, Nucleic Acid Research 2020). We have used this method in a research output currently under review for PLOS genetics nd available as preprint in Biorxiv (doi.org/10.1101/871640 ). |
URL | https://doi.org/10.1093/nar/gkaa009 |
Title | ChIP-seq and RNA-seq from mouse branchial arches |
Description | The ArrayExpress accession numbers for the data sets are: E-MTAB-8608 - ChIP-seq for Pbx on mouse second and posterior branchial arches at E11.5 E-MTAB-8607- ChIP-seq for Hoxa3 on mouse posterior branchial arches at E11.5 E-MTAB-7766: ChIP-seq for Hoxa2 on mouse second branchial arch (BA2) at embryonic day (E) 11.5 E-MTAB-8606 ChIP-seq for Hoxa2 on mouse posterior branchial arches at E11.5 E-MTAB-7767: Meis ChIP-seq on mouse first branchial arch (BA1) and posterior branchial arches connected to outflow tract of the heart (PBA/OFT) at embryonic day (E) 11.5 E- E-MTAB-7966: H3K27Ac_BA1_ChIP-seq, mouse E-MTAB-7963 RNA-seq analysis of the first branchial arch of the mouse embryos at E10.5 and E11.5 |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | The datasets have been released in January 2020 |
URL | https://www.ebi.ac.uk/arrayexpress/experiments |
Title | H3K27Ac ChIP-seq in PBA/OFT and BA2 |
Description | ChiP-seq to detect enhancers in in individual branchial/pharyngeal arches H3K27Ac ChIP-seq_E11.5 PBA/OFT_rep1 H3K27Ac ChIP-seq_E11.5 PBA/OFT_rep2 H3K27Ac ChIP-seq_E11.5 BA2_rep1 H3K27Ac ChIP-seq_E11.5 BA2_rep2 |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | The data has been submitted to ArrayExpress and will become publicly available pending publication of their related manuscript: A tissue-specific, Gata6-driven transcriptional program instructs remodeling of the mature arterial tree by Losa et al. |
URL | https://www.ebi.ac.uk/arrayexpress/ |
Title | Meis ChIP-seq in BA2 and PBA/OFT |
Description | ChIP-seq of Meis in individual branchial/pharyngeal arches Meis ChIP-seq_E11.5 PBA/OFT_rep1 ArrayExpress E-MTAB-5536 Meis ChIP-seq_E11.5 PBA/OFT_rep2 ArrayExpress E-MTAB-5536 Meis ChIP-seq_E11.5 BA2_rep1 ArrayExpress E-MTAB-5536 Meis ChIP-seq_E11.5 BA2_rep2 ArrayExpress E-MTAB-5536 |
Type Of Material | Database/Collection of data |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | The data has been submitted to ArrayExpress and will become publicly available pending publication of their related manuscript: A tissue-specific, Gata6-driven transcriptional program instructs remodeling of the mature arterial tree by Losa et al. |
URL | https://www.ebi.ac.uk/arrayexpress/ |
Description | Hox and TALE |
Organisation | University of Massachusetts |
Department | University of Massachusetts Medical School |
Country | United States |
Sector | Academic/University |
PI Contribution | This is an ongoing collaboration between my group and the group of Professor Charles Sagerstrom. My group helped in the generation and analysis of ChIP-seq datasets |
Collaborator Contribution | The Sagerstrom group helped with experiments in zebrafish, including the generation of mutants and transient transgenics to evaluate the activity of regulatory regions in the genome |
Impact | 1. Ladam F, Stanney W, Donaldson IJ, Yildiz O, Bobola N, Sagerström CG (2018). TALE factors use two distinct functional modes to control an essential zebrafish gene expression program. Elife doi: 10.7554/eLife.36144. 2. Losa M, Latorre V, Andrabi M, Ladam F, Sagerstrom C, Novoa A, Zarrineh P, Bridoux L, Hanley N, Mallo M, Bobola N*. A tissue-specific, Gata6-driven transcriptional program instructs remodeling of the mature arterial tree (2017). Elife doi: 10.7554/eLife.31362. 3. Amin S, Donaldson IJ, Zannino DA, Hensman J, Rattray M, Losa M, Spitz F, Ladam F, Sagerström C, Bobola N* (2015). Hoxa2 selectively enhances Meis binding to change a branchial arch ground state. Dev Cell 32:265-77. |
Start Year | 2014 |
Description | Role of PBX as pioneer transcription factor |
Organisation | University of California, San Francisco |
Department | School of Medicine (UCSF) |
Country | United States |
Sector | Academic/University |
PI Contribution | I was visiting Scholar within the Program in Craniofacial Biology in the laboratory of Prof. Licia Selleri from March to May 2018. We started experiments on the colony of PBX mutant mice and initiated our collaborative project. I met with several Faculty members and gave seminars at UCSF and also UMass (Boston). |
Collaborator Contribution | Prof. Selleri enabled me to collect materials from PBX combined mutants by covering the costs of the mutant PBX strains and providing help from her group members. These material is currently being analysed. |
Impact | There are no outcomes available from this collaboration yet. |
Start Year | 2018 |
Description | Role of binding site-flanking DNA conformation in genome-wide recognition by development-associated transcription factors |
Organisation | Jawaharlal Nehru University, India |
Country | India |
Sector | Academic/University |
PI Contribution | My group hosted a PhD student from JNU (Delhi) in February 2020 and contributed to the online symposium 'Transcriptional dynamics in developmental biology" 20-21 August 2020. My group organised an online symposium on 'Genome regulation in development and disease' with 300 participants on 26th February 2021. |
Collaborator Contribution | Our partner organised the online symposium 'Transcriptional dynamics in developmental biology" 20-21 August 2020. |
Impact | Online symposium 'Transcriptional dynamics in developmental biology' 20-21 August 2020. Online symposium on 'Genome regulation in development and disease' 26th February 2021. This collaboration is multi-disciplinary and involves computer science/bioinformatics and developmental biology |
Start Year | 2019 |
Description | International symposium 'Genome regulation in development and disease' |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | We organised the workshop with the support of UK-India Education and Research Initiative (UKIERI) with the aim of enhancing educational linkages between India and the UK. We have reached about 300 participants, many of which were postgraduate students at India and UK based institutions. |
Year(s) Of Engagement Activity | 2021 |
Description | International symposium, HOX and TALE in Stem Cells, Developmental and Cancer Biology, Nottingham, UK, September 2019 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | I co-organised this meeting with Drs Alexander Thompson, Cinzia Allegrucci and Paloma Ordonez. The meeting was attended by the main experts on homeodomain proteins across Europe. The workshop provided an opportunity for undergraduate and postgraduate students to be exposed to excellent scientific presentations as well as informal discussions. |
Year(s) Of Engagement Activity | 2019 |