Mechanisms of chromatin regulation by lamins

Spatial organization of chromatin within the 3-dimensional space of the nucleus is increasingly recognized as an important additional pathway for gene regulation during various cellular processes. Besides chromatin-regulating proteins that affect wrapping of DNA around nucleosomes or DNA looping, lamins are increasingly emerging as important additional chromatin regulators. Lamins are well known as structural nuclear proteins that form a stable filamentous meshwork at the nuclear envelope, providing mechanical stability. These lamin filaments also link tightly packed chromatin regions to the nuclear periphery, thereby contributing to silencing of genes. Our lab has been focusing on the regulation and functions of a different, poorly studied pool of lamins, which is mobile and distributes throughout the entire nuclear space. We recently showed that these mobile lamins interact with and are regulated by a specific chromatin-binding protein, called lamin-associated polypeptide 2 (LAP2) alpha. Importantly, mobile lamins and LAP2alpha, in stark contrast to the peripheral stable lamin filaments, associate with open chromatin, and changes in the levels of the lamin-LAP2alpha complex caused dramatic changes in chromatin organization. These observations led to the hypothesis that lamin-LAP2alpha complexes are involved in regulating general chromatin accessibility for chromatin modifying complexes, but the molecular mechanism remained elusive. In this project we will elucidate at mechanistic detail how LAP2alpha and lamins affect chromatin using a combination of genome-wide and proteome-wide molecular biological and bioinformatic approaches. Firstly, we will confirm changes in chromatin accessibility upon LAP2alpha depletion, using two different assays combined with deep sequencing (ATAC-seq and MNase-seq), and correlate these changes with changes in binding of lamins to chromatin. Secondly, we will identify which domains in LAP2alpha regulate chromatin accessibility, by expressing LAP2alpha mutants lacking either the chromatin- or lamin-binding domains in LAP2alpha-depleted cells. Thirdly, using chromatin-immunoprecipitation combined with mass spectrometry we will identify LAP2alpha and lamin-interacting chromatin proteins and test whether the functions of the identified candidates change upon LAP2alpha depletion. These data will provide information about cellular pathways involved in LAP2alpha/lamin-mediated chromatin regulation. Lastly, we plan to test the physiological relevance of these pathways for muscle differentiation in in vitro cell culture systems containing or lacking LAP2alpha, and correlate observed changes in chromatin accessibility and lamin-chromatin binding with changes in gene expression and muscle differentiation. Overall, this study will provide novel mechanistic insight into the role of lamins and LAP2alpha in chromatin organization and gene expression during cell differentiation. In view of the involvement of lamin mutations in diseases such as premature aging syndromes, for which changes in chromatin have been reported, our data may also shed light on potential disease mechanisms.

Spatial organization of chromatin within the 3-dimensional struture of the nucleus is an important pathway for the regulation of cell-type specific genes. As a specific example, during muscle regeneration 3D chromatin-organization regulates the generation of muscle-specific proteins to allow formation of new muscle fibers from muscle precursor cells. Structural proteins in the nucleus, called lamins, are well known to affect 3D chromatin organization. One group of lamins forms a stable filamentous protein network at the nuclear periphery that anchors inactive chromatin regions to the periphery contributing to the silencing of non-muscle genes. In contrast, another group of lamins forms highly dynamic complexes with the lamin-binding protein, lamin-associated polypeptide 2 (LAP2) alpha, in the nuclear interior. These lamins bind to active chromatin regions and seem to be involved in the regulation of muscle-specific genes during muscle differentiation. However, neither the mechanisms how lamins and LAP2alpha interact with active chromatin, nor their effect on gene regulation are understood at mechanistic level. Our project addressed both of these important questions. By introducing mutant versions of lamins and LAP2alpha into cells we identified the sub-regions in the proteins involved in binding to each other and in their association with chromatin. We found that a complex cooperation of various protein domains contributes to and regulates these interactions. Most importantly, LAP2alpha negatively affected chromatin binding of lamins by competing for binding sites on chromatin and by recruiting lamins into non-chromatin bound complexes in the nucleus. To address the functions of LAP2alpha and lamins in the regulation of muscle-specific genes, we set up an in vitro system to investigate differentiation of muscle precursor cells into muscle fibers and investigated the interaction of lamins and LAP2alpha with chromatin on a genome-wide level. We found that both LAP2alpha and lamins bind to chromatin regions away from muscle-specific genes in muscle precursor cells, while LAP2alpha, but not lamins, translocates to regions of muscle specific genes during early stages of muscle differentiation, positively affecting the activation of these genes by unknown mechanisms. Deletion of LAP2alpha led to defects in the expression of a subset of these genes and, importantly to a spreading of lamins to muscle genes. Our results show that LAP2alpha regulates muscle-specific gene expression by at least two mechanisms, firstly by promoting efficient activation of muscle specific genes and secondly, by preventing the spreading of lamins to these genes. Overall, our project provides novel insights into mechanisms how lamin chromatin association is regulated and how this affects cell-type specific gene regulation. These findings are not only important for a better understanding of lamin functions, but may also shed light on potential ways to treat muscle diseases caused by mutations in lamins, such as Emery Dreifuss muscular dystrophy.