Nard Kubben

1 S1 Typical healthy and progeria cell nuclei 2 3 Figure S1. Representative images of typical healthy (top row) and progeria (bottom row) nuclei. 4 Images are shown in four different channels: progerin, DAPI, lamin B1, and γH2AX. The outline of each 5 nucleus (shown in green) was first extracted from the DAPI channel (nuclear shape) and then mapped onto 6 the other three channels. As the images show, typical progeria nuclei have pronounced progerin expression, 7 blebbed nuclear outlines, decreased lamin B1 expression, and high levels of DNA damage (γH2AX). 8 S2 Sorting out the mismatch of images across different channels 9 In Fig 1 (a), Fig 6 and Fig S1, we noticed that nucleus in Lamin B1 channel tends to be 10 larger than its counterpart in DAPI channel. In addition, there is also a shift in the image 11 when we tried to overlay the outline segmented from DAPI channel directly onto Lamin 12 B1 channel. Since our intensity measurements (measurements for Lamin B1, Progerin 13 and γH2AX channel) are based on the outlines segmented in DAPI channel (we did this 14 because cells used in our experiment are perturbed in lamin B1, progerin and γH2AX 15 expression, causing them not reliable for outline segmentation), these two problems can 16 lead to serious mistake. In order to sort out these two issues, we compared measurements 17 between their values calculated based on outline segmented from DAPI channel and 18 outline segmented in lamin B1 channel. A random sample of nuclei in GFP-progerin 19 repressed control are used for this comparison, and four measurements (square root of 20 area, boundary point intensity, mean intensity and standard deviation of intensity) are 21 compared. Plotted in Fig S2 are the results. Each dot in Fig S2 represents a nucleus. As 22 shown in Fig S2 (a), the size of nucleus measured in Lamin B1 channel is constantly 23 larger than DAPI channel, with the average nuclear radius calculated in Lamin B1 24 channel ~ 600 nm longer than in DAPI channel. This difference may be due to the fact 25 that DAPI attaches to DNA while Lamin B1 stain directly attaches to lamina that 26 supports nuclear membrane. As to the shifting, since different cameras are used to 27 capture images in different channels, even though the alignment of cameras was auto-28 corrected, there can still be slight shifts of direction. As shown in Fig S2 (b)-(c), the 29

The past decades have provided remarkable insights into how the eukaryotic cell nucleus and the genome within it are organized. The combined use of imaging, biochemistry and molecular biology approaches has revealed several basic principles of nuclear architecture and function, including the existence of chromatin domains of various sizes, the presence of a large number of non-membranous intranuclear bodies, non-random positioning of genes and chromosomes in 3D space, and a prominent role of the nuclear lamina in organizing genomes. Despite this tremendous progress in elucidating the biological properties of the cell nucleus, many questions remain. Here, we highlight some of the key open areas of investigation in the field of nuclear organization and genome architecture with a particular focus on the mechanisms and principles of higher-order genome organization, the emerging role of liquid phase separation in cellular organization, and the functional role of the nuclear lamina in physiological processes.

Ageing is the predominant risk factor for many common diseases. Human premature ageing diseases are powerful model systems to identify and characterize cellular mechanisms that underpin physiological ageing. Their study also leads to a better understanding of the causes, drivers and potential therapeutic strategies of common diseases associated with ageing, including neurological disorders, diabetes, cardiovascular diseases and cancer. Using the rare premature ageing disorder Hutchinson-Gilford progeria syndrome as a paradigm, we discuss here the shared mechanisms between premature ageing and ageing-associated diseases, including defects in genetic, epigenetic and metabolic pathways; mitochondrial and protein homeostasis; cell cycle; and stem cell-regenerative capacity.

Diseases caused by mutations in lamins A and C (laminopathies) suggest a crucial role for A-type lamins in different cellular processes. Laminopathies mostly affect tissues of mesenchymal origin. As transforming growth factor-b1 (TGF-b1) signalling impinges on the retinoblastoma protein (pRB) and SMADs, we tested the hypothesis that lamins modulate cellular responses to TGF-b1 signalling, via the regulation of these transcription factors in mesenchymal cells. Here, we report that A-type lamins are essential for the inhibition of fibroblast proliferation by TGF-b1. TGF-b1 dephosphorylated pRB through PP2A, both of which, we show, are associated with lamin A/C. In addition, lamin A/C modulates the effect of TGF-b1 on collagen production, a marker of mesenchymal differentiation. Our findings implicate lamin A/C in control of gene activity downstream of TGF-b1, via nuclear phosphatases such as PP2A. This biological function provides a novel explanation for the observed mesenchymal dysfunction in laminopathies.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functioning. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved compounds, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts through lowering the expression of progerin and other A-type lamins. These findings establish a complementary approach to anti-progeroid drug discovery.

Lamina-associated polypeptide 2α (LAP2α) localizes throughout the nucleoplasm and interacts with the fraction of lamins A/C that is not associated with the peripheral nuclear lamina. The LAP2α-lamin A/C complex negatively affects cell proliferation. Lamins A/C are encoded by LMNA, a single heterozygous mutation of which causes Hutchinson-Gilford progeria syndrome (HGPS). This mutation generates the lamin A variant progerin, which we show here leads to loss of LAP2α and nucleoplasmic lamins A/C, impaired proliferation, and down-regulation of extracellular matrix components. Surprisingly, contrary to wild-type cells, ectopic expression of LAP2α in cells expressing progerin restores proliferation and extracellular matrix expression but not the levels of nucleoplasmic lamins A/C. We conclude that, in addition to its cell cycle-inhibiting function with lamins A/C, LAP2α can also regulate extracellular matrix components independently of lamins A/C, which may help explain the proliferation-promoting function of LAP2α in cells expressing progerin.

Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.

Mice overexpressing SIRT6 live longer than wild-type mice while SIRT6 knockout mice exhibit similar degenerative phenotypes as individuals with Hutchinson-Gilford progeria syndrome (HGPS). Thus, we sought to test whether levels of SIRT6 are reduced in cells from individuals with HGPS and whether restored SIRT6 expression may impede premature aging phenotypes. Levels of endogenous SIRT6 and progerin in HGPS and normal fibroblasts were assessed by Western blotting and immunofluorescence. A tetracycline-inducible system was utilized to test whether progerin causes a rapid reduction in SIRT6 protein. SIRT6 was overexpressed in HGPS cells via lentiviral infection with biological endpoints including senescence-associated β-galactosidase (SA-β-gal) positivity, frequency of nuclear atypia, the number of 53BP1-positive DNA damage foci and growth rates. Typical HGPS fibroblasts express lower levels of SIRT6 than fibroblasts from normal and atypical HGPS donors. Experimental induction of progerin did not cause a detectable reduction of SIRT6 protein. However, overexpression of SIRT6 in HGPS cells was associated with a reduced frequency of SA-β-gal positivity, fewer misshapen nuclei, fewer DNA damage foci, and increased growth rates. Typical HGPS fibroblasts exhibit reduced levels of SIRT6 protein via a mechanism that remains to be elucidated. Our findings suggest that restoring SIRT6 expression in HGPS cells may partially impede senescence and the formation of dysmorphic nuclei. © 2015 S. Karger AG, Basel.

Polycomb group (PcG) proteins are part of a transcriptional memory system in
higher eukaryotes, and are necessary to maintain the transcriptional repressed states
of HOX genes. PcG proteins also have a well-known effect on the cell-cycle via
repression of the Cdkn2a/INK4A locus. In order to repress genes the EED/EZH2 PcG
complex deactelyates the histone H3 N-terminal tail, and induces trimethylation of
histone H3 at lysine 27 (H3K27
3M), known to be bound by the human Polycomb
Repressive Complex 1 (hPRC1). Little is known about the regulation of PcG proteins
themselves. PcG proteins are regulated both cell-cycle dependently and
independently. Cell-cycle dependent phosphorylation of the PcG protein Bmi1
correlates inversely with its chromatin association.53 Cell-cycle independent
regulation involves the mitogen activated protein kinase (MAPK) cascades, all
inducing a MAPK activated protein kinase called 3pK, which is an in vivo kinase for
Bmi1.
In the present study we found that stress signaling results in loss of
pericentromeric association of the PcG proteins Bmi1, Ring1A, Ring1B and HPc2.
The PcG protein HPH1 shows a nuclear redistribution upon activation of MAPK
signaling. MAPK induced (cell-cycle independent) phosphorylation of the PcG protein
Bmi1 in addition was shown to result in chromatin dissociation, while HPH1 only
detaches in part form chromatin under these conditions. Immunofluorescence data
indicate that MAPK induced phsophorylation of H3 at serine 28 might be involved in
chromatin dissociation of PcG proteins from the H3K27
3M bound hPRC1 complex.
Upon chromatin dissociation interactions with 3pK are maintained intact.
Preliminary data show that ubiquitination might play a role in the PcG
transcriptional memory system. Overexpression of TAP (Tandem Affinity Purification)
tagged Ring1B (an in vitro ubiquitin E3 ligase) resulted in a 7 kDa increase by 3pK
immunodetection on a Western blot, also detected with an ubiquitin specific antibody,
and pulled down with antisera against 3pK. Further experiments are necessary to
verify whether Ring1B is involved in mono-ubiquitination of 3pK, and to test whether
the hPRC1 complex is involved in histone ubiquitination.