Lamina – associated chromatin in the context of the mammalian genome folding

S. V. Ulianov, Y. Y. Shevelyov, S. V. Razin © 2016 S. V. Ulianov et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577


Introduction
The entire nuclear space in mammals is substantially compartmentalized [1].Chromosomes occupy dis tinct territories, whose internal structure is spatially organized at multiple levels.Local and long-range contacts between genomic regions are driven by sto chastic motion of chromatin fiber, specific associa tions of functionally related gene loci, direct pro tein-protein interactions resulting in loop formation, and by the co-occurrence of remote chromosomal segments within nuclear bodies and specific nuclear structures [2].At the whole-chromosome level, mammalian chromatin is partitioned into predomi nantly active and generally repressed compartments, formed by long-range interactions of topologically associating domains (TADs) whose formation, in turn, appears to be driven by the looping between CTCF/cohesin-occupied regions [3].
The nuclear lamina (NL) is the largest structure inside the nucleus.The NL represents a fibrillary protein layer adjacent to inner nuclear membrane and composed of several types of lamins and laminassociated proteins.Components of the NL were found to be directly bound to chromatin and chroma tin-associated regulatory factors.In mammals, about 30-40% of the genome interact with the nuclear lamina [4].Constitutive lamina-associated regions Review ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32. N 5. P 327-333 doi: http://dx.doi.org/10.7124/bc.00092E of the genome (LADs) are typically gene-poor and AT-rich inactive genomic regions ranged between 0.1 and 10 Mb in size and are characterized by high level of H3K9 mono-, di-and three-methylation along with the Polycomb-associated repressive mark H3K27me3 [4,5].A large cohort of studies performed using fluorescence in situ hybridization and various biochemical techniques have revealed that the nuclear periphery in a vicinity of the NL rep resents generally inactive nuclear compartment and accumulates gene loci undergoing transcriptional re pression during development and differentiation [6].
A number of reports on the role of the nuclear lamina in the genome folding were published in last few years.Here, we briefly review several recent ad vances in understanding the chromatin spatial orga nization and its relationships with the chromatin re cruiting to the nuclear lamina.

The overall scheme of the chromatin spatial organization in mammals
Recent progress in the exploration of the animal ge nome spatial structure achieved using various highthroughput 3C-based techniques such as 4C, 5C, Hi-C and capture-C [7] has revealed a complex pat tern of local and long-distance spatial interactions within the interphase chromatin, and the basic prin ciples of the genome folding were disclosed [1,8].At the whole-genome level, the spatial clustering of small chromosomes and large chromosomes with each other was observed in human cells [9] (Fig. 1A).These data corroborate classical cytological obser vations and the results of fluorescence in situ hybrid ization showing that chromosomes occupy distinct, largely non-overlapped chromosome territories within the eukaryotic cell nucleus, and that small gene-rich chromosomes are typically located within the central part of the nucleus whereas large chromo somes are located at the nuclear periphery [10].At the chromosomal level, the interphase chroma tin in mammals is partitioned into A and B chromatin compartments [9] (Fig. 1B).The A-compartment is formed by pairwise long-range (up to throughout the entire chromosome) interactions of gene-dense highly transcribed regions enriched with a broad pat tern of active epigenetic marks.Interacting partners could be single TADs (see below) or arrays of TADs having a length up to dozens of megabases.In con trast, the B-compartment is formed by long-range interactions of inactive parts of the genome and gene deserts.The chromatin compartment profile is con siderably variable among different cell types.During differentiation of the human embryonic stem cells (ESC) a large reconfiguration of chromatin compart ments and an expansion of the B-compartment were observed [11].Genes that were upregulated upon differentiation were preferentially transferred from B to A compartment, whereas downregulated genes predominantly changed the compartment from A to B. Global reorganization of chromatin compart ments was also observed in senescent cells [12,13].Hence, the chromatin compartment profile reflects the functional state of the genome.
Increase of a Hi-C map resolution to approxi mately 50 Kb has revealed the presence of self-in teracting regions 100-1000 Kb in length located side by side along the chromosome and interacting with each other relatively weak[ly] [14, 15] (Fig. 1C).Such regions were initially called topologically as sociating domains (TADs), or contact domains (CDs), and are commonly interpreted as chromatin globules.TADs have a typical size of 100-1000 Kb in mammals and about 50-200 Kb in Drosophila [14,16,17].Mammalian TAD boundaries are en riched with housekeeping and tRNA genes, SINE repetitive elements and CTCF-binding sites [14].In Drosophila, TADs harbor predominantly repressed genomic regions whereas TAD boundaries and in ter-TADs contain active genes (predominantly housekeeping) [17,18].TAD boundaries in mam mals possess prominent enhancer-blocking activity.It has been shown that communication via chromatin loop formation between enhancers and target pro moters typically occurs within the same TAD [19], and TADs colocalize with the so-called "regulatory" domains that delimit zones of enhancer influence [20].Thus, in terms of function, mammalian TADs represent the transcription regulatory units of the ge  Although TAD boundaries are critical genom ic elements preventing abnormal enhancer-promoter communication [21], they do not completely insu late TADs from each other: contact frequency be tween adjacent TADs is only about 2 fold lower than the intra-TAD contact frequency [1].
The further increase of the Hi-C maps resolution up to 1 Kb allowed revealing the abundant presence of CTCF-anchored chromatin loops forming the socalled "loop domains" with a median size of 185 Kb located inside the megabase-sized TADs [22] (Fig. 1D).Approximately 10000 such loops were found in the human genome.About 30% of these loops bring the promoters and enhancers together, and genes associated with the loops are expressed at sig nificantly higher level than the genes whose promot ers are not involved into looping interactions.Interestingly, according to different estimations, 60-90 % of loops [22,23] are formed between conver gent CTCF binding sites that hints the possible mech anism of loop formation based on CTCF protein structural features.The recently proposed model of DNA loop extrusion successfully explains the ob served Hi-C data [24][25][26].However, the molecular machine that actually performs the extrusion (and consequently provides enhancer-promoter communi cation) is currently not found.The main candidates are RNA-polymerase II and the condensin complex [26,27].Along with loop domains, the so-called "or dinary" domains were also observed.Despite the fact [that] the formation of these domains could not be ex plained directly by loop extrusion, the indirect mecha nism could be suggested: the genomic region located between two loop domains is spatially segregated from them that may lead to the increased contact fre quency inside this region as compared to its contact frequency with the flanking loop domains.Thus, CTCF/cohesin-anchored loops represent the basic level of the large-scale chromatin topology in mam mals and are directly involved into long-range tran scriptional regulation.Interestingly, CTCF-anchored loops are not robustly detected in the Drosophila ge nome, and TAD boundaries in Drosophila are not considerably enriched with CTCF binding sites [18].
It denotes that mechanisms of TAD formation may be different in mammals and insects.Recently, we have proposed a model implementing internucleosomal in teractions of non-acetylated repressed chromatin (predominantly deposited within TADs in Drosophila) as the driving force for the TAD formation and main tenance in Drosophila [18].Notably, the same mecha nism could be responsible for the compaction of the extrusion-driven loops into globular structures in mammalian genomes.

The role of the nuclear lamina in chromatin spatial organization
A considerable portion of the mammalian genome (about 30-40 %) is associated with the nuclear lam ina [4].The mechanical aspects of chromatin tether ing to the NL are not fully understood, but there are at least two models [28]: zipping structure and point ed anchors.According to the first model, the whole LAD is recruited to the NL that is supported by the observation that large LADs are typically attached to the NL via long contact runs.The second model pos tulates the existence of a limited number of anchor points within a LAD that cooperatively provide LAD attachment to the NL.The main candidates on the role of such anchors are binding sites for tran scriptional repressors [29][30][31].However, in the both models, H3K9 and H3K27 methylation appear to be crucial for the LAD deposition at the NL, because the readers for these epigenetic marks are located within or are recruited to the lamina [32].
There are several controversial reports on the role of nuclear lamina in the maintenance of the inter phase chromatin structure in mammals.Human fi broblasts expressing dominant-negative form of Lamin-A (progerin) demonstrate a considerable loss of spatial compartmentalization of active and inac tive genome regions as revealed by Hi-C analysis, [an] altered pattern of H3K27me3 distribution and substantial changes of gene expression [33].Microscopic studies have revealed that the loss of Lamin-B1 in mouse fibroblasts results in relocation of a gene-poor chromosome 18 from the lamina to the nuclear interior [34], and in a human colon can cer cell line Lamin-B1 deficiency leads to decon densation of chromosome territories [35].On the other hand, it has been shown that double knockdown of Lamin B1/B2 virtually does not affect the LAD profile and gene expression in mouse ESC [36].To this end, some other proteins localized with in inner nuclear membrane could be responsible for the chromatin positioning at nuclear periphery.The most likely candidates are Lamin-B receptor (LBR) and LEM-proteins such as EMD which were found to interact with chromatin in vivo [37][38][39].

LAD dynamics: lessons from single-cell studies
Dynamic interactions between the nuclear lamina (NL) and interphase chromatin were extensively studied in Bas van Steensel`s laboratory.The first clear evidence for highly dynamic nature of the NLchromatin contacts has been obtained using the m6 A-Tracer technology based on the expression of the fu sion of GFP protein with the DpnI restriction enzyme recognizing methylated adenine in GATC context [40].As adenine-6-methylation is a stable covalent modification, it is inheritable in cell generations al lowing one to track the fate of LADs throughout the cell cycle and after cell division in a living cell ex pressing lamin fused with bacterial Dam-methylase (the enzyme used in DamID technology to methylate adenine in GATC context).It has been shown that chromatin attached to the nuclear lamina possesses remarkably constrained mobility and generally does not migrate to the nuclear interior during interphase.However, LADs stochastically reshuffle after mitosis and some of them could be found in a vicinity of nu cleoli in daughter cells.The next breakthrough tech nology providing the further progress in understand ing the NL-chromatin interaction mechanisms and dynamics is a recently developed single-cell DamID approach [41].The current version of this method is suitable for studying the NL-chromatin contacts in single cells at a resolution of 100 Kb.The results ob tained indicate that about 15% of the genome com posed of constitutive gene-poor LADs associates with the NL in the majority of cells.This finding sug gests the presence of a "scaffold" structure presum ably involved in the overall shaping of the chromo some spatial configuration.In contrast, about 30 % of the genome exhibit a high cell-to-cell variability in the interaction with the nuclear lamina.Interestingly, distantly located loci often establish the contacts with the nuclear lamina in a coordinated manner.Further more, it was found that at distances up to 20 Mb the Hi-C profile moderately correlates with the degree of NL-chromatin contacts.It is tempting to assume that spatial interactions of remote genomic regions with each other may direct the coordinated recruitment of functionally-related loci to the nuclear lamina and thus provide coordinated gene repression.

Concluding remarks
In sum, the nuclear lamina plays a remarkable role in the genome folding and regulation.The further un derstanding of the mechanisms involved into chro matin tethering to the nuclear lamina could be con siderably improved by applying new microscopic and biochemical techniques such as super-resolu tion live-cell imaging and combination of singlecell DamID technique with Hi-C analysis of chro matin configuration in the same cell.

Fig. 1 .
Fig. 1.A schematic representa tion of the mammalian chroma tin spatial organization at differ ent levels, and corresponding illustrative Hi-C maps.A -Chromosomes occupy distinct territories within cell nucleus.Large chromosomes are typi cally located at the nuclear pe riphery, and small ones are de posited within the nuclear inte rior that is manifested in the enrichment of Hi-C-captured contacts within clusters of large and small chromosomes.In the illustrative Hi-C map, shown on the left panel, color intensity represents interaction frequency between the whole chromo somes.B -Active and repressed chromosome regions are largely segregated from each other within the chromosome territo ry forming active A-and re pressed B-compart ment.Color intensity on the illustrative Hi-C represents interaction fre quency within chromatin com partments between extended re gions of the chromosome.C -At megabase-and submega base-scale, chromatin is parti tioned into self-interacting topologically associating do mains (TADs) commonly inter preted as chromatin globules.Color intensity on the illustra tive Hi-C represents interaction frequency between 50 Kb ge nomic bins.D -Inside TADs, CTCF-binding sites interact with each other forming loop domains ranged between ~20-200 Kb in size.These loops of ten bring enhancers and pro moters together providing posi tive transcription regulation.Color intensity on the illustra tive Hi-C represents interaction frequency between 10 Kb ge nomic bins.