Worcester, MA, 01605-0103, USA 7 ​ Gladstone Institutes, University of California, San Francisco, CA ​ 94158, ​ USA *equal contribution # ​ Correspondance: GF - ​ geoff.fudenberg@gmail.com ​ , IS - ​ irina.solovei@lrz.uni-muenchen.de ​ , LM -leonid@mit.edu The mammalian cell nucleus displays a remarkable spatial segregation of active euchromatic from inactive heterochromatic genomic regions. In conventional nuclei, euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery. In contrast, rod photoreceptors in nocturnal mammals have inverted nuclei, with a dense heterochromatic core and a thin euchromatic outer shell. This inverted architecture likely converts rod nuclei into microlenses to facilitate nocturnal

Repository Citation Falk M, Feodorova Y, Naumova N, Massachusetts Institute of Technology, Illumina Incorporated, Leonhardt H, Ludwig Maximilians Universitat, Munchen, Dekker J, University of California, San Francisco, Ludwig Maximilians Universitat, Munchen, Massachusetts Institute of Technology. (2018). Heterochromatin drives organization of conventional and inverted nuclei [preprint]. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/244038. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1527

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery1,2. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina2,4, preferential attraction of similar chromatin to each other1,4–12, higher levels of chromatin mobility in active chromatin13–15 and transcription-related clustering of euchromatin16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin–lamina interactions in conventional nuclei18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals19,20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases. Attractions between heterochromatic regions are essential for phase separation of the active and inactive genome in inverted and conventional nuclei, whereas chromatin–lamina interactions are necessary to build the conventional genomic architecture from these segregated phases.

Peripheral heterochromatin in mammalian nuclei is tethered to the nuclear envelope by at least two mechanisms here referred to as the A- and B-tethers. The A-tether includes lamins A/C and additional unknown components presumably INM protein(s) interacting with both lamins A/C and chromatin. The B-tether includes the inner nuclear membrane (INM) protein Lamin B-receptor, which binds B-type lamins and chromatin. Generally, at least one of the tethers is always present in the nuclear envelope of mammalian cells. Deletion of both causes the loss of peripheral heterochromatin and consequently inversion of the entire nuclear architecture, with this occurring naturally in rod photoreceptors of nocturnal mammals. The tethers are differentially utilized during development, regulate gene expression in opposite manners, and play an important role during cell differentiation. Here we aimed to identify the unknown chromatin binding component(s) of the A-tether. We analyzed 10 mouse tissues by immunostaining with antibodies against 7 INM proteins and found that every cell type has specific, although differentially and developmentally regulated, sets of these proteins. In particular, we found that INM protein LEMD2 is concomitantly expressed with A-type lamins in various cell types but is lacking in inverted nuclei of rod cells. Truncation or deletion of Lmna resulted in the downregulation and mislocalization of LEMD2, suggesting that the two proteins interact and pointing at LEMD2 as a potential chromatin binding mediator of the A-tether. Using nuclei of mouse rods as an experimental model lacking peripheral heterochromatin, we expressed a LEMD2 transgene alone or in combination with lamin C in these cells and observed no restoration of peripheral heterochromatin in either case. We conclude that in contrary to the B-tether, the A-tether has a more intricate composition and consists of multiple components that presumably vary, at differing degrees of redundancy, between cell types and differentiation stages.

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes.

Diurnality, associated with enhanced visual acuity and color vision, is typical of most modern Primates. However, it remains a matter of debate when and how many times primates re-acquired diurnality or returned to nocturnality. We analyzed the features specific to nocturnal and diurnal vision that were recently found in the nuclei of mammalian rod photoreceptor cells in 11 species representing various groups of the Primates and related tree shrew and colugo. In particular, heterochromatin in rod nuclei of nocturnal mammals is clustered in the center of rod nuclei (inverted architecture), whereas rods of diurnal mammals retain rods with peripheral heterochromatin (conventional architecture). Rod nuclei of the nocturnal owl monkey have a state transitional to the inverted one. Surprisingly, rod nuclei of the tarsier have a conventional nuclear architecture typical for diurnal mammals, strongly implying that recent Tarsiiformes returned to nocturnality from the diurnal state. Diurnal lemurs retain inverted rod nuclei typical of nocturnal mammals, which conforms to the notion that the ancestors of all Lemuroidea were nocturnal. Data on the expression of proteins indispensable for peripheral heterochromatin maintenance (and, respectively, conventional or inverted nuclear organization) in rod cells support the view that the primate ancestors were nocturnal and transition to diurnality occurred independently in several primate and related groups: Tupaia, diurnal lemurs, and, at least partially independently, in Simiiformes (monkeys and apes) and Tarsiiformes.

Background: Methyl-CpG binding protein 2 (MECP2) is a protein that specifically binds methylated DNA, thus regulating transcription and chromatin organization. Mutations in the gene have been identified as the principal cause of Rett syndrome, a severe neurological disorder. Although the role of MECP2 has been extensively studied in nervous tissues, still very little is known about its function and cell type specific distribution in other tissues.Results: Using immunostaining on tissue cryosections, we characterized the distribution of MECP2 in 60 cell types of 16 mouse neuronal and non-neuronal tissues. We show that MECP2 is expressed at a very high level in all retinal neurons except rod photoreceptors. The onset of its expression during retina development coincides with massive synapse formation. In contrast to astroglia, retinal microglial cells lack MECP2, similar to microglia in the brain, cerebellum, and spinal cord. MECP2 is also present in almost all non-neural cell types, with the exception of intestinal epithelial cells, erythropoietic cells, and hair matrix keratinocytes. Our study demonstrates the role of MECP2 as a marker of the differentiated state in all studied cells other than oocytes and spermatogenic cells. MECP2-deficient male (Mecp2(-/y)) mice show no apparent defects in the morphology and development of the retina. The nuclear architecture of retinal neurons is also unaffected as the degree of chromocenter fusion and the distribution of major histone modifications do not differ between Mecp2(-/y) and Mecp2(wt) mice. Surprisingly, the absence of MECP2 is not compensated by other methyl-CpG binding proteins. On the contrary, their mRNA levels were downregulated in Mecp2(-/y) mice.Conclusions: MECP2 is almost universally expressed in all studied cell types with few exceptions, including microglia. MECP2 deficiency does not change the nuclear architecture and epigenetic landscape of retinal cells despite the missing compensatory expression of other methyl-CpG binding proteins. Furthermore, retinal development and morphology are also preserved in Mecp2 null mice. Our study reveals the significance of MECP2 function in cell differentiation and sets the basis for future investigations in this direction.

To improve light propagation through the retina, the rod nuclei of nocturnal mammals are uniquely changed compared to the nuclei of other cells. In particular, the main classes of chromatin are segregated in them and form regular concentric shells in order; inverted in comparison to conventional nuclei. A broad study of the epigenetic landscape of the inverted and conventional mouse retinal nuclei indicated several differences between them and several features of general interest for the organization of the mammalian nuclei. In difference to nuclei with conventional architecture, the packing density of pericentromeric satellites and LINE-rich chromatin is similar in inverted rod nuclei; euchromatin has a lower packing density in both cases. A high global chromatin condensation in rod nuclei minimizes the structural difference between active and inactive X chromosome homologues. DNA methylation is observed primarily in the chromocenter, Dnmt1 is primarily associated with the euchromatic shell. Heterochromatin proteins HP1-alpha and HP1-beta localize in heterochromatic shells, whereas HP1-gamma is associated with euchromatin. For most of the 25 studied histone modifications, we observed predominant colocalization with a certain main chromatin class. Both inversions in rod nuclei and maintenance of peripheral heterochromatin in conventional nuclei are not affected by a loss or depletion of the major silencing core histone modifications in respective knock-out mice, but for different reasons. Maintenance of peripheral heterochromatin appears to be ensured by redundancy both at the level of enzymes setting the epigenetic code (writers) and the code itself, whereas inversion in rods rely on the absence of the peripheral heterochromatin tethers (absence of code readers).

Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

This article is aimed at providing comparative quantitative data about postnatal mammalian retina development, and at searching for some general rules at both the descriptive and the mechanistic level. In mammals the eye continues to grow, and the retina continues to expand, much after the end of retinal cytogenesis. Thus, although the total number of retinal cells remains constant after cessation of mitotic activity (and the end of 'physiological cell death'), the retinal surface area increases by a factor of two or more. In most mammals, ocular growth exceeds retinal expansion: the neural retina lines 70–80% of the inner ocular surface at the beginning but only about 40–60% in adults. Differential local expansion of the retina (the peripheral area increases more than the central one) can be explained by 'passive stretching' of the retinal tissue by the growing eyeball; it depends on the different biomechanical properties of the peripheral vs. central retinal tissue. The increasing retinal surface area allows for a re-distribution of cells such that the thickness of the (particularly, outer) nuclear layer(s) decreases proportional to the areal expansion. This causes a considerable developmental reduction of the number of cell nuclei 'stacked above each other' by a factor of more than two, and requires a translocation of the somata against their neighbors. We provide a physico-mathematical model of these oblique 'down-sliding' movements of the photoreceptor cell somata along the Müller cell process in the center of their columnar cell unit.

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.

For studying the 3D structure formed by consecutive genomic regions of chromosomes we propose using statistical shape theory in conjunction with registration methods In contrast to earlier work, where the 3D chromatin structure was analyzed indirectly, we here directly exploit the 3D locations of genomic regions to determine the large-scale structure of chromatin fiber Our study is based on 3D microscopy images of the X-chromosome where four consecutive genomic regions (BACs) have been simultaneously labeled by multicolor FISH. To allow unique reconstruction of the 3D shape, image data with sets of four consecutive BACs have been acquired with an overlap of three BACs between the different sets. We have statistically analyzed the data and it turned out that for all datasets the 3D structure is non-random. In addition, we found that the shapes of active and inactive X-chromosomal genomic regions are statistically independent Moreover, we reconstructed the 3D structure of chromatin in a small genomic region based on five BACs resulting from two overlapping four BACs We also found that spatial normalization of cell nuclei using non-rigid image registration has a significant influence on the location of the genomic regions.

The spatial organization of the genome plays an important role in the regulation of nuclear functions and undergoes large scale changes during differentiation. These changes in the nuclear distribution of chromatin are, in a complex way, related to transcriptional status and epigenetic modifications. Recent studies emphasize the roles that gene promoters and alterations in replication timing play in the spatial reorganization of chromatin during cell differentiation. Changes in the association of chromatin regions with the nuclear lamina also emerge as a significant factor of transcriptional regulation. New results suggest that the spatial organization of chromatin in embryonic stem cells may be important for maintenance of the pluripotent state, whereas the nuclear architecture of differentiated cells facilitates formation of transcriptionally active zones with shared transcription and splicing machinery.

Interphase nuclei have a conserved architecture: heterochromatin occupies the nuclear periphery, whereas euchromatin resides in the nuclear interior. It has recently been found that rod photoreceptor cells of nocturnal mammals have an inverted architecture, which transforms these nuclei in microlenses and supposedly facilitates a reduction in photon loss in the retina. This unique deviation from the nearly universal pattern throws a new light on the nuclear organization. In the article we discuss the implications of the studies of the inverted nuclei for understanding the role of the spatial organization of the nucleus in nuclear functions.

We show that the nuclear architecture of rod photoreceptor cells differs fundamentally in nocturnal and diurnal mammals. The rods of diurnal retinas possess the conventional architecture found in nearly all eukaryotic cells, with most heterochromatin situated at the nuclear periphery and euchromatin residing toward the nuclear interior. The rods of nocturnal retinas have a unique inverted pattern, where heterochromatin localizes in the nuclear center, whereas euchromatin, as well as nascent transcripts and splicing machinery, line the nuclear border. The inverted pattern forms by remodeling of the conventional one during terminal differentiation of rods. The inverted rod nuclei act as collecting lenses, and computer simulations indicate that columns of such nuclei channel light efficiently toward the light-sensing rod outer segments. Comparison of the two patterns suggests that the conventional architecture prevails in eukaryotic nuclei because it results in more flexible chromosome arrangements, facilitating positional regulation of nuclear functions.

AbstractDie Augen nachtaktiver Säugetiere enthalten besonders viele der für das Nachtsehen zuständigen, hochempfindlichen Stäbchen‐Fotorezeptoren. Damit können sie Licht wahrnehmen, dessen Intensität millionenfach unter der des Tageslichts liegt. Neue Untersuchungen zeigen, dass sich der nächtliche Lebensstil und die damit verbundenen visuellen Herausforderungen bis auf die Organisation der Zellkerne in den Stäbchen ausgewirkt haben. Dicht gepackte inaktive Bereiche und weniger dicht gepackte aktive Bereiche der DNA sind in den Stäbchenkernen nachtaktiver Säuger anders verteilt als in den Körperzellen fast aller Organismen, einschließlich der Stäbchen tagaktiver Säuger. Die Zellkerne der nachtaktiven Säuger fungieren als Sammellinsen, die das eintreffende Licht bündeln.

While cells are mostly transparent they are phase objects that differ in shape and refractive index. Any image that is projected through layers of cells will normally be distorted by refraction, reflection, and scattering. Strangely, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-sensitive photoreceptor cells, with each photon having a chance of being scattered. Here we report how nature has optimized this apparently unfavourable situation. We investigated the optical properties of retinal tissue and individual Müller cells, which are radial glial cells spanning the entire thickness of the retina. Using confocal microscopy, quantitative refractometry, and a modified fiber-based dual-beam laser trap, we found that these cells act as optical fibers and guide light, which would otherwise be scattered, from the retinal surface to the photoreceptor cells. Their parallel arrangement in the retina is reminiscent of fiber-optic plates used for low-distortion image transfer. Behind the Müller cells, there seems to be a specific adaptation of the rod photoreceptor nuclei for improved light transmission through the outer nuclear layer of nocturnal animals. These nuclei have an inverted chromatin structure that turns them into micro-lenses channeling the light through the ONL. These findings ascribe a new function to glial cells, demonstrate the first nuclear adaptation for an optical function, and shed new light on the inverted retina as an optical system.