DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina.

A large fraction of the mammalian genome is organized into inactive chromosomal domains along the nuclear lamina. The mechanism by which these lamina associated domains (LADs) are established remains to be elucidated. Using genomic repositioning assays, we show that LADs, spanning the developmentally regulated IgH and Cyp3a loci contain discrete DNA regions that associate chromatin with the nuclear lamina and repress gene activity in fibroblasts. Lamina interaction is established during mitosis and likely involves the localized recruitment of Lamin B during late anaphase. Fine-scale mapping of LADs reveals numerous lamina-associating sequences (LASs), which are enriched for a GAGA motif. This repeated motif directs lamina association and is bound by the transcriptional repressor cKrox, in a complex with HDAC3 and Lap2ß. Knockdown of cKrox or HDAC3 results in dissociation of LASs/LADs from the nuclear lamina. These results reveal a mechanism that couples nuclear compartmentalization of chromatin domains with the control of gene activity.

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole-cell mechanics.

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the lamin­LINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics.

Initiation of allelic exclusion by stochastic interaction of Tcrb alleles with repressive nuclear compartments.

Studies of antigen-receptor loci have linked directed monoallelic association with pericentromeric heterochromatin to the initiation or maintenance of allelic exclusion. Here we provide evidence for a fundamentally different basis for T cell antigen receptor-beta (Tcrb) allelic exclusion. Using three-dimensional immunofluorescence in situ hybridization, we found that germline Tcrb alleles associated stochastically and at high frequency with the nuclear lamina or with pericentromeric heterochromatin in developing thymocytes and that such interactions inhibited variable-to-diversity-joining (V(beta)-to-D(beta)J(beta)) recombination before beta-selection. The introduction of an ectopic enhancer into Tcrb resulted in fewer such interactions and impaired allelic exclusion. We propose that initial V(beta)-to-D(beta)J(beta) recombination events are generally monoallelic in developing thymocytes because of frequent stochastic, rather than directed, interactions of Tcrb alleles with repressive nuclear compartments. Such interactions may be essential for Tcrb allelic exclusion.

Regulation of B cell fate commitment and immunoglobulin heavy-chain gene rearrangements by Ikaros.

The transcription factor Ikaros is essential for B cell development. However, its molecular functions in B cell fate specification and commitment have remained elusive. We show here that the transcription factor EBF restored the generation of CD19(+) pro-B cells from Ikaros-deficient hematopoietic progenitors. Notably, these pro-B cells, despite having normal expression of the transcription factors EBF and Pax5, were not committed to the B cell fate. They also failed to recombine variable gene segments at the immunoglobulin heavy-chain locus. Ikaros promoted heavy-chain gene rearrangements by inducing expression of the recombination-activating genes as well as by controlling accessibility of the variable gene segments and compaction of the immunoglobulin heavy-chain locus. Thus, Ikaros is an obligate component of a network that regulates B cell fate commitment and immunoglobulin heavy-chain gene recombination.

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.

Higher order chromatin organization in cancer.

In spite of our increased understanding of how genomes are dysregulated in cancer and a plethora of molecular diagnostic tools, the front line and 'gold standard' detection of cancer remains the pathologist's detection of gross changes in cellular and tissue structure, most strikingly nuclear dis-organization. In fact, for over 140 years it has been noted that nuclear morphology is often disrupted in cancer. Even today, nuclear morphology measures include nuclear size, shape, DNA content (ploidy) and 'chromatin organization'. Given the importance of nuclear shape to diagnoses of cancer phenotypes, it is surprising and frustrating that we currently lack a detailed understanding to explain these changes and how they might arise and relate to molecular events in the cell. It is an implicit hypothesis that perturbation of chromatin and epigenetic signatures may lead to alterations in nuclear structure (or vice versa) and that these perturbations lie at the heart of cancer genesis. In this review, we attempt to synthesize research leading to our current understanding on how chromatin interactions at the nuclear lamina, epigenetic modulation and gene regulation may intersect in cancer and offer a perspective on critical experiments that would help clarify how nuclear architecture may contribute to the cancerous phenotype. We also discuss the historical understanding of nuclear structure in normal cells and as a diagnostic in cancer.

Lamins: The backbone of the nucleocytoskeleton interface.

The nuclear lamina (NL) is a crucial component of the inner nuclear membrane (INM) and consists of lamin filaments and associated proteins. Lamins are type V intermediate filament proteins essential for maintaining the integrity and mechanical properties of the nucleus. In human cells, 'B-type' lamins (lamin B1 and lamin B2) are ubiquitously expressed, while 'A-type' lamins (lamin A, lamin C, and minor isoforms) are expressed in a tissue- and development-specific manner. Lamins homopolymerize to form filaments that localize primarily near the INM, but A-type lamins also localize to and function in the nucleoplasm. Lamins play central roles in the assembly, structure, positioning, and mechanics of the nucleus, modulating cell signaling and influencing development, differentiation, and other activities. This review highlights recent findings on the structure and regulation of lamin filaments, providing insights into their multifaceted functions, including their role as "mechanosensors", delving into the emerging significance of lamin filaments as vital links between cytoskeletal and nuclear structures, chromatin organization, and the genome.

Choreography of lamina-associated domains: structure meets dynamics.

Lamina-associated domains are large regions of heterochromatin positioned at the nuclear periphery. These domains have been implicated in gene repression, especially in the context of development. In mammals, LAD organization is dependent on nuclear lamins, inner nuclear membrane proteins, and chromatin state. In addition, chromatin readers and modifier proteins have been implicated in this organization, potentially serving as molecular tethers that interact with both nuclear envelope proteins and chromatin. More recent studies have focused on teasing apart the rules that govern dynamic LAD organization and how LAD organization, in turn, relates to gene regulation and overall 3D genome organization. This review highlights recent studies in mammalian cells uncovering factors that instruct the choreography of LAD organization, re-organization, and dynamics at the nuclear lamina, including LAD dynamics in interphase and through mitotic exit, when LAD organization is re-established, as well as intra-LAD subdomain variations.

The Nuclear Lamina.

Lamins interact with a host of nuclear membrane proteins, transcription factors, chromatin regulators, signaling molecules, splicing factors, and even chromatin itself to form a nuclear subcompartment, the nuclear lamina, that is involved in a variety of cellular processes such as the governance of nuclear integrity, nuclear positioning, mitosis, DNA repair, DNA replication, splicing, signaling, mechanotransduction and -sensation, transcriptional regulation, and genome organization. Lamins are the primary scaffold for this nuclear subcompartment, but interactions with lamin-associated peptides in the inner nuclear membrane are self-reinforcing and mutually required. Lamins also interact, directly and indirectly, with peripheral heterochromatin domains called lamina-associated domains (LADs) and help to regulate dynamic 3D genome organization and expression of developmentally regulated genes.

Nucleoplasmic lamin C rapidly accumulates at sites of nuclear envelope rupture with BAF and cGAS.

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

The shifting shape of genomes: dynamics of heterochromatin interactions at the nuclear lamina.

The nucleus is a highly structured organelle with many chromatin and protein compartments that partition the genome into regulatory domains. One such a compartment within the mammalian nucleus is the microenvironment underlying the nuclear envelope (NE) where intermediate filament proteins, lamins, act as a link between cytoskeletal and inner nuclear membrane (INM) proteins, chromatin binders and modifiers, and heterochromatin. These dynamic interactions regulate many cellular processes and, when they are perturbed, can lead to genome dysregulation and disease.

Mapping the micro-proteome of the nuclear lamina and lamina-associated domains.

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2ß to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

Lamin C is required to establish genome organization after mitosis.

BACKGROUND: The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. RESULTS: Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. CONCLUSIONS: Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Assembling a gene regulatory network for specification of the B cell fate.

The generation of B lymphocyte precursors is dependent on the combinatorial action of the transcription factors PU.1, Ikaros, E2A, EBF, and Pax-5. Loss of PU.1 results in a severe reduction in Flk2+, IL-7R+ lymphoid progenitors as well as impaired expression of EBF and Pax-5. Restoration of EBF expression facilitates rapid generation of pro-B cells from PU.1-/- progenitors. Molecular analysis suggests that PU.1 directly participates in regulation of the EBF gene. Although PU.1 is dispensable for expression of most early B lineage genes, it is required for CD45R/B220. Using EBF-/- mutant progenitors, we show that EBF induces Pax-5 and the early program of B lineage gene expression. Importantly, Pax-5 does not rescue B cell development from either PU.1-/- or EBF-/- progenitors. Pax-5 expression and function are contingent on EBF. Based on these results, we propose a hierarchical regulatory network for specification and commitment to the B cell fate.

Molecular pathways and mechanisms regulating the recombination of immunoglobulin genes during B-lymphocyte development.

The hallmark of B-cell development is the ordered recombination of immunoglobulin (Ig) genes. Recently, considerable progress has been achieved in assembling gene regulatory networks comprised of signaling components and transcription factors that regulate B-cell development. In this chapter we synthesize experimental evidence to explain how such signaling pathways and transcription factors can orchestrate the ordered recombination of immunoglobulin (Ig) genes. Recombination of antigen-receptor loci is regulated both by the developmentally controlled expression of the Rag1 and Rag2 genes and the accessibility of particular loci and their gene segments to recombination. A new framework has emerged that invokes nuclear compartmentalization, large-scale chromatin dynamics and localized changes in chromatin structure in regulating the accessibility of Ig loci at specific stages of B-cell development. We review this emergent framework and discuss new experimental approaches that will be needed to explore the underlying molecular mechanisms.

A Lamina-Associated Domain Border Governs Nuclear Lamina Interactions, Transcription, and Recombination of the Tcrb Locus.

Tcrb locus V(D)J recombination is regulated by positioning at the nuclear periphery. Here, we used DamID to profile Tcrb locus interactions with the nuclear lamina at high resolution. We identified a lamina-associated domain (LAD) border composed of several CTCF-binding elements that segregates active non-LAD from inactive LAD regions of the locus. Deletion of the LAD border causes an enhancer-dependent spread of histone H3 lysine 27 acetylation from the active recombination center into recombination center-proximal LAD chromatin. This is associated with a disruption to nuclear lamina association, increased chromatin looping to the recombination center, and increased transcription and recombination of recombination center-proximal gene segments. Our results show that a LAD and LAD border are critical components of Tcrb locus gene regulation and suggest that LAD borders may generally function to constrain the activity of nearby enhancers.

An Accessible Proteogenomics Informatics Resource for Cancer Researchers.

Proteogenomics has emerged as a valuable approach in cancer research, which integrates genomic and transcriptomic data with mass spectrometry-based proteomics data to directly identify expressed, variant protein sequences that may have functional roles in cancer. This approach is computationally intensive, requiring integration of disparate software tools into sophisticated workflows, challenging its adoption by nonexpert, bench scientists. To address this need, we have developed an extensible, Galaxy-based resource aimed at providing more researchers access to, and training in, proteogenomic informatics. Our resource brings together software from several leading research groups to address two foundational aspects of proteogenomics: (i) generation of customized, annotated protein sequence databases from RNA-Seq data; and (ii) accurate matching of tandem mass spectrometry data to putative variants, followed by filtering to confirm their novelty. Directions for accessing software tools and workflows, along with instructional documentation, can be found at z.umn.edu/canresgithub. Cancer Res; 77(21); e43-46. ©2017 AACR.

Tagged Chromosomal Insertion Site System: A Method to Study Lamina-Associated Chromatin.

The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization of the genome, we developed a new tool-the tagged chromosomal insertion site (TCIS) system-to identify and study minimal DNA sequences that drive nuclear compartmentalization and applied this system to specifically study the role of cis elements in targeting DNA to the nuclear lamina. The TCIS system allows Cre-recombinase-mediated site-directed integration of any DNA fragment into a locus tagged with lacO arrays, thus enabling both functional molecular studies and positional analysis of the altered locus. This system can be used to study the minimal DNA sequences that target the nuclear periphery (or other nuclear compartments), allowing researchers to understand how genome-wide results obtained, for example, by DNA adenine methyltransferase identification, chromosome conformation capture (HiC), or related methods, connect to the actual organization of DNA and chromosomes at the single-cell level. Finally, TCIS allows one to test roles for specific proteins in chromatin reorganization and to determine how changes in nuclear environment affect chromatin state and gene regulation at a single locus.

Finding the Middlemen in Genome Organization.

Chromatin domains associated with the nuclear lamina are generally heterochromatic and transcriptionally repressed. How they are recruited to and maintained at the nuclear periphery remains unclear. A recent study by Gonzalez-Sandoval et al. (2015) in Cell identifies a chromatin-binding protein that links repressive chromatin with the inner nuclear membrane.

Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.

Nuclear organization has been implicated in regulating gene activity. Recently, large developmentally regulated regions of the genome dynamically associated with the nuclear lamina have been identified. However, little is known about how these lamina-associated domains (LADs) are directed to the nuclear lamina. We use our tagged chromosomal insertion site system to identify small sequences from borders of fibroblast-specific variable LADs that are sufficient to target these ectopic sites to the nuclear periphery. We identify YY1 (Ying-Yang1) binding sites as enriched in relocating sequences. Knockdown of YY1 or lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1 proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation and histone H3 lysine di- and trimethylation. Our results also reveal that endogenous loci appear to be dependent on lamin A/C, YY1, H3K27me3, and H3K9me2/3 for maintenance of lamina-proximal positioning.