Title: Lamina-Associated Domains: Links with Chromosome Architecture, Heterochromatin, and Gene Repression
Abstract: In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation. In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation. In metazoan cell nuclei, the inner nuclear membrane is lined by the nuclear lamina (NL), a fibrous layer consisting primarily of type V intermediate filament proteins named lamins. Early electron microscopy revealed the tight apposition of a layer of condensed chromatin adjacent to the NL (Fawcett, 1966Fawcett D.W. On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates.Am. J. Anat. 1966; 119: 129-145Crossref PubMed Scopus (231) Google Scholar) (Figure 1A). Later, DNA fluorescence in situ hybridization (FISH) demonstrated that specific genomic loci are preferentially located at the nuclear periphery, often when these loci exhibit low transcriptional activity (reviewed in Lanctôt et al., 2007Lanctôt C. Cheutin T. Cremer M. Cavalli G. Cremer T. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions.Nat. Rev. Genet. 2007; 8: 104-115Crossref PubMed Scopus (629) Google Scholar). During the past decade, genome-wide mapping methods have identified genomic regions that are in close contact with the NL, termed lamina-associated domains (LADs). LADs are of particular interest for two broad reasons. First, their NL-anchoring helps to establish interphase chromosome topology and thus the overall genome spatial organization. Second, most of the several thousands of genes in LADs are expressed at very low levels, suggesting a role in gene repression. Here, we summarize the features of LADs, the dynamics of their interactions with the NL and recent progress in identifying the molecular mechanisms underlying their interactions with the NL. We also discuss current insights into the functional significance of LADs with respect to transcriptional regulation and the links with other nuclear compartments. We conclude by raising key questions for future investigation. LADs are defined as genomic regions that make molecular contact with the NL. They have been identified primarily using the DamID technology, in which bacterial DNA adenine methyltransferase (Dam) is tethered to a NL protein (typically Lamin B1) leading to adenine methylation of DNA regions that contact the NL protein (Pickersgill et al., 2006Pickersgill H. Kalverda B. de Wit E. Talhout W. Fornerod M. van Steensel B. Characterization of the Drosophila melanogaster genome at the nuclear lamina.Nat. Genet. 2006; 38: 1005-1014Crossref PubMed Scopus (405) Google Scholar). This modification can be visualized by microscopy (Figures 1B and 1C) or mapped genome-wide (Figure 1D). LADs can also be mapped by chromatin immunoprecipitation (ChIP) (Handoko et al., 2011Handoko L. Xu H. Li G. Ngan C.Y. Chew E. Schnapp M. Lee C.W. Ye C. Ping J.L. Mulawadi F. et al.CTCF-mediated functional chromatin interactome in pluripotent cells.Nat. Genet. 2011; 43: 630-638Crossref PubMed Scopus (493) Google Scholar), but this has been technically challenging for reasons that are only partially understood (Gesson et al., 2016Gesson K. Rescheneder P. Skoruppa M.P. von Haeseler A. Dechat T. Foisner R. A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha.Genome Res. 2016; 26: 462-473Crossref PubMed Scopus (124) Google Scholar, Lund et al., 2015Lund E.G. Duband-Goulet I. Oldenburg A. Buendia B. Collas P. Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin.Nucleus. 2015; 6: 30-39Crossref PubMed Scopus (55) Google Scholar). LADs have been mapped in D. melanogaster, C. elegans, and mammalian cell lines (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar, Ikegami et al., 2010Ikegami K. Egelhofer T.A. Strome S. Lieb J.D. Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2.Genome Biol. 2010; 11: R120Crossref PubMed Scopus (140) Google Scholar, Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, Pickersgill et al., 2006Pickersgill H. Kalverda B. de Wit E. Talhout W. Fornerod M. van Steensel B. Characterization of the Drosophila melanogaster genome at the nuclear lamina.Nat. Genet. 2006; 38: 1005-1014Crossref PubMed Scopus (405) Google Scholar). Mouse and human cells have ∼1,000–1,500 LADs, typically 10 kb–10 Mb in size (∼0.5 Mb median), that are distributed along all chromosomes (Figure 1D). They cover more than one-third of the mouse and human genome in individual cell types, making LADs one of the most prominent features of the epigenome. In D. melanogaster, which has a much more compact genome, LADs are ∼5-fold smaller. However, the average gene number per LAD is similar in flies and mammals, suggesting that LAD organization has co-evolved with gene spacing (van Bemmel et al., 2010van Bemmel J.G. Pagie L. Braunschweig U. Brugman W. Meuleman W. Kerkhoven R.M. van Steensel B. The insulator protein SU(HW) fine-tunes nuclear lamina interactions of the Drosophila genome.PLoS ONE. 2010; 5: e15013Crossref PubMed Scopus (73) Google Scholar). In C. elegans, NL-interacting chromatin domains are enriched at the distal parts of each chromosome (González-Aguilera et al., 2014González-Aguilera C. Ikegami K. Ayuso C. de Luis A. Íñiguez M. Cabello J. Lieb J.D. Askjaer P. Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans.Genome Biol. 2014; 15: R21Crossref PubMed Scopus (37) Google Scholar, Ikegami et al., 2010Ikegami K. Egelhofer T.A. Strome S. Lieb J.D. Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2.Genome Biol. 2010; 11: R120Crossref PubMed Scopus (140) Google Scholar). As expected from the observed tight association of condensed chromatin with the NL, LADs possess several molecular features typical of heterochromatin (Table 1). Most genes in LADs are transcriptionally silent or express at low levels (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar, Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). Furthermore, they overlap with regions that replicate late during S-phase (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar, Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, Pope et al., 2014Pope B.D. Ryba T. Dileep V. Yue F. Wu W. Denas O. Vera D.L. Wang Y. Hansen R.S. Canfield T.K. et al.Topologically associating domains are stable units of replication-timing regulation.Nature. 2014; 515: 402-405Crossref PubMed Scopus (539) Google Scholar). LADs also have a low overall gene density and include most “gene deserts,” defined as gene-free genomic regions >1 Mb. LADs are enriched for histone modifications H3K9me2 and H3K9me3 typical of heterochromatin (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar, Wen et al., 2009Wen B. Wu H. Shinkai Y. Irizarry R.A. Feinberg A.P. Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells.Nat. Genet. 2009; 41: 246-250Crossref PubMed Scopus (458) Google Scholar). The facultative heterochromatin mark H3K27me3 is also enriched at LAD boundaries of some cell types (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar, Harr et al., 2015Harr J.C. Luperchio T.R. Wong X. Cohen E. Wheelan S.J. Reddy K.L. Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.J. Cell Biol. 2015; 208: 33-52Crossref PubMed Scopus (181) Google Scholar). Human pericentromeric heterochromatin is also typically included in LADs, as are a subset of telomeric regions (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar). Finally, LADs are not enriched in cytosine methylation. In fact, in colorectal cancer cell genomes, large DNA hypomethylated regions show a strong overlap with LADs (Berman et al., 2011Berman B.P. Weisenberger D.J. Aman J.F. Hinoue T. Ramjan Z. Liu Y. Noushmehr H. Lange C.P. van Dijk C.M. Tollenaar R.A. et al.Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains.Nat. Genet. 2011; 44: 40-46Crossref PubMed Scopus (477) Google Scholar), but this association is not yet understood.Table 1Features of Mammalian LADsLADInter-LADGene densitylowaMore pronounced in cLADs compared to fLADs (Meuleman et al., 2013).highGene expressionlowhighHi-C compartmentBAReplication timinglateearlyRetroelementsLINEaMore pronounced in cLADs compared to fLADs (Meuleman et al., 2013).SINESequence A/T contenthighaMore pronounced in cLADs compared to fLADs (Meuleman et al., 2013).lowHistone marksH3K9me2, H3K9me3, (H3K27me3)aH3K4me1, H3K4me3, H3K27ac, other active marksPericentric heterochromatinfrequentrareNucleolus associationfrequentinfrequenta More pronounced in cLADs compared to fLADs (Meuleman et al., 2013Meuleman W. Peric-Hupkes D. Kind J. Beaudry J.B. Pagie L. Kellis M. Reinders M. Wessels L. van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.Genome Res. 2013; 23: 270-280Crossref PubMed Scopus (280) Google Scholar). Open table in a new tab DamID in various mammalian cell types has shown that some LADs are cell-type invariant (constitutive LADs [cLADs]), while others interact with the NL in only certain cell types (facultative LAD [fLADs]) (Meuleman et al., 2013Meuleman W. Peric-Hupkes D. Kind J. Beaudry J.B. Pagie L. Kellis M. Reinders M. Wessels L. van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.Genome Res. 2013; 23: 270-280Crossref PubMed Scopus (280) Google Scholar, Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). cLADs largely coincide with AT-rich isochores, are rich in LINE (long interspersed nuclear element) and poor in SINE (short interspersed nuclear element) repetitive elements, and form the most gene-poor subset of LADs. cLAD genomic positions and sizes, but not their actual DNA sequences, are strongly conserved between mouse and human (Meuleman et al., 2013Meuleman W. Peric-Hupkes D. Kind J. Beaudry J.B. Pagie L. Kellis M. Reinders M. Wessels L. van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.Genome Res. 2013; 23: 270-280Crossref PubMed Scopus (280) Google Scholar). We speculate that cLADs collectively may form a structural “backbone,” tethering chromosomes to the NL at specific positions and thereby guiding the overall folding of interphase chromosomes. fLADs make up over half of all LADs, are more gene-dense than cLADs, and their positions are less conserved between mouse and human (Meuleman et al., 2013Meuleman W. Peric-Hupkes D. Kind J. Beaudry J.B. Pagie L. Kellis M. Reinders M. Wessels L. van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.Genome Res. 2013; 23: 270-280Crossref PubMed Scopus (280) Google Scholar). During differentiation of mouse cells, hundreds of genes change position relative to the NL, demonstrating the flexibility and dynamics of genome intranuclear spatial organization. Detachment from the NL is frequently accompanied by gene activation while attachment to the NL is frequently accompanied by gene inactivation (Peric-Hupkes et al., 2010Peric-Hupkes D. Meuleman W. Pagie L. Bruggeman S.W. Solovei I. Brugman W. Gräf S. Flicek P. Kerkhoven R.M. van Lohuizen M. et al.Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.Mol. Cell. 2010; 38: 603-613Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar, Robson et al., 2016Robson M.I. de Las Heras J.I. Czapiewski R. Lê Thành P. Booth D.G. Kelly D.A. Webb S. Kerr A.R. Schirmer E.C. Tissue-specific gene repositioning by muscle nuclear membrane proteins enhances repression of critical developmental genes during myogenesis.Mol. Cell. 2016; 62: 834-847Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Below, we will discuss the causality relationships between gene repression and NL interactions in more detail. An extreme form of LAD relocation occurs in the retinal rod cells of nocturnal animals (Solovei et al., 2009Solovei I. Kreysing M. Lanctôt C. Kösem S. Peichl L. Cremer T. Guck J. Joffe B. Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution.Cell. 2009; 137: 356-368Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). In fully differentiated rod cells, most of the heterochromatin normally located at the nuclear periphery coalesces in the center of the nucleus. LINE elements were also found in this central area in rod cells, suggesting that even most cLADs move to the nuclear center. Chromatin within interphase nuclei is mobile, but the motion of an individual chromosome locus is usually confined to a short range of <1 μm (reviewed in Chuang and Belmont, 2007Chuang C.H. Belmont A.S. Moving chromatin within the interphase nucleus-controlled transitions?.Semin. Cell Dev. Biol. 2007; 18: 698-706Crossref PubMed Scopus (49) Google Scholar). Similar observations have been made for LADs in human cells. DNA regions contacting the NL were labeled in vivo through the inducible expression of a Dam-LaminB1 fusion protein and then tracked in live cells throughout a single interphase through their tagging by a fluorescent m6A-binding protein. LADs dynamically interact with the NL, but over a period of hours move only within a layer <1 μm thick (Figure 1B), corresponding to the heterochromatin adjacent to the NL as seen by electron microscopy (Kind et al., 2013Kind J. Pagie L. Ortabozkoyun H. Boyle S. de Vries S.S. Janssen H. Amendola M. Nolen L.D. Bickmore W.A. van Steensel B. Single-cell dynamics of genome-nuclear lamina interactions.Cell. 2013; 153: 178-192Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Thus, LADs form the layer of condensed chromatin lining the nuclear envelope but are mobile and only intermittently contact the NL (Figure 1C). When m6A-tagged LADs were tracked through mitosis, many LADs that were associated with the NL in the mother cell were instead located deep in the nuclear interior of the daughter cells (Kind et al., 2013Kind J. Pagie L. Ortabozkoyun H. Boyle S. de Vries S.S. Janssen H. Amendola M. Nolen L.D. Bickmore W.A. van Steensel B. Single-cell dynamics of genome-nuclear lamina interactions.Cell. 2013; 153: 178-192Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). This suggests LAD nuclear position is partially randomized after each mitosis. Certain genes appear to move to and from the nuclear periphery in a circadian rhythm (Zhao et al., 2015Zhao H. Sifakis E.G. Sumida N. Millán-Ariño L. Scholz B.A. Svensson J.P. Chen X. Ronnegren A.L. Mallet de Lima C.D. Varnoosfaderani F.S. et al.PARP1- and CTCF-mediated interactions between active and repressed chromatin at the lamina promote oscillating transcription.Mol. Cell. 2015; 59: 984-997Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), but this is probably not a general principle. More detailed views have emerged from single-cell DamID maps obtained from over 100 individual G1 cells: certain LADs interact with the NL in almost every cell, while others do so in only a fraction of the cells (Kind et al., 2015Kind J. Pagie L. de Vries S.S. Nahidiazar L. Dey S.S. Bienko M. Zhan Y. Lajoie B. de Graaf C.A. Amendola M. et al.Genome-wide maps of nuclear lamina interactions in single human cells.Cell. 2015; 163: 134-147Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Each LAD has its own characteristic NL contact frequency. Interestingly, LADs contacting the NL in most cells tend to coincide with cLADs and have extremely low gene content (<1 gene/Mb). Thus, a subset of LADs, predominantly cLADs and comprising ∼15% of the genome, may act as specialized genomic regions that robustly anchor chromosomes to the NL. From a molecular perspective, LADs are huge: a typical LAD of 500 kb consists of ∼2,500 nucleosomes. How do such large chromatin domains interact with the NL? Single-cell DamID at a resolution of 100 kb has shown that contacts with the NL typically involve continuous stretches of chromatin up to several Mb in length (Kind et al., 2015Kind J. Pagie L. de Vries S.S. Nahidiazar L. Dey S.S. Bienko M. Zhan Y. Lajoie B. de Graaf C.A. Amendola M. et al.Genome-wide maps of nuclear lamina interactions in single human cells.Cell. 2015; 163: 134-147Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), suggesting multivalent interactions. Given that the NL and chromatin are both polymeric structures, multivalent interaction is perhaps not surprising. At a larger scale, LADs on the same chromosome likely bind cooperatively to the NL. LADs on chromosomes with high LAD densities interact more robustly with the NL than those on chromosomes with low LAD densities (Kind et al., 2015Kind J. Pagie L. de Vries S.S. Nahidiazar L. Dey S.S. Bienko M. Zhan Y. Lajoie B. de Graaf C.A. Amendola M. et al.Genome-wide maps of nuclear lamina interactions in single human cells.Cell. 2015; 163: 134-147Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Visualization of H3K9me2 domains on a single human chromosome showed that these domains, presumably LADs, formed a chromosomal surface touching the NL (Chen et al., 2014bChen X. Yammine S. Shi C. Tark-Dame M. Göndör A. Ohlsson R. The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell.Epigenetics. 2014; 9: 1439-1445Crossref PubMed Scopus (13) Google Scholar). Concerted interactions by multiple LADs may stably secure chromosomes to the NL. To what extent are NL interactions encoded in the DNA sequence? Several studies used microscopy to assay the autonomous targeting of stably integrated LAD-derived DNA sequences to the nuclear periphery. LADs at the human IGH and CYP3A loci contain several DNA regions of 4–32 kb that each conferred NL association when integrated ectopically into a single genomic location. In one instance, this was narrowed down to a simple repeat of GA dinucleotides (Zullo et al., 2012Zullo J.M. Demarco I.A. Piqué-Regi R. Gaffney D.J. Epstein C.B. Spooner C.J. Luperchio T.R. Bernstein B.E. Pritchard J.K. Reddy K.L. Singh H. DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina.Cell. 2012; 149: 1474-1487Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar); however, this sequence motif is not generally enriched in LADs (Guelen et al., 2008Guelen L. Pagie L. Brasset E. Meuleman W. Faza M.B. Talhout W. Eussen B.H. de Klein A. Wessels L. de Laat W. van Steensel B. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.Nature. 2008; 453: 948-951Crossref PubMed Scopus (1320) Google Scholar). Dissecting an ∼200 kb human genomic region encompassing the beta-globin (HBB) locus identified three peripheral targeting regions ranging in size from 6–32 kb (Bian et al., 2013Bian Q. Khanna N. Alvikas J. Belmont A.S. β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications.J. Cell Biol. 2013; 203: 767-783Crossref PubMed Scopus (71) Google Scholar). Each of these regions could redirect an ∼110 kb HBB region from pericentric heterochromatin to the NL; the largest was capable of targeting to the nuclear periphery by itself. A similar analysis of two fLADs identified many fragments (ranging from 0.9 kb to tens of kb) that could promote peripheral localization (Harr et al., 2015Harr J.C. Luperchio T.R. Wong X. Cohen E. Wheelan S.J. Reddy K.L. Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.J. Cell Biol. 2015; 208: 33-52Crossref PubMed Scopus (181) Google Scholar). Because the integration sites were not mapped in this study, it is unclear whether these elements are sufficient for peripheral targeting or rather require a specific context. Nevertheless, the observation again that multiple non-overlapping regions in a LAD exhibit peripheral targeting activity supports the model that LAD-NL interactions are typically multivalent. How LAD-derived sequences drive NL interactions remains enigmatic but may be linked to their chromatin state. Significant progress has been made toward the identification of proteins that facilitate the anchoring of LADs to the NL. Several reports indicate that H3K9 methylation has a prominent role. In C. elegans, transgene repeats adopt a heterochromatic state and are positioned at the nuclear periphery. H3K9 methyltransferases MET-2 and SET-25 are required for this peripheral localization (Towbin et al., 2012Towbin B.D. González-Aguilera C. Sack R. Gaidatzis D. Kalck V. Meister P. Askjaer P. Gasser S.M. Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery.Cell. 2012; 150: 934-947Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). Depletion of both enzymes relocated a transgene array to the nuclear interior and caused a partial loss of NL interactions along most chromosomes. In mammalian cells disruption or inhibition of G9A, a methyltransferase-mediating H3K9me2 deposition weakened NL-LAD association (Bian et al., 2013Bian Q. Khanna N. Alvikas J. Belmont A.S. β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications.J. Cell Biol. 2013; 203: 767-783Crossref PubMed Scopus (71) Google Scholar, Chen et al., 2014bChen X. Yammine S. Shi C. Tark-Dame M. Göndör A. Ohlsson R. The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell.Epigenetics. 2014; 9: 1439-1445Crossref PubMed Scopus (13) Google Scholar, Harr et al., 2015Harr J.C. Luperchio T.R. Wong X. Cohen E. Wheelan S.J. Reddy K.L. Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.J. Cell Biol. 2015; 208: 33-52Crossref PubMed Scopus (181) Google Scholar, Kind et al., 2013Kind J. Pagie L. Ortabozkoyun H. Boyle S. de Vries S.S. Janssen H. Amendola M. Nolen L.D. Bickmore W.A. van Steensel B. Single-cell dynamics of genome-nuclear lamina interactions.Cell. 2013; 153: 178-192Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Analysis of the ∼1 Mb chromosome region encompassing the human HBB locus suggests that both H3K9me2 and H3K9me3 modifications contribute in different LAD regions to anchoring this chromosome region to the NL. Depletion of the two H3K9me3 methyltransferases, Suv39H1 and Suv39H2, caused NL detachment of human HBB BAC transgenes, while inhibition of G9A caused NL detachment of BAC transgenes containing a LAD region several hundred kb distant from the HBB locus (Bian et al., 2013Bian Q. Khanna N. Alvikas J. Belmont A.S. β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications.J. Cell Biol. 2013; 203: 767-783Crossref PubMed Scopus (71) Google Scholar). Only the triple SUV39H1, SUV39H2, and G9A knockdown could peel the endogenous HBB locus and most of an adjacent, ∼1 Mb LAD from the NL, indicating that these enzymes promote NL association in a redundant manner (Bian et al., 2013Bian Q. Khanna N. Alvikas J. Belmont A.S. β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications.J. Cell Biol. 2013; 203: 767-783Crossref PubMed Scopus (71) Google Scholar). A study in C. elegans uncovered a mechanistic link between H3K9 methylation and the NL. A small protein named CEC-4 was found to be anchored in the inner nuclear membrane and to bind mono-, di-, and tri-methylated H3K9 through its chromodomain. Loss of cec-4 detached a heterochromatic transgene array from the NL and reduced genome-wide NL contacts similarly to a met-2/set-25 double mutant (Gonzalez-Sandoval et al., 2015Gonzalez-Sandoval A. Towbin B.D. Kalck V. Cabianca D.S. Gaidatzis D. Hauer M.H. Geng L. Wang L. Yang T. Wang X. et al.Perinuclear anchoring of H3K9-methylated chromatin stabilizes induced cell fate in C. elegans embryos.Cell. 2015; 163: 1333-1347Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). cec-4 does not have obvious orthologs in mammals, but PRR14 is a human protein that may play a somewhat similar LAD-tethering role (Poleshko et al., 2013Poleshko A. Mansfield K.M. Burlingame C.C. Andrake M.D. Shah N.R. Katz R.A. The human protein PRR14 tethers heterochromatin to the nuclear lamina during interphase and mitotic exit.Cell Rep. 2013; 5: 292-301Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). PRR14 contains both a NL targeting domain and a separate domain that binds HP1α, a heterochromatin protein known to bind H3K9me2/3. PRR14 loads onto chromosomes immediately after mitosis via its interaction with HP1α, while later in interphase it requires Lamin A/C for its localization at the NL. Depletion of PRR14 caused a wrinkled NL morphology and partial loss of H3K9me3 at the nuclear periphery, although it has not yet been investigated whether LAD-NL interactions are disrupted. The histone mark H3K27me3 may also be partially linked to LADs. Knockdown of EZH2, the methyltransferase responsible for deposition of this mark, caused reduced NL interactions of an ectopically integrated LAD fragment (Harr et al., 2015Harr J.C. Luperchio T.R. Wong X. Cohen E. Wheelan S.J. Reddy K.L. Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.J. Cell Biol. 2015; 208: 33-52Crossref PubMed Scopus (181) Google Scholar). Further studies are needed to confirm a direct role of H3K27me3 in specific LAD-NL interactions. Perhaps related to this histone modification is the reported role of the DNA-binding factor Ying-Yang 1 (YY1). Artificial tethering of YY1 to a reporter locus caused local accumulation of H3K27me3 and relocation of the locus to the nuclear periphery, while knockdown of YY1 shifted several LADs to the nuclear interior. It is puzzling, however, that the consensus recognition motif of YY1 is globally enriched in DNA outside LADs (Harr et al., 2015Harr J.C. Luperchio T.R. Wong X. Cohen E. Wheelan S.J. Reddy K.L. Directed