The role of the nuclear lamina in epigenetic regulation

Lamins directly and indirectly tether chromatin to the nuclear envelope. The observation that human A- and B-type lamins interact with DNA in vitro (Shoeman and Traub, 1990; Gruenbaum and Foisner, 2015), led to the belief that lamins directly sequester chromatin to the nuclear periphery. This view is supported by findings in Drosophila cells, where multiple genes become detached from the nuclear lamina in response to the depletion of a single B-type lamin (Shevelyov et al., 2009; Kohwi et al., 2013), and in C.elegans, where a transgene array can be sequestered to the nuclear lamina by the only lamin, LMN-1 (Mattout et al., 2011). It was later demonstrated that in mammalian cells, peripheral heterochromatin is anchored at the nuclear envelope through a Lamin A/C-dependent mechanism but is complemented by a ‘Lamin B receptor’ (LBR)-dependent one (Yokochi et al., 2009; Kind et al., 2013; Solovei et al., 2013). This redundancy likely explains, why a depletion of all lamins does not significantly alter the landscape of lamina-located genomic loci or the expression of lamina-associated genes in mouse embryonic stem cells (Amendola and van Steensel, 2015; Zheng, Kim and Zheng, 2015; Zheng et al., 2018). In fact, in addition to LBR, several non-lamin proteins, including ‘Barrier-to- autointegration’ (BAF), the nuclear lamina component ‘Proline-rich 14’ (PRR14) and the transmembrane protein emerin, have been reported to contribute to chromatin tethering to the nuclear envelope (Berk, Tifft and Wilson, 2013; Poleshko et al., 2013; Amendola and van Steensel, 2015; Jamin and Wiebe, 2015; Zheng, Kim and Zheng, 2015).

Crucially, heterochromatin plays a central role in directing DNA to the nuclear lamina. Both PRR14 and LBR bind to HP1 (Olins et al., 2010; Poleshko et al., 2013), a heterochromatin mark known to be associated with H3K9me2/3 (Bannister et al., 2001; Lachner et al., 2001). In line

1. Introduction 1.2 The nuclear lamina - a fibrous layer with complex functions

‘Euchromatic Histone Lysine Methyltransferase 2’ (EHMT2/G9A), the enzymes that catalyze methylation of H3K9, relaxes or abolishes chromatin-lamina interactions, indicating that methylation of this histone mark is critical for lamina binding (Pinheiro et al., 2012; Bian et al., 2013; Kind et al., 2013; Harr et al., 2015). Likewise, the heterochromatic histone marks H3K27me3 and H4K20me3 have been reported to be involved in lamina tethering, although their role remains less well defined (Olins et al., 2010; Harr et al., 2015). It is important to note, however, that lamins can also interact with euchromatin, as the nucleoplasmic fraction of Lamin A/C associates with euchromatic regions through an interaction with ‘Lamina-associated polypeptide 2α’ (LAP2α) (Gesson et al., 2016).

Genomic regions in contact with the nuclear lamina are known as ‘Lamina-associated domains’ (LADs) (Figure 3). Mammalian nuclei contain between 1,000 and 1,500 of such domains, typically 10 kb - 10 Mb in size (van Steensel and Belmont, 2017). In some human and murine cell types, they can constitute up to one-third of the genome, thus making them an important characteristic of mammalian epigenomes (van Steensel and Belmont, 2017). LADs are enriched for AT-rich DNA and are characterized by low gene density, low transcriptional levels and an enrichment of the heterochromatin marks H3K9me2/3, as well as H3K27me3 at their boundaries (Figure 3) (Guelen et al., 2008; Wen et al., 2009; Peric-Hupkes et al., 2010; Kind et al., 2013; Harr et al., 2015). Some LADs are conserved between different cell types (constitutive LADs), whereas others vary (facultative LADs) (Peric-Hupkes et al., 2010; Meuleman et al., 2013). Constitutive LADs are enriched for AT-rich DNA sequences and ‘Long interspersed nuclear elements’ (LINEs), and are especially gene-poor (Meuleman et al., 2013; van Steensel and Belmont, 2017). Although the underlying DNA sequences differ, the genomic position and sizes of constitutive LADs are highly conserved between human and mouse genomes (Meuleman et al., 2013), suggesting that they represent a structural backbone anchoring chromatin to the nuclear lamina at specific positions (van Steensel and Belmont, 2017). Facultative LADs, on the other hand, are more gene-dense and less conserved (Meuleman et al., 2013). Importantly, many LADs also vary between mother and daughter cells, indicating a certain randomization after mitosis (Figure 3) (Kind et al., 2013). In fact, single-cell- based experiments with human myeloid leukemia cells have revealed that every LAD has a specific contact frequency at the nuclear lamina (Kind et al., 2015). One explanation for this phenomenon comes from the observation, that LADs partially overlap with nucleoli-associated domains and that the two can switch positions after mitosis (van Koningsbruggen et al., 2010; Kind et al., 2013; van Steensel and Belmont, 2017).

1. Introduction 1.2 The nuclear lamina - a fibrous layer with complex functions

An important question with regard to the LAD interactome is how relative gene positioning affects gene expression. While 5-10 % of LAD-related genes are expressed at high levels, the large majority of them are transcriptionally silent (Guelen et al., 2008; Peric-Hupkes et al., 2010). Consistently, genes gaining nuclear lamina contact during differentiation often get downregulated, whereas genes released to the nuclear interior become activated (Pickersgill et

al., 2006; Peric-Hupkes et al., 2010; Lund et al., 2013; Robson et al., 2016). These dynamics

seem to be dependent on the heterochromatic nature of LADs, as artificial tethering of reporter genes to the nuclear lamina results in their downregulation (Akhtar et al., 2013), and depletion of H3K9me2 leads to an upregulation of LAD-located genes in mouse embryonic stem cells (Yokochi et al., 2009).

Interestingly, despite their heterochromatic nature, LADs do not contain high levels of cytosine methylation. In fact, they largely overlap with ‘Partially methylated domains’ (PMDs), i.e., expansive genomic regions with <70 % average methylation, and thus differ strongly from ‘Highly methylated domains’ (HMDs), which feature >70 % average methylation levels (Lister et

al., 2009; Schroeder et al., 2011). PMDs have been shown to become hypomethylated as a

consequence of accumulated cell divisions during cell culture, as well as in aging and cancer

Figure 3: Schematic model of conserved and variable lamina-associated domains (LADs). Genomic regions in contact with the nuclear lamina are enriched for AT-rich DNA and are characterized by low gene density, low transcriptional levels and an enrichment of the heterochromatin marks H3K9me2/3, as well as H3K27me3 at their boundaries. Some LADs are conserved between different cell types, whereas others vary. Additionally, many LADs are shuffled between mother and daughter cells, giving each LAD its own contact

1. Introduction 1.2 The nuclear lamina - a fibrous layer with complex functions

(Lister et al., 2009; Berman et al., 2012; Salhab et al., 2018; Zhou et al., 2018). This phenomenon has been attributed to both their replication late in S-phase and the absence of H3K36me3 (Aran et al., 2011; Salhab et al., 2018; Zhou et al., 2018).