The cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and metazoans, is remarkable in the constancy of their double-membraned structure in both somatic and germ cells. Such remarkable structural conservation underscores common and ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis in some animals, little is known about the evolution of nuclear envelope remodeling dynamics during oogenesis. Here, we uncovered a novel form of nuclear envelope remodeling as oocytes are formed in the flatworm Schmidtea mediterranea. From zygotene to metaphase II, both nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, likely involving de novo membrane synthesis. 3-D electron microscopy reconstructions demonstrated that the NE transforms itself into numerous double-membraned vesicles similar in membrane architecture to NE doublets in mammalian oocytes after germinal vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential for the maintenance of stem cells in this and other organisms. Our data contribute a new model to the canonical view of NE dynamics and suggest important roles of NE remodeling in planarian oogenesis.

Developing cellular models to study progressive disorders like Alzheimer’s is a challenge due to the unknown initiator of the disease and its slow progression. Recent developments in stem cell technology have made it possible to generate human-specific models such as organoids to study brain development and disease. Organoids are miniature organs that are artificially grown in vitro. Brain organoids are 3D tissues that closely resemble developing human brain. Their important advantage is that they are derived from human pluripotent stem cells. In this pilot study, we are using brain organoids isolated at different days of their growth in combination with quantitative proteomics using isobaric tandem mass tags to define changes in protein contents and amounts within the developing brain, with the goal to apply this tool to study progressive disease pathology.

The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient-poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary culture of liver cells Support Protocol 1: Maintenance of A. mexicanus primary liver cells Support Protocol 2: Banking of A. mexicanus primary liver cells Support Protocol 3: Recovery of A. mexicanus primary liver cells Support Protocol 4: Primary liver cell cloning Support Protocol 5: Characterization of A. mexicanus primary liver cell growth pattern Basic Protocol 2: Lentiviral transduction of A. mexicanus primary liver cells.

Investigating the function of delicate mammalian eyes often requires chemical fixation, histological sectioning, immunohistochemistry (IHC) and in situ hybridization (ISH). One of the long-standing challenges in the ocular histology field is the limited success of maintaining intact morphology via cryo- or paraffin procedures. Although our latest protocol significantly improved the morphology of mouse eyeball sections, the window technique is time-consuming and requires extensive practice to avoid damage while making windows. In this study, we present a novel glyoxal fixative that is suitable for a freeze-substitution approach to improve both morphology and molecular target preservation of mouse eyes. The method prevents morphology distortion in all tested eyeballs. Therefore, it suits a variety of research needs from morphological examination to investigation of single-molecule RNA expression, using hematoxylin and eosin (H&E) stain, IHC, and ISH assays on either frozen (cryo) or paraffin-infiltrated tissue sections. In addition, this method can be easily performed in many histology laboratories.

It can be challenging to maintain tissue integrity using established histology pro-tocols. Here, we describe a protocol composed of Hartman's fixation, window technique, microwave-based tissue processing, optimized depigmentation, and antigen retrieval pretreatment. This is followed by the ViewRNA single -molecule fluorescence in situ hybridization and immunofluorescence techniques to optimize routine histological staining and molecular histology multiplexing assays. Our protocol is highly reproducible in any laboratory and may decrease animal usage and lab resource expenditure.For complete details on the use and execution of this protocol, please refer to Pang et al. (2021).

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. beta-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of beta-catenin in restraining germline and somatic lineage differentiation genes. We show that beta-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1 gamma. Our data indicate that these complexes help beta-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of beta-catenin in preserving lineage differentiation integrity in ground state ESCs.

The BaseScope™ assay is a novel, highly sensitive RNA in situ hybridization (ISH) technique, allowing detection of short RNA sequences as well as discrimination between single-nucleotide alterations. Multiplexing BaseScope™ ISH with immunofluorescence assay has proven challenging because the diffusion of colorimetric dyes such as Fast Red in aqueous solutions degrades spatial resolution. In this study, we explore alkaline phosphatase-based fluorescent signal detection methods and integrate it with BaseScope™ RNA ISH. We found that Fast Blue BB/NAMP can be used in BaseScope™ ISH for signal detection. Additionally, we found that the calcium binding fluorochromes calcein and xylenol orange can be used to localize alkaline phosphatase activity in both immunohistochemistry (IHC) and BaseScope™ ISH assays. When applied to mouse brain and intestine tissue sections, we successfully detected circular RNA molecules and cell proliferation antigen Ki-67 proteins. This technological advance expanded the substrate selection of alkaline phosphatase-based BaseScope™ RNA ISH to allow researchers and clinical professionals accurately assess RNA targets with immunofluorescent multiplexing.

The cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and metazoans, is remarkable in the constancy of both their double-membraned structure and protein composition. Such remarkable structural conservation underscores common and ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis in some animals, little is known about the evolution of nuclear envelope break down (NEBD) during cell division. Here, we uncovered a novel form of NEBD in primary oocytes of the flatworm Schmidtea mediterranea . From zygotene to metaphase II, both nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, likely involving de novo membrane synthesis. 3-D electron microscopy reconstructions demonstrated that the NE transforms itself into numerous double-membraned vesicles similar in membrane architecture to NE doublets in mammalian oocytes after germinal vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential for the maintenance of stem cells in this and other organisms. Our data contribute a new model to the canonical view of NE dynamics and support that NEBD is an evolutionarily adaptable trait in multicellular organisms.

Biologists have long been fascinated with the organization and function of intricate protein complexes. Therefore, techniques for precisely imaging protein complexes and the location of proteins within these complexes are critically important and often require multidisciplinary collaboration. A challenge in these explorations is the limited resolution of conventional light microscopy. However, a new microscopic technique has circumvented this resolution limit by making the biological sample larger, thus allowing for super-resolution of the enlarged structure. This ‘expansion’ is accomplished by embedding the sample in a hydrogel that, when exposed to water, uniformly expands. Here, we present a protocol that transforms thick expansion microscopy (ExM) hydrogels into sections that are physically expanded four times, creating samples that are compatible with the super-resolution technique structured illumination microscopy (SIM). This super-resolution ExM method (ExM–SIM) allows the analysis of the three-dimensional (3D) organization of multiprotein complexes at ~30-nm lateral ( xy ) resolution. This protocol details the steps necessary for analysis of protein localization using ExM–SIM, including antibody labeling, hydrogel preparation, protease digestion, post-digestion antibody labeling, hydrogel embedding with tissue-freezing medium (TFM), cryosectioning, expansion, image alignment, and particle averaging. We have used this approach for 3D mapping of in situ protein localization in the Drosophila synaptonemal complex (SC), but it can be readily adapted to study thick tissues such as brain and organs in various model systems. This procedure can be completed in 5 d.

In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.

Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes.

Although a variety of affinity purification mass spectrometry (AP-MS) strategies have been used to investigate complex interactions, many of these are susceptible to artifacts because of substantial overexpression of the exogenously expressed bait protein. Here we present a logical and systematic workflow that uses the multifunctional Halo tag to assess the correct localization and behavior of tagged subunits of the Sin3 histone deacetylase complex prior to further AP-MS analysis. Using this workflow, we modified our tagging/expression strategy with 21.7% of the tagged bait proteins that we constructed, allowing us to quickly develop validated reagents. Specifically, we apply the workflow to map interactions between stably expressed versions of the Sin3 subunits SUDS3, SAP30, or SAP30L and other cellular proteins. Here we show that the SAP30 and SAP30L paralogues strongly associate with the core Sin3 complex, but SAP30L has unique associations with the proteasome and the myelin sheath. Next, we demonstrate an advancement of the complex NSAF (cNSAF) approach, in which normalization to the scaffold protein SIN3A accounts for variations in the proportion of each bait capturing Sin3 complexes and allows a comparison among different baits capturing the same protein complex. This analysis reveals that although the Sin3 subunit SUDS3 appears to be used in both SIN3A and SIN3B based complexes, the SAP30 subunit is not used in SIN3B based complexes. Intriguingly, we do not detect the Sin3 subunits SAP18 and SAP25 among the 128 high-confidence interactions identified, suggesting that these subunits may not be common to all versions of the Sin3 complex in human cells. This workflow provides the framework for building validated reagents to assemble quantitative interaction networks for chromatin remodeling complexes and provides novel insights into focused protein interaction networks.

The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism.

Promoter proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II has been shown to correlate with recruitment of a Super‐Elongation Complex or SEC that contains the transcription elongation factors ELL/EAF and P‐TEFb, as well as a collection of additional proteins encoded by genes involved in translocations with the MLL gene in leukemia. Our lab has recently obtained evidence for a role of human Mediator in recruiting SEC and another ELL/EAF‐containing complex to genes. In particular, we have shown that a conserved N‐terminal domain in the Mediator subunit Med26 provides docking sites for ELL/EAF, P‐TEFb, and associated proteins as well as the general initiation factor TFIID. Our findings are consistent with the model that Med26 functions as a molecular switch that interacts first with the Pol II initiation complex through direct interactions with TFIID and then “exchanges” TFIID for complexes containing elongation factors ELL/EAF and P‐TEFb to facilitate the transition of Pol II into the elongation stage of transcription.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

Multidimensional protein identification technology (MudPIT) is a platform integrating multidimensional liquid chromatography and tandem mass spectrometry for protein profiling. As MudPIT becomes increasingly popular, the full characterization of large datasets remains challenging. A total of 36 MudPIT technical replicates from two biological samples were acquired on XPs and LTQs ion-trap tandem mass spectrometers. This study evaluated the applicability of label-free quantitative proteomic information by fitting mean and standard deviation of normalized spectral abundance factors (NSAFs) to a power law global error model (PLGEM). Protein fractions from affinity purifications of 293 cells expressing various Flag-tagged human Mediator subunits were enzymatically digested and analyzed by MudPIT. A total of 475 unique proteins were identified from 36 runs, resulting in a 36 × 475 matrix of relative abundance (NSAF values). We observed a “goodness of fit” when PLGEM was applied to the entire 36 × 475 dataset, and subsets of the data containing only NSAF values from XP or from LTQ, indicating that NSAF datasets are compatible with this modeling scheme. Specifically, log-log plots of standard deviation vs. mean NSAFs revealed a linear dependence, with slopes ranging from 0.66 to 0.75 and an adjusted r2 equaled to 0.99. In addition, the residuals were found to be normally distributed across the entire dynamic range of average NSAF values. Similar ranges and distribution were also observed in Affymetrix GeneChip datasets. This study demonstrates the utilities of NSAFs and PLGEM in statistically analyzing MudPIT datasets. With additional modeling using datasets from more complex samples or with “spiked in” experiments, this approach has a potential to become an integral data-mining tool for proteomics datasets acquired from one mass spectrometer as well as across multiple instrumentation platforms.

Extracts prepared from the isolated nuclei of cultured cells have been instrumental in dissecting the mechanisms by which transcription and mRNA processing occur. These extracts are able to recapitulate accurate transcription initiation and splicing in vitro, which has been useful in direct functional studies. They also serve as the starting material for purification of proteins that can then be reassembled in functional studies or examined in more detail biochemically. This unit describes the preparation of nuclear extracts from cultured cells and optimized production of transcriptionally active extracts from HeLa cells. Additional protocols describe optimization of the method to increase the yield of specific proteins, adaptation of the method for downstream applications such as affinity purification, and preparation of the cytoplasmic (S-100) fraction.

Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks.