Periodic Reporting for period 1 - I.am.a.LAD (From chromatin fibers to lamina-associated domains: what are the recognition determinants?)
Reporting period: 2021-12-01 to 2023-11-30
About 2 meters of genomic DNA are confined in a 10 µm cell nucleus. Genome folding needs to be controlled at multiple levels to achieve such a level of compaction. One such mechanism involves contacts of the genome with the nuclear lamina (NL), a proteinaceous meshwork that coats the inner nuclear envelope. Genome-NL interactions occur through long stretches of genomic sequences named lamina-associated domains (LADs). Conversely, inter-LADs (iLADs) represent genomic sequences not in contact with the NL. In most human and mouse cell types, there are about 1,000 of these LADs, each spanning 0.1 - 10 Mb and collectively covering about 40% of the genome. LADs are massive structures: a LAD of 3 Mb consists of ~1 mm of linear DNA or roughly 150 µm of nucleosomal 10 nm fibre. However, how the tethering of such a gigantic molecule to the NL is organized, and how it is instructed by its own DNA sequence remains a mystery. The goal of the study is to identify new LAD determinants, driving LAD recognition and targeting to the NL.
Genome organization deregulation is often associated with cancer development. By elucidating fundamental principles governing genome organization, I hope that my research will enable the identification of new targets that could play a role in cancer development or progression. Moreover, a non-functional NL can lead to the development of diseases called laminopathies. However, the extent to which LADs are affected and how they contribute to disorder development remains unknown. By unveiling mechanisms triggering LAD recognition and targeting to the NL, this project will also open new perspectives towards the understanding of laminopathy development.
To gain insight into the forces driving genome–nuclear lamina interactions, I first developed a technique to locally scramble LAD and iLAD sequences. This method consists in 1) randomly relocating loxP sites in the genome by hopping of the Sleeping Beauty (SB) transposable element, and 2) creating local recombinations between them (i.e. deletions and inversions) by Cre-lox recombination. I generated 12 cell lines, each harbouring distinct LAD-iLAD recombinations, and then explored their new LAD pattern. My results show that LAD-NL interactions are multivalent. Interestingly, I identified tethering elements as more potent than others. Those have an autonomous affinity for the NL and can boost the NL association of flanking sequences. Finally, I also show that neighbouring LADs can cooperate, if close enough in the linear space, to boost their association with the NL.
Genome organization deregulation is often associated with cancer development. By elucidating fundamental principles governing genome organization, I hope that my research will enable the identification of new targets that could play a role in cancer development or progression. Moreover, a non-functional NL can lead to the development of diseases called laminopathies. However, the extent to which LADs are affected and how they contribute to disorder development remains unknown. By unveiling mechanisms triggering LAD recognition and targeting to the NL, this project will also open new perspectives towards the understanding of laminopathy development.
To gain insight into the forces driving genome–nuclear lamina interactions, I first developed a technique to locally scramble LAD and iLAD sequences. This method consists in 1) randomly relocating loxP sites in the genome by hopping of the Sleeping Beauty (SB) transposable element, and 2) creating local recombinations between them (i.e. deletions and inversions) by Cre-lox recombination. I generated 12 cell lines, each harbouring distinct LAD-iLAD recombinations, and then explored their new LAD pattern. My results show that LAD-NL interactions are multivalent. Interestingly, I identified tethering elements as more potent than others. Those have an autonomous affinity for the NL and can boost the NL association of flanking sequences. Finally, I also show that neighbouring LADs can cooperate, if close enough in the linear space, to boost their association with the NL.
I initiated the project by developing a technique to locally scramble LAD and iLAD sequences (see attached Figure 1, top panel). It consists of three main steps: 1) genomic integration of a cassette consisting of a SB transposon and two loxP sites, 2) SB hopping, to relocate one of the loxP sites within LAD sequences, and 3) Cre-mediated recombination between the loxP sites, creating either genomic inversions or deletions. After optimizing both SB hopping and Cre-mediated recombination, I generated long-range deletions and inversions ranging from 2.5 kb to 2 Mb and spanning both LAD and iLAD regions. Cre recombination was remarkably efficient (~10% at 2 Mb). This technique, performed in mouse embryonic stem cells, is now fully implemented in the lab.
I established a collection of 12 cell lines, dissecting two neighboring LADs in the mouse genome. I then assessed the LAD pattern in those recombined clones by pA-DamID, a technique already established in the lab. My data show that LADs are tethered to the NL by multiple elements. Interestingly, some elements are more potent than others. While the latter only has a modest affinity for the NL, the former autonomously interacts with the NL and even boosts the association of flanking sequences to the NL. Each of the two LADs inspected contained such potent subregions, suggesting that they could be a common tethering mechanism for LADs at the NL.
I then wondered whether entire LADs could cooperate to strengthen their association with the NL. I show that provided that they are close enough in the linear space, neighboring LADs can indeed cooperate to promote their association with the NL.
LADs are heterochromatic structures often associated with repressive histone marks such as H3K9me2 and H3K9me3. We therefore investigated whether the observed changes in NL interactions were mirrored by changes in H3K9me3. I performed pA-DamID to probe H3K9me3 deposition in several recombined and control cells. Interestingly, the changes in NL association were only partially mirrored by changes in H3K9me3.
Finally, I investigated the functional consequences of NL association and H3K9me3 changes by performing RNA-seq experiments in multiple recombined and control cells. As expected, gain in lamina association correlated with gene downregulation. However, it correlated slightly better with H3K9me3 changes. My findings suggest that changes in H3K9me3 are a slightly more important predictor of the changes in gene expression than changes in NL interactions. In conclusion, scrambling of LAD and iLAD sequences alters NL interactions, H3K9me3, and gene activity.
The project and associated findings were presented multiple times over the years at international conferences, through both posters and oral presentations. It included prestigious ones such as Cold Spring Harbor: Nuclear Organization & Function and Gordon Research Conferences. To better disseminate my results, I also wrote a scientific paper that is open to read by the scientific community on the public repository BioRxiv. I intend to publish those exciting results in a high-impact Open Access journal as soon as possible. To ensure transparency and favour data reproducibility, lab journals will be released along with the publication. Besides, to spread my findings as soon as possible, I used the lab website to update the community about my project, before its publication.
I established a collection of 12 cell lines, dissecting two neighboring LADs in the mouse genome. I then assessed the LAD pattern in those recombined clones by pA-DamID, a technique already established in the lab. My data show that LADs are tethered to the NL by multiple elements. Interestingly, some elements are more potent than others. While the latter only has a modest affinity for the NL, the former autonomously interacts with the NL and even boosts the association of flanking sequences to the NL. Each of the two LADs inspected contained such potent subregions, suggesting that they could be a common tethering mechanism for LADs at the NL.
I then wondered whether entire LADs could cooperate to strengthen their association with the NL. I show that provided that they are close enough in the linear space, neighboring LADs can indeed cooperate to promote their association with the NL.
LADs are heterochromatic structures often associated with repressive histone marks such as H3K9me2 and H3K9me3. We therefore investigated whether the observed changes in NL interactions were mirrored by changes in H3K9me3. I performed pA-DamID to probe H3K9me3 deposition in several recombined and control cells. Interestingly, the changes in NL association were only partially mirrored by changes in H3K9me3.
Finally, I investigated the functional consequences of NL association and H3K9me3 changes by performing RNA-seq experiments in multiple recombined and control cells. As expected, gain in lamina association correlated with gene downregulation. However, it correlated slightly better with H3K9me3 changes. My findings suggest that changes in H3K9me3 are a slightly more important predictor of the changes in gene expression than changes in NL interactions. In conclusion, scrambling of LAD and iLAD sequences alters NL interactions, H3K9me3, and gene activity.
The project and associated findings were presented multiple times over the years at international conferences, through both posters and oral presentations. It included prestigious ones such as Cold Spring Harbor: Nuclear Organization & Function and Gordon Research Conferences. To better disseminate my results, I also wrote a scientific paper that is open to read by the scientific community on the public repository BioRxiv. I intend to publish those exciting results in a high-impact Open Access journal as soon as possible. To ensure transparency and favour data reproducibility, lab journals will be released along with the publication. Besides, to spread my findings as soon as possible, I used the lab website to update the community about my project, before its publication.
My results so far provide a better understanding of LAD biology. It is now clear that there are multiple and diverse interactions between LADs and the NL. Furthermore, my findings provide new insights into the link between NL association, H3K9me3 deposition, and gene expression.
In the upcoming months, I plan to investigate the mechanisms by which LAD potent subregions interact with the NL and boost the NL association of flanking sequences. Additionally, I intend to explore the causal relationship between NL association, H3K9me3 deposition and, gene expression.
My findings will lead to a better understanding of LAD formation, and more broadly, of 3D genome organization. Since genome organization is often disrupted in cancer, I hope that my research will help identify new targets that could play a role in cancer development or progression. Alternatively, this project will also open new perspectives towards the understanding of the development of laminopathies, a class of diseases that originate from a non-functional NL. To summarize, this basic science-oriented project will pave the way for many promising applicative studies.
In the upcoming months, I plan to investigate the mechanisms by which LAD potent subregions interact with the NL and boost the NL association of flanking sequences. Additionally, I intend to explore the causal relationship between NL association, H3K9me3 deposition and, gene expression.
My findings will lead to a better understanding of LAD formation, and more broadly, of 3D genome organization. Since genome organization is often disrupted in cancer, I hope that my research will help identify new targets that could play a role in cancer development or progression. Alternatively, this project will also open new perspectives towards the understanding of the development of laminopathies, a class of diseases that originate from a non-functional NL. To summarize, this basic science-oriented project will pave the way for many promising applicative studies.