Heterochromatin drives organization of conventional and inverted nuclei [preprint]

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 vision, and may relate to the absence of particular proteins that tether heterochromatin to the lamina. However, both the mechanism of inversion and the role of interactions between different types of chromatin and the lamina in nuclear organization remain unknown. To elucidate this mechanism we performed Hi-C and microscopy on cells with inverted nuclei and their conventional counterparts. Strikingly, despite the inversion evident in microscopy, both types of nuclei display similar Hi-C maps. To resolve this paradox we developed a polymer model of chromosomes and found a universal mechanism that reconciles Hi-C and microscopy for both inverted and conventional nuclei. Based solely on attraction between heterochromatic regions, this mechanism is sufficient to drive phase separation of euchromatin and heterochromatin and faithfully reproduces the 3D organization of inverted nuclei. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. To further test our models, we eliminated lamina interactions in models of conventional nuclei and found that this triggers a spontaneous process of inversion that qualitatively reproduces the pathway of morphological changes during nuclear inversion in vivo. Together, our experiments and modeling suggest that interactions among heterochromatic regions are central to phase separation of the active and inactive genome in inverted and conventional nuclei, while interactions with the lamina are essential for building the conventional architecture from these segregated phases. Ultimately our data suggest that an inverted organization constitutes the default state of nuclear architecture

DNA methylation reader MECP2:Cell type- and differentiation stage-specific protein distribution

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 Mecp2wt 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

Small chromosomal regions position themselves autonomously according to their chromatin class

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) genedepleted 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

Viewing Nuclear Architecture through the Eyes of Nocturnal Mammals

© 2019 Elsevier Ltd The cell nucleus is a remarkably well-organized organelle with membraneless but distinct compartments of various functions. The largest of them, euchromatin and heterochromatin, are spatially segregated in such a way that the transcriptionally active genome occupies the nuclear interior, whereas silent genomic loci are preferentially associated with the nuclear envelope. This rule is broken by rod photoreceptor cells of nocturnal mammals, in which the two major compartments have inverted positions. The inversion and dense compaction of heterochromatin converts these nuclei into microlenses that focus light and facilitate nocturnal vision. As is often the case in biology, when a mutation helps to understand normal processes and structures, inverted nuclei have served as a tool to unravel general principles of nuclear organization, including mechanisms of heterochromatin tethering to the nuclear envelope, autonomous behavior of small genomic segments, and euchromatin–heterochromatin segregation

Immunohistochemical phenotype of colorectal carcinoma in patients with KRAS mutation and mismatch repair status

Introduction: Aberrant expression of CK7/CK20/CDX2 is reported in percentage of colorectal carcinomas (CRC). Aim: The objective of this study was to investigate specific morphological and immunohistochemical characteristics of colorectal carcinoma with KRAS mutation status and microsatellite instability. Materials and methods: Seventy-one patients with CRC and examined KRAS mutation status were included in the investigation. Immunohistochemistry was performed using antibodies to CK7, CK20, CDX2, PMS2, and MSH6. An automatic immunostainer (Ventana BenchMark GX) was used following the manufacturer’ instructions. Fisher’s exact test was used for statistical analysis (p value <0.05). Results: Immunohistochemical analysis was performed for CK7, CK20, CDX2, PMS2, and MSH6. Aberrant expression of the typical immunohistochemical profile CK7/CK20/CDX2 was observed in 50% of the cases. The highest sensitivity and specificity were established for CDX2, with 93% of the cases demonstrating positive nuclear expression in the tumor cells. As for the microsatellite status, 20% of the examined colorectal cancers showed loss in expression for one or both of the mismatch repair proteins - PMS2 and MSH6, which was associated with loss of expression for CK20 and CDX2 as well. Downhill correlation was found also between CK20 expression and the presence of mutation in the gene for KRAS. Conclusions: Our results may support the heterogeneity of colorectal carcinoma. Statistically significant correlation was found between the expression of CK20 and CDX2 and microsatellite deficient and KRAS mutant colorectal cancers. This may lead to application of immunohistochemical screening panel for selection of patients with CRC for genetic testing. Further studies on large cohorts correlating different immunohistochemical profiles to molecular subtypes of colorectal carcinoma are needed for better understanding of the pathogenesis and behavior of colorectal carcinoma

Immunohistochemical expression of CK20, CK7, and CDX2 in colorectal carcinoma in correlation with pathomorphological characteristics

Introduction: Colorectal carcinoma is the third most common cancer worldwide. The usual immunophenotype of colorectal adenocarcinoma is CDX2 positive, CK20 positive, and CK7 negative. Aberrant expression is reported in a variety of colorectal carcinomas but its relation to morphological variables and survival data is still unclear. Aim: The aim of this study was to investigate the correlation between the aberrant immunostaining of colorectal carcinoma and different clinicopathological characteristics. Materials and methods: Immunohistochemical expression of CK20, CK7, and CDX2 was evaluated in 71 cases of colorectal carcinoma. Statistical analysis was performed to identify correlations between the morphological characteristics and the immunoprofile of colorectal carcinoma. Results: Positive cytoplasmic and/or membranous signal for CK20 was observed in 66.2% of colorectal carcinomas. CK7 positive immunostaining was seen in 7% of the cases. In terms of combined expression of CK20 and CK7, the proportion of immunoprofile CK20+/CK7− was the highest, accounting for 46 out of 71 colorectal carcinomas, followed by CK20−/CK7−, then CK20−/CK7+ and CK20+/CK7+. Concerning CDX2, the majority of colorectal carcinomas (87.3%) showed positive staining. Statistically significant correlation was established between CDX2 expression and histologic grade and depth of tumour invasion. Loss of CK20 positivity was associated with higher histologic grade. No association between CK7 expression and histopathologic features was established. Conclusions: The results support the heterogeneity of colorectal cancer. Over 35% of the cases in this study showed deviations from the expected immunoprofile. This should be taken into consideration when diagnosing colorectal carcinoma in metastatic regions

Glioblastoma Multiforme Classified As Mesenchymal Subtype

ВВЕДЕНИЕ: В качестве неблагоприятных прогностических факторов при первичных глиобластомах в последнее время обсуждаются не только клинические показатели, но и различные клеточные, генетические и иммунологические маркеры. ЦЕЛЬ: Работа ставит себе целью анализировать случай с первичной мультиформенной глиобластомой и краткой выживаемостью после оперативной интер- венции, а также и определить неблагоприятные прогностические маркеры. ПРЕДСТАВЛЕНИЕ СЛУЧАЯ: Авторы представляют случай 71-оголетнего мужчины с доказанной мультиформенной глиобластомой и постоперативной выживаемостью в 48 дней. Из-за непродолжительной выживаемости пациент не подвергнут телегамматерапии и адювантной терапии Темозоломид- ом. С помощью молекулярно- биологических и иммунологических анализов определены транскрипционные и сывороточные уровни TNF-α, IL-6, YKL-40 и CD44. Устанавливаются экстремно высокие транскрипционные уровни генов CD44, IL-6 и YKL-40, увеличенная экспрессия TNF-α, сопровожденные повышенной сывороточной концентрацией IL-6, TNF-α и YKL-40 и пониженной сывороточной концентрацией CD44. ЗАКЛЮЧЕНИЕ: Молекулярно-биологически

Heterochromatin drives compartmentalization of inverted and conventional nuclei

The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome(1,2). In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery(1,2). Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments(3), and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina(2,4), preferential attraction of similar chromatin to each other(1,4-12), higher levels of chromatin mobility in active chromatin(13-15) and transcription-related clustering of euchromatin(16,17). However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei(18). The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals(19,20) 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