Brian C. Capell

Human laminopathies: nuclei gone genetically awry

Few genes have generated as much recent interest as LMNA, LMNB1 and LMNB2, which encode the components of the nuclear lamina. Over 180 mutations in these genes are associated with at least 13 known diseases — the laminopathies. In particular, the study of LMNA, its products and the phenotypes that result from its mutation have provided important insights into subjects ranging from transcriptional regulation, the cell biology of the nuclear lamina and mechanisms of ageing. Recent studies have begun the difficult task of correlating the genotypes of laminopathies with their phenotypes, and potential therapeutic strategies using existing drugs, modified oligonucleotides and RNAi are showing real promise for the treatment of these diseases.

A lamin A protein isoform overexpressed in Hutchinson–Gilford progeria syndrome interferes with mitosis in progeria and normal cells

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by dramatic premature aging. Classic HGPS is caused by a de novo point mutation in exon 11 (residue 1824, C → T) of the LMNA gene, activating a cryptic splice donor and resulting in a mutant lamin A (LA) protein termed “progerin/LAΔ50” that lacks the normal cleavage site to remove a C-terminal farnesyl group. During interphase, irreversibly farnesylated progerin/LAΔ50 anchors to the nuclear membrane and causes characteristic nuclear blebbing. Progerin/LAΔ50s localization and behavior during mitosis, however, are completely unknown. Here, we report that progerin/LAΔ50 mislocalizes into insoluble cytoplasmic aggregates and membranes during mitosis and causes abnormal chromosome segregation and binucleation. These phenotypes are largely rescued with either farnesyltransferase inhibitors or a farnesylation-incompetent mutant progerin/LAΔ50. Furthermore, we demonstrate that small amounts of progerin/LAΔ50 exist in normal fibroblasts, and a significant percentage of these progerin/LAΔ50-expressing normal cells are binucleated, implicating progerin/LAΔ50 as causing similar mitotic defects in the normal aging process. Our findings present evidence of mitotic abnormality in HGPS and may shed light on the general phenomenon of aging.

A farnesyltransferase inhibitor prevents both the onset and late progression of cardiovascular disease in a progeria mouse model

Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic form of human premature aging. Death occurs at a mean age of 13 years, usually from heart attack or stroke. Almost all cases of HGPS are caused by a de novo point mutation in the lamin A (LMNA) gene that results in production of a mutant lamin A protein termed progerin. This protein is permanently modified by a lipid farnesyl group, and acts as a dominant negative, disrupting nuclear structure. Treatment with farnesyltransferase inhibitors (FTIs) has been shown to prevent and even reverse this nuclear abnormality in cultured HGPS fibroblasts. We have previously created a mouse model of HGPS that shows progressive loss of vascular smooth muscle cells in the media of the large arteries, in a pattern that is strikingly similar to the cardiovascular disease seen in patients with HGPS. Here we show that the dose-dependent administration of the FTI tipifarnib (R115777, Zarnestra) to this HGPS mouse model can significantly prevent both the onset of the cardiovascular phenotype as well as the late progression of existing cardiovascular disease. These observations provide encouraging evidence for the current clinical trial of FTIs for this rare and devastating disease.

Autophagy mediates degradation of nuclear lamina

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3–lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3–lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

Mechanisms of Cardiovascular Disease in Accelerated Aging Syndromes

In the past several years, remarkable progress has been made in the understanding of the mechanisms of premature aging. These rare, genetic conditions offer valuable insights into the normal aging process and the complex biology of cardiovascular disease. Many of these advances have been made in the most dramatic of these disorders, Hutchinson–Gilford progeria syndrome. Although characterized by features of normal aging such as alopecia, skin wrinkling, and osteoporosis, patients with Hutchinson–Gilford progeria syndrome are affected by accelerated, premature arteriosclerotic disease that leads to heart attacks and strokes at a mean age of 13 years. In this review, we highlight recent advances in the biology of premature aging uncovered in Hutchinson–Gilford progeria syndrome and other accelerated aging syndromes, advances that provide insight into the mechanisms of cardiovascular diseases ranging from atherosclerosis to arrhythmias.

Cytoplasmic chromatin triggers inflammation in senescence and cancer

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS–STING (cyclic GMP–AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin–cGAS–STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Protein farnesylation inhibitors cause donut-shaped cell nuclei attributable to a centrosome separation defect

Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson–Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced “donut formation” and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.

CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma

UNLABELLED Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here, we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E)-activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Furthermore, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma. SIGNIFICANCE Although BRAF(V600E) mutations cause melanocytes to initially proliferate into benign moles, mechanisms responsible for their eventual growth arrest are unknown. Using melanocytes from human moles, we show that BRAF activation leads to a CDKN2B induction that is critical for restraining BRAF oncogenic effects, and when lost, contributes to melanoma.

Human longevity and common variations in the LMNA gene: a meta-analysis

A mutation in the LMNA gene is responsible for the most dramatic form of premature aging, Hutchinson–Gilford progeria syndrome (HGPS). Several recent studies have suggested that protein products of this gene might have a role in normal physiological cellular senescence. To explore further LMNA’s possible role in normal aging, we genotyped 16 SNPs over a span of 75.4 kb of the LMNA gene on a sample of long‐lived individuals (LLI) (US Caucasians with age ≥ 95 years, N = 873) and genetically matched younger controls (N = 443). We tested all common nonredundant haplotypes (frequency ≥ 0.05) based on subgroups of these 16 SNPs for association with longevity. The most significant haplotype, based on four SNPs, remained significant after adjustment for multiple testing (OR = 1.56, P = 2.5 × 10−5, multiple‐testing‐adjusted P = 0.0045). To attempt to replicate these results, we genotyped 3619 subjects from four independent samples of LLI and control subjects from (i) the New England Centenarian Study (NECS) (N = 738), (ii) the Southern Italian Centenarian Study (SICS) (N = 905), (iii) France (N = 1103), and (iv) the Einstein Ashkenazi Longevity Study (N = 702). We replicated the association with the most significant haplotype from our initial analysis in the NECS sample (OR = 1.60, P = 0.0023), but not in the other three samples (P > 0.15). In a meta‐analysis combining all five samples, the best haplotype remained significantly associated with longevity after adjustment for multiple testing in the initial and follow‐up samples (OR = 1.18, P = 7.5 × 10−4, multiple‐testing‐adjusted P = 0.037). These results suggest that LMNA variants may play a role in human lifespan.

Genome-Wide Epigenetics

WhaT is epiGeneTics? The term “epigenetics” was coined by Conrad Waddington to describe “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (Goldberg et al., 2007). Very broadly, the word has come to refer to the study of the regulation of genes, their expression, and how that translates into particular phenotypes, independent of any change to the underlying DNA sequence. More simply stated, epigenetics is the study of functionally relevant changes in gene expression (with subsequent changes in cellular phenotype) that result from mechanisms other than from changes in the underlying DNA nucleotide sequence. Despite the fact that there is no change in the nucleo tide sequence, epigenetic modifications may be heritable and can be passed down to subsequent generations through cell replication and division of alternative chromatin states. This “turning on or off” of genes explains why, despite having the same underlying DNA sequence, a keratinocyte looks and behaves so differently than a hepatocyte and why the epigenetic state is carried over to maintain celland tissue-type specification. Although a given cell’s (or individual’s) genome remains relatively stable over time, the epigenome can and does vary depending on a number of factors, including environmental conditions. These processes allow for many “good” functions, including normal organism development; however, aberrant epigenetic mechanisms are implicated in different disease processes, including malignancies. This article provides a brief overview of the field of epigenetics and offers a glimpse into some of the major techniques used to study it, with a particular focus on chromatin immunoprecipitation followed by sequencing (ChIP-seq), the current standard method for studying proteins and other epigenetic factors that bind to DNA. At the heart of epigenetic control is the organization of DNA into chromatin. This begins with 147 base pairs of DNA wrapped around eight histone proteins, which include the core histones H2A, H2B, H3, and H4 (Figure 1). Each of these histone octamers is referred to as a nucleosome. The nucleosomes are packaged tightly into even more compact fibers known as chromatin. Through this complex structure, epigenetic regulation occurs primarily through four mechanisms. First, DNA can undergo direct chemical modification by cytosine methylation, which is a general marker of gene silencing. Second, posttranslational modifications of the core histones can occur, primarily through methylation, acetylation, ubiquitylation, and phosphorylation, making up the primary chromatin structure (Figure 1). Acting in concert with these two aspects of the epigenetic machinery, noncoding RNAs contribute to the regulation of these processes (Greer and Shi, 2012). Finally, the chromatin is then packaged, via long-range interactions, into a higher-order structure within the cell nucleus. All of these organizational steps serve to modulate DNA accessibility and thus control gene expression. More open regions of chromatin, or euchromatin, are poised for activation by the transcriptional machinery, whereas more ADVANTAGES OF CHIP-SEQ