Active RB causes visible changes in nuclear organization

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state.

Non-invasive Chromatin Deformation and Measurement of Differential Mechanical Properties in the Nucleus

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C /μm) can lead to substantial intra-nuclear chromatin displacements (>1 μm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high rigidity and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

When Down Is Up: Heterochromatin, Nuclear Organization and X Upregulation

Organisms with highly differentiated sex chromosomes face an imbalance in X-linked gene dosage. Male Drosophila solve this problem by increasing expression from virtually every gene on their single X chromosome, a process known as dosage compensation. This involves a ribonucleoprotein complex that is recruited to active, X-linked genes to remodel chromatin and increase expression. Interestingly, the male X chromosome is also enriched for several proteins associated with heterochromatin. Furthermore, the polytenized male X is selectively disrupted by the loss of factors involved in repression, silencing, heterochromatin formation or chromatin remodeling. Mutations in many of these factors preferentially reduce male survival or enhance the lethality of mutations that prevent normal recognition of the X chromosome. The involvement of primarily repressive factors in a process that elevates expression has long been puzzling. Interestingly, recent work suggests that the siRNA pathway, often associated with heterochromatin formation and repression, also helps the dosage compensation machinery identify the X chromosome. In light of this finding, we revisit the evidence that links nuclear organization and heterochromatin to regulation of the male X chromosome.

The Mammalian Locus Coeruleus Complex—Consistencies and Variances in Nuclear Organization

Descriptions of the nuclear parcellation of the locus coeruleus complex have been provided in approximately 80 mammal species spanning the phylogenetic breadth of this class. Within the mammalian rostral hindbrain, noradrenergic neurons (revealed with tyrosine hydroxylase and dopamine-ß-hydroxylase immunohistochemistry) have been observed within the periventricular grey matter (A4 and A6 nuclei) and parvicellular reticular nucleus (A5 and A7 nuclei), with the one exception to date being the tree pangolin, where no A4/A6 neurons are observed. The alphanumeric nomenclature system, developed in laboratory rodent brains, has been adapted to cover the variation observed across species. Cross-species homology is observed regarding the nuclear organization of noradrenergic neurons located in the parvicellular reticular nucleus (A5 and A7). In contrast, significant variations are observed in the organization of the A6 neurons of the locus coeruleus proper. In most mammals, the A6 is comprised of a moderate density of neurons, but in Murid rodents, primates, and megachiropteran bats, the A6 exhibits a very high density of neurons. In primates and megachiropterans, there is an additional moderate density of A6 neurons located rostromedial to the high-density portion. These variations are of importance in understanding the translation of findings in laboratory rodents to humans.

Emerging Roles of Repetitive and Repeat-Containing RNA in Nuclear and Chromatin Organization and Gene Expression

Genomic repeats have been intensely studied as regulatory elements controlling gene transcription, splicing and genome architecture. Our understanding of the role of the repetitive RNA such as the RNA coming from genomic repeats, or repetitive sequences embedded in mRNA/lncRNAs, in nuclear and cellular functions is instead still limited. In this review we discuss evidence supporting the multifaceted roles of repetitive RNA and RNA binding proteins in nuclear organization, gene regulation, and in the formation of dynamic membrane-less aggregates. We hope that our review will further stimulate research in the consolidating field of repetitive RNA biology.

Analysis of the Expression Pattern of Cajal-Retzius Cell Markers in the Xenopus laevis Forebrain

Cajal-Retzius cells are essential for cortical development in mammals, and their involvement in the evolution of this structure has been widely postulated, but very little is known about their progenitor domains in non-mammalian vertebrates. Using in situhybridization and immunofluorescence techniques we analyzed the expression of some of the main Cajal-Retzius cell markers such as Dbx1, Ebf3, ER81, Lhx1, Lhx5, p73, Reelin, Wnt3a, Zic1, and Zic2 in the forebrain of the anuran Xenopus laevis, because amphibians are the only class of anamniote tetrapods and show a tetrapartite evaginated pallium, but no layered or nuclear organization. Our results suggested that the Cajal-Retzius cell progenitor domains were comparable to those previously described in amniotes. Thus, at dorsomedial telencephalic portions a region comparable to the cortical hem was defined in Xenopus based on the expression of Wnt3a, p73, Reelin, Zic1, and Zic2. In the septum, two different domains were observed: a periventricular dorsal septum, at the limit between the pallium and the subpallium, expressing Reelin, Zic1, and Zic2, and a related septal domain, expressing Ebf3, Zic1, and Zic2. In the lateral telencephalon, the ventral pallium next to the pallio-subpallial boundary, the lack of Dbx1 and the unique expression of Reelin during development defined this territory as the most divergent with respect to mammals. Finally, we also analyzed the expression of these markers at the prethalamic eminence region, suggested as Cajal-Retzius progenitor domain in amniotes, observing there Zic1, Zic2, ER81, and Lhx1 expression. Our data show that in anurans there are different subtypes and progenitor domains of Cajal-Retzius cells, which probably contribute to the cortical regional specification and territory-specific properties. This supports the notion that the basic organization of pallial derivatives in vertebrates follows a comparable fundamental arrangement, even in those that do not have a sophisticated stratified cortical structure like the mammalian cerebral cortex.