Multi-level transcriptomic analysis of LMNA -related dilated cardiomyopathy identifies disease-driving processes.

LMNA- related dilated cardiomyopathy ( LMNA -DCM) is one of the most severe forms of DCM. The incomplete understanding of the molecular disease mechanisms results in lacking treatment options, leading to high mortality amongst patients. Here, using an inducible, cardiomyocyte-specific lamin A/C depletion mouse model, we conducted a comprehensive transcriptomic study, combining both bulk and single nucleus RNA sequencing, and spanning LMNA -DCM disease progression, to identify potential disease drivers. Our refined analysis pipeline identified 496 genes already misregulated early in disease. The expression of these genes was largely driven by disease specific cardiomyocyte sub-populations and involved biological processes mediating cellular response to DNA damage, cytosolic pattern recognition, and innate immunity. Indeed, DNA damage in LMNA -DCM hearts was significantly increased early in disease and correlated with reduced cardiomyocyte lamin A levels. Activation of cytosolic pattern recognition in cardiomyocytes was independent of cGAS, which is rarely expressed in cardiomyocytes, but likely occurred downstream of other pattern recognition sensors such as IFI16. Altered gene expression in cardiac fibroblasts and immune cell infiltration further contributed to tissue-wide changes in gene expression. Our transcriptomic analysis further predicted significant alterations in cell-cell communication between cardiomyocytes, fibroblasts, and immune cells, mediated through early changes in the extracellular matrix (ECM) in the LMNA -DCM hearts. Taken together, our work suggests a model in which nuclear damage in cardiomyocytes leads to activation of DNA damage responses, cytosolic pattern recognition pathway, and other signaling pathways that activate inflammation, immune cell recruitment, and transcriptional changes in cardiac fibroblasts, which collectively drive LMNA -DCM pathogenesis.

Targeting the ADPKD methylome using nanoparticle-mediated combination therapy.

DNA methylation aberrancies are found in autosomal dominant polycystic kidney disease (ADPKD), which suggests the methylome to be a promising therapeutic target. However, the impact of combining DNA methylation inhibitors (DNMTi) and ADPKD drugs in treating ADPKD and on disease-associated methylation patterns has not been fully explored. To test this, ADPKD drugs, metformin and tolvaptan (MT), were delivered in combination with DNMTi 5-aza-2'-deoxycytidine (Aza) to 2D or 3D cystic Pkd1 heterozygous renal epithelial cells (PKD1-Het cells) as free drugs or within nanoparticles to enable direct delivery for future in vivo applications. We found Aza synergizes with MT to reduce cell viability and cystic growth. Reduced representation bisulfite sequencing (RRBS) was performed across four groups: PBS, Free-Aza (Aza), Free-Aza+MT (F-MTAza), and Nanoparticle-Aza+MT (NP-MTAza). Global methylation patterns showed that while Aza alone induces a unimodal intermediate methylation landscape, Aza+MT recovers the bimodality reminiscent of somatic methylomes. Importantly, site-specific methylation changes associated with F-MTAza and NP-MTAza were largely conserved including hypomethylation at ADPKD-associated genes. Notably, we report hypomethylation of cancer-associated genes implicated in ADPKD pathogenesis as well as new target genes that may provide additional therapeutic effects. Overall, this study motivates future work to further elucidate the regulatory mechanisms of observed drug synergy and apply these combination therapies in vivo.

Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer.

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.

Examining age-dependent DNA methylation patterns and gene expression in the male and female mouse hippocampus.

DNA methylation is a well-characterized epigenetic modification involved in numerous molecular and cellular functions. Methylation patterns have also been associated with aging mechanisms. However, how DNA methylation patterns change within key brain regions involved in memory formation in an age- and sex-specific manner remains unclear. Here, we performed reduced representation bisulfite sequencing (RRBS) from mouse dorsal hippocampus - which is necessary for the formation and consolidation of specific types of memories - in young and aging mice of both sexes. Overall, our findings demonstrate that methylation levels within the dorsal hippocampus are divergent between sexes during aging in genomic features correlating to mRNA functionality, transcription factor binding sites, and gene regulatory elements. These results define age-related changes in the methylome across genomic features and build a foundation for investigating potential target genes regulated by DNA methylation in an age- and sex-specific manner.

DNA methylation analysis reveals epimutation hotspots in patients with dilated cardiomyopathy-associated laminopathies.

BACKGROUND: Mutations in LMNA, encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. RESULTS: Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA-related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutations were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. CONCLUSIONS: Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.

Stochastic modeling reveals kinetic heterogeneity in post-replication DNA methylation.

DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.