Chromatin regulation by lamina-independent lamins

Lamins are intermediate filament proteins that assemble into the lamina, a protein scaffold at the nuclear periphery in metazoan cells. The nuclear lamina defines the shape and mechanical properties of the nucleus, anchors heterochromatin to the inner nuclear membrane and affects gene expression and signaling. Lamins are divided into A- and B-types. While B-type lamins are tightly associated with the inner nuclear membrane, A-type lamins are also present as a non-membrane bound and lamina-independent pool throughout the nuclear interior. In this project we test the newly emerging hypothesis that the poorly characterized nucleoplasmic pool of A-type lamins fulfills important functions in chromatin organization and cell fate decision. We have previously identified a specific interaction partner and regulator of nucleoplasmic A-type lamins, termed lamina-associated polypeptide 2 (LAP2) alpha. Deletion of LAP2alpha in mice caused loss of nucleoplamsic A-type lamins and led to a misregulation of tissue progenitor cells. However, the molecular mechanisms are still unclear. Here we will investigate the dynamics and regulation of lamin A/C LAP2alpha interactions during the cell cycle using high-end light microscopy. In addition, we will test the effect of LAP2alpha on in vitro lamin assembly. Furthermore, we aim at analyzing the genome-wide association of A-type lamins and LAP2alpha with chromatin and test how the proteins mutually affect their chromatin binding properties. Analyses of RNA profiles in wild-type versus LAP2alpha deficient cells will reveal how chromatin-binding of LAP2alpha and/or A-type lamins affect gene expression. Using a novel proximity based BioID assay we plan to identify proteins that associate with LAP2alpha and/or A-type lamins on chromatin. Mouse embryonic stem cells, which normally express A-type lamins at very low and LAP2alpha at high levels, will be used to address the physiological relevance of nucleoplasmic lamin-LAP2alpha complexes. Therefore, the chromatin properties and differentiation potential of these cells will be tested upon lamin A over- expression or LAP2alpha downregulation. Based on our preliminary data we also hypothesize that the altered nucleoplasmic pool of A-type lamins caused by disease-linked lamin A/C mutations affects chromatin organization, thereby contributing to the disease phenotype. Thus, we will include two disease-linked mutants in our analyses, the congenital muscular dystrophy-linked delK32 lamin A mutant and the premature ageing-linked progerin lamin variant, which localize exclusively to the nucleoplasm or lamina, respectively. This study is expected to provide completely new insights into molecular mechanisms of lamin-linked chromatin organization and significantly advance our understanding of lamin functions in health and disease.

Lamins are proteins that form a scaffold structure, the nuclear lamina, at the nuclear envelope of mammalian cells. The lamina has been shown to attach inactive genomic regions, called heterochromatic lamina associated domains, to the nuclear envelope to mediate gene silencing. Our research has been focusing on a different pool of lamins, which is not part of the stable lamina scaffold but exists in a highly dynamic complex in the nuclear interior. These nucleoplasmic lamins have received little attention and are underexplored, although emerging data point towards exciting unique roles of this lamin pool in chromatin organization and in lamin-linked diseases, such as the progeria premature aging disease. This project aimed at the analysis of the dynamic behavior and regulation of the nucleoplasmic lamin pool and its role in chromatin organization and progeria disease. We had previously shown that a specific binding protein of lamins, called LAP2alpha, is essential for the existence of the dynamic lamin pool in the nuclear interior, and we hypothesized that in the absence of LAP2alpha nucleoplasmic lamins are completely lost. Using high-end microscopic live cell imaging we surprisingly find here that nucleoplasmic lamins are not lost in LAP2alpha deficient cells, but formed higher order structures that are far less dynamic. We also show that the dynamic behavior of the nucleoplasmic lamins is essential for chromatin regulation in the nuclear interior, which is increasingly recognized as an important additional level of gene expression control. In addition, we find that the nucleoplasmic pool of lamins, together with LAP2alpha, bound not only to heterochromatic genomic regions as previously shown but also to open active chromatin. Removal of LAP2alpha, causing reduced lamin dynamic behavior, changed this chromatin-association pattern completely and affected gene expression. Our data clearly hit new scientific ground, as they show that the role of lamins in chromatin organization and gene regulation is much more complex than thought before. The new hypothesis suggests that lamins do not only contribute to gene silencing in heterochromatin but may serve important functions in controlling gene expression in open, active genomic regions by regulating chromatin structure. Finally we demonstrated the physiological relevance of our finding by testing these proteins in progeria disease. We find that in diseased cells LAP2alpha and nucleoplasmic lamins are lost, leading to growth arrest. Surprisingly, re-expression of LAP2alpha rescued cell growth, most likely through chromatin changes and upregulation of important extracellular matrix genes. Overall, our project identified novel roles of lamins and LAP2alpha in gene regulation. This significantly extends our knowledge on lamin function and may be relevant for premature aging disease and therefore help to develop new therapies for patients.