The functional evolution of Hox1 proteins
By: Bio Patrika
Tags: Gene, Scicomm, Author Interview, Evolution

Dr. Narendra Pratap Singh’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. Singh is currently a Postdoc the lab of Prof. Robb Krumlauf at Stowers Institute for Medical Research, Kansas City, USA. He published a paper titled “A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins” as the first author in Genes & Development journal (2020).

How would you explain your paper’s key results to the non-scientific community?

Many gene duplications have been observed during the emergence of more complex animals like humans. However, the contribution of these duplications in the emergence of vertebrates from a simple invertebrate animal are poorly understood. To investigate the role of such evolutionary event, we focused on highly conserved HOX proteins that are required for determining body plan in animals. We used the modern gene-editing technologies (that was not possible in the past) to replace a labial Hox gene in the fruit fly with the three related genes in the mouse-HOXA1, B1, and D1. Remarkably, we found that only the mouse HOXA1 in fruit flies restores its original function, but B1 and D1 do not, suggesting that A1 retains an ancestral function that has survived 600 million years of evolution. In-depth analysis revealed that a six amino acid (2% of whole protein) region is critical for the ancestral function of A1, which is important for modulating interactions with other proteins.

The function of HOXB1 and HOXD1 have diverged from the ancestor and neo-functionalized to have roles in facial expression of mammals (absent in invertebrates) and hair follicle development, respectively. This study shows how genes conserve the essential function and diverge to generate novelties. The work paves the way for additional studies on the evolution of protein activity as well as further exploration into the gene duplication and divergence.

Figure 1. The invertebrate labial gene got duplicated into HOXA1, B1, C1 and D1 during vertebrate evolution. The mouse HOXA1 has retained the ancestral function (blue) while HOXB1 (red) and HOXD1 (yellow) are neo-functionalized. The HOXC1 gene was lost in the mammalian lineage (dotted box). B) Different regions of HOXA1 protein are color coded from N to C terminal of the protein. The ancestral activity of HOXA1 protein is distributed across the protein (mentioned on the left side). The C-terminal motif region (CTM: red) is critical for the conserved ancestral activity while the DNA binding homeodomain (HD) and the linker region (LK) have moderate ancestral activity and the N-terminal has only a weak ancestral activity.

“This study shows how genes conserve the essential function and diverge to generate novelties.”

What are the possible consequences of these findings for your research area?

Genome sequencing projects were expected to uncover the emergence of the novel genes/proteins that led to emergence of vertebrates in evolution. However, genome analysis revealed that a large number of genes are common among animals. In addition, in vitro analysis revealed that the homologous transcription factors have very similar DNA binding properties and probably identical functions. The protein sequence analysis and in vitro studies were not adequate to explain the cause of functional diversity among transcription factors. In this study we used functional approaches, where we knocked-in open reading frames of mouse Hox1 orthologs (HOXA1, B1, and D1) at labial locus to replace Drosophila Labial protein using CRISPR/Cas9 technology. We observed that only HOXA1 in fruit flies could restore labial function, but B1 and D1 have diverged and lost most of the ancestral function. This suggests remarkable conservation of ancestral function in HOXA1 through 600 million years of evolution. Further experiments using chimeric proteins revealed a six amino acid sequence critical for the ancestral function of A1, which is important for modulating its interactions with cofactor PBX1. This implies that protein sequence similarity is not always a good predictor of function and small changes during evolution can have a profound impact.

DNA binding properties of Hox proteins are mediated by the highly conserved homeodomain. Here we observed that changes in just six amino acids outside of the homeodomain modifies the function and DNA binding properties through interactions with the cofactor. This implies that protein-protein interaction domains rather than critical DNA binding domains could be a common target of mutations during evolution.

“[…] protein sequence similarity is not always a good predictor of function and small changes during evolution may have a profound impact.”

What was the exciting moment (eureka moment) during your research?

It was unexpected to observe a high degree of labial-like ancestral activity in mouse HOXA1 protein despite 600 million years of evolution.

What do you hope to do next?

During this study, I standardized an effective functional approach to study the function of related proteins. There were many firsts in this study; I want to further expand my analysis to other closely related transcription factors to understand general mechanisms evolution has used to bring novelties in animal body plans.

Where do you seek scientific inspiration?

The immense animal diversity in nature inspires me to study it. During my research journey, I worked with great mentors who trained and inspired me to achieve higher goals.

How do you intend to help Indian science improve?

I am now in the job market and looking for an Assistant Professor position to start my research program. I am looking for positions in India as well to contribute to teaching and research.

Reference

Singh N P, De Kumar B, Paulson A, Parrish M E, Zhang Y, Florens L, Conaway JW, Si K, Krumlauf R. A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins. Genes Dev. 2020. doi: 10.1101/gad.342329.120.

Email: nps@stowers.org

Author research interests.

My long-term research interest lies in understanding the functional evolution of transcription factors (TFs). Several families of TFs evolved early in animal evolution and then expanded during evolution of vertebrate lineage by gene duplication events. These gene duplications are considered as a critical step for emergence of vertebrates from simple invertebrate animals. I am currently expanding the CRISPR/Cas9 gene editing tools in flies to test many evolutionary conserved homologous TFs for their functional conservation and diversification. This will help understand the mechanisms evolution has selected for emergence of novelties in animal body plan.