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In the intricate realm of cellular biology, the identity and function of individual cells and tissues transcend the uniformity of DNA sequences shared by all cells in an organism. Rather, it is the intricate interplay of active genes at a given place and time that determines cellular identity and function. These active genes, transcribed from the DNA template into distinct messenger RNA (mRNA) molecules, encode the proteins essential for cellular function.
At specific genomic regions known as promoters, complex molecular machinery orchestrates the transcription of DNA sequences into mRNA. Interestingly, most genes harbor multiple potential start and end sites for transcription, enabling the production of diverse mRNA variants. This phenomenon of expressing one gene in different forms expands the repertoire and functionality of the genome manifold. However, it also adds a layer of complexity to genome research.
In a groundbreaking study, scientists at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg sought to unravel the extent to which genes employ different start and end sites for transcription, their combinations, and whether they vary under different conditions.
Valérie Hilgers, a research group leader at the MPI-IE, highlights the technical challenge of comprehensively examining this question, stating, "We have to 'read' each and every mRNA molecule from all genes from the very beginning to the very end. This is a humongous task that has not been undertaken before."
To tackle this monumental endeavor, the scientists employed a modified next-generation sequencing technology capable of profiling individual mRNAs. In conventional short-read sequencing, each mRNA is fragmented, amplified, and sequenced, with bioinformatic techniques subsequently reconstructing the fragments into a continuous sequence. To obtain full-length mRNA information for the entire genome across various Drosophila tissues, including the brain, the Hilgers team collaborated with the Deep Sequencing Facility of the MPI to optimize long-read sequencing technologies. Carlos Alfonso-Gonzalez, the first author of the publication, explains, "Long-read sequencing allows for the retrieval of much longer sequencing reads than widely used standard sequencing. However, we even had to optimize this technology and increase the typical read length by several fold to obtain full-length mRNA information in our different model systems."
In addition to studying Drosophila, the Hilgers Lab incorporated cerebral organoids, referred to as "mini-brains," derived from induced pluripotent stem cells, as a human nervous system model. The data collected through this extensive sequencing effort provided unparalleled insights into the transcription of individual genes. Hilgers elucidates their remarkable finding, stating, "We realized that far from start sites (TSSs) and end sites (TESs) being randomly combined one to another, we found that often, sites of transcription start are specifically linked to distinct sites of transcription end." This causal linkage between TSSs and TESs was demonstrated in ovaries, where artificially activating a TSS typically exclusive to the brain overrode the normal TES and induced the utilization of the brain TES. This discovery underscores the critical role of TSSs in shaping the unique RNA landscape of each tissue, thus influencing tissue identity.
Notably, the researchers observed a distinct phenomenon—certain TSSs exhibited unexpected dominance behavior, overpowering conventional signals for transcription termination, out-competing other TSSs, and leading to the selection of specific TESs. These commanding TSSs were aptly named "dominant promoters" by the team. Additionally, they discovered that the interactions between these dominant promoters and their associated gene ends were guided by distinct epigenetic signatures. Importantly, the findings from Drosophila brain cells were corroborated in the human brain organoids, indicating that promoter dominance may serve as a conserved, and potentially universal, mechanism for regulating protein production and cellular functionality.
The implications of this novel mechanism are far-reaching. Through a comprehensive analysis of sequence conservation, the researchers at Freiburg uncovered an intriguing co-evolution between TSSs and TESs. Over millions of years of evolution across different species, they observed that changes in individual nucleotides at gene start sites associated with dominant promoters were accompanied by corresponding changes at the corresponding gene end sites. Hilgers explains the significance of this observation, stating, "We interpret this observation as a 'push' through evolution, to sustain the interaction between both extremities of the gene, which implies significant importance of these couplings for animal fitness."
These findings shed light on the intricate regulatory mechanisms underlying gene transcription and the resulting diversity of mRNA variants. By understanding the interplay between promoters, start sites, and end sites, scientists gain a deeper understanding of how genes are expressed and how this expression contributes to cellular identity and function. Moreover, the identification of dominant promoters provides insights into the hierarchical control of gene expression, where certain promoters hold sway over others, ultimately shaping the RNA landscape and influencing tissue-specific characteristics.
Read the original publication in Cell.
At specific genomic regions known as promoters, complex molecular machinery orchestrates the transcription of DNA sequences into mRNA. Interestingly, most genes harbor multiple potential start and end sites for transcription, enabling the production of diverse mRNA variants. This phenomenon of expressing one gene in different forms expands the repertoire and functionality of the genome manifold. However, it also adds a layer of complexity to genome research.
In a groundbreaking study, scientists at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg sought to unravel the extent to which genes employ different start and end sites for transcription, their combinations, and whether they vary under different conditions.
Valérie Hilgers, a research group leader at the MPI-IE, highlights the technical challenge of comprehensively examining this question, stating, "We have to 'read' each and every mRNA molecule from all genes from the very beginning to the very end. This is a humongous task that has not been undertaken before."
To tackle this monumental endeavor, the scientists employed a modified next-generation sequencing technology capable of profiling individual mRNAs. In conventional short-read sequencing, each mRNA is fragmented, amplified, and sequenced, with bioinformatic techniques subsequently reconstructing the fragments into a continuous sequence. To obtain full-length mRNA information for the entire genome across various Drosophila tissues, including the brain, the Hilgers team collaborated with the Deep Sequencing Facility of the MPI to optimize long-read sequencing technologies. Carlos Alfonso-Gonzalez, the first author of the publication, explains, "Long-read sequencing allows for the retrieval of much longer sequencing reads than widely used standard sequencing. However, we even had to optimize this technology and increase the typical read length by several fold to obtain full-length mRNA information in our different model systems."
In addition to studying Drosophila, the Hilgers Lab incorporated cerebral organoids, referred to as "mini-brains," derived from induced pluripotent stem cells, as a human nervous system model. The data collected through this extensive sequencing effort provided unparalleled insights into the transcription of individual genes. Hilgers elucidates their remarkable finding, stating, "We realized that far from start sites (TSSs) and end sites (TESs) being randomly combined one to another, we found that often, sites of transcription start are specifically linked to distinct sites of transcription end." This causal linkage between TSSs and TESs was demonstrated in ovaries, where artificially activating a TSS typically exclusive to the brain overrode the normal TES and induced the utilization of the brain TES. This discovery underscores the critical role of TSSs in shaping the unique RNA landscape of each tissue, thus influencing tissue identity.
Notably, the researchers observed a distinct phenomenon—certain TSSs exhibited unexpected dominance behavior, overpowering conventional signals for transcription termination, out-competing other TSSs, and leading to the selection of specific TESs. These commanding TSSs were aptly named "dominant promoters" by the team. Additionally, they discovered that the interactions between these dominant promoters and their associated gene ends were guided by distinct epigenetic signatures. Importantly, the findings from Drosophila brain cells were corroborated in the human brain organoids, indicating that promoter dominance may serve as a conserved, and potentially universal, mechanism for regulating protein production and cellular functionality.
The implications of this novel mechanism are far-reaching. Through a comprehensive analysis of sequence conservation, the researchers at Freiburg uncovered an intriguing co-evolution between TSSs and TESs. Over millions of years of evolution across different species, they observed that changes in individual nucleotides at gene start sites associated with dominant promoters were accompanied by corresponding changes at the corresponding gene end sites. Hilgers explains the significance of this observation, stating, "We interpret this observation as a 'push' through evolution, to sustain the interaction between both extremities of the gene, which implies significant importance of these couplings for animal fitness."
These findings shed light on the intricate regulatory mechanisms underlying gene transcription and the resulting diversity of mRNA variants. By understanding the interplay between promoters, start sites, and end sites, scientists gain a deeper understanding of how genes are expressed and how this expression contributes to cellular identity and function. Moreover, the identification of dominant promoters provides insights into the hierarchical control of gene expression, where certain promoters hold sway over others, ultimately shaping the RNA landscape and influencing tissue-specific characteristics.
Read the original publication in Cell.