Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo

1988 ◽  
Vol 174 (1) ◽  
pp. 199-214 ◽  
Author(s):  
Xiang Lu ◽  
Marijana Kopun ◽  
Dieter Werner
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Cycle Phase ◽  
Cdna Libraries ◽  
Cycle Phase ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells

Coordinating Self-Renewal and Proliferation via MLL1-HOX Pathways and Beyond

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-28-SCI-28
Author(s):  
Patricia Ernst ◽  
Erika L. Artinger ◽  
Bibhu P. Mishra ◽  
Kristin M. Zaffuto ◽  
Bin E. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Transcriptional Regulators ◽  
Conditional Knockout ◽  
Specific Gene ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Specific Gene Expression ◽  
Lineage Specific Gene

Abstract Abstract SCI-28 Epigenetic regulation of gene expression plays a central role in normal hematopoietic stem cell (HSC) maintenance and leukemogenesis. The histone methyltransferase, MLL1, is essential for the maintenance of HSCs and is a common target of chromosomal translocations that result in acute leukemia. To discover genetic networks regulated by MLL1 in HSCs, we identified genes that were acutely deregulated upon Mll1 loss in HSCs, using a conditional knockout approach and lineage-negative, c-Kit+, Sca-1+, CD48-negative (LSK/CD48neg) cells. The majority of genes that changed were proliferation-associated genes, upregulated in Mll1−/− LSK/CD48neg cells. This reflected the fact that Mll1-deficient HSCs exhibit increased proliferation in vivo, a phenotype previously documented using the Mx1-cre inducible model. To determine whether the increased proliferation was cell-intrinsic, we performed single cell proliferation studies in serum-free medium containing SCF, IL-11, and Flt3L. We found that Mll1−/− LSK/CD48neg single cells entered the cell cycle earlier and that each cell cycle was shorter than wild-type controls. Evidence for failure to suppress lineage-specific gene expression was also observed; specifically, five percent of the upregulated genes encoded erythroid-specific proteins. These included erythroid transcriptional regulators such as GATA1 and KLF1, but also structural proteins such as spectrin, KEL, and EpoR. The relationship between erythroid-lineage genes and Mll1 was unique, since no other lineage-specific programs were upregulated in Mll1−/− LSK/CD48neg cells. Among the genes downregulated upon Mll1 loss, the largest category was comprised of transcriptional regulators, including Mecom, Pbx1, and Prdm16, which are known to control HSC self-renewal and quiescence. As observed in many other tissues, Mll1−/− LSK/CD48neg cells also exhibited reduced Hoxa9 expression. Interestingly, not all identified MLL1 target genes required menin, a cofactor thought to participate in directing MLL1 to particular genomic loci in vivo, and not all targets were Mll1-dependent in nonhematopoietic tissues. Chromatin immunoprecipitation and functional studies suggest that the identified genes act within a series of parallel pathways as direct transcriptional targets of MLL1. Interestingly, reexpression of Prdm16 alone could rescue Mll1-deficient cells from rapid attrition in bone marrow chimeras. Furthermore, Prdm16 corrected the hyperproliferation phenotype of Mll1−/− LSK/CD48neg cells. These data demonstrate that MLL1 coordinately regulates proliferation, lineage-specific gene expression programs, and self-renewal. By elucidating the normal MLL1-dependent transcriptional network within HSCs, we show that this pathway is overlapping but distinguishable from the leukemogenic pathway, suggesting that targeted therapy with minimal side effects on hematopoiesis will be feasible. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of Different Fixatives on β-Galactosidase Activity

2002 ◽  
Vol 50 (10) ◽  
pp. 1421-1424 ◽  
Author(s):  
Wenbin Ma ◽  
Keith Rogers ◽  
Berton Zbar ◽  
Laura Schmidt
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Specific Gene ◽  
Galactosidase Activity ◽  
Time Points ◽  
Specific Gene Expression ◽  
Carnoy’S Solution ◽  
Reporter Mice ◽  
Different Temperatures

β-Galactosidase (β-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal β-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed β-Gal activity.

scholarly journals Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain

2008 ◽  
Vol 105 (46) ◽  
pp. 18012-18017 ◽  
Author(s):  
Jun Kohyama ◽  
Takuro Kojima ◽  
Eriko Takatsuka ◽  
Toru Yamashita ◽  
Jun Namiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Mammalian Brain ◽  
Cytokine Signaling ◽  
Specific Gene ◽  
Neural Cells ◽  
Specific Gene Expression

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

scholarly journals Role for a YY1-Binding Element in Replication-Dependent Mouse Histone Gene Expression

1998 ◽  
Vol 18 (12) ◽  
pp. 7106-7118 ◽  
Author(s):  
Katherine A. Eliassen ◽  
Amy Baldwin ◽  
Eric M. Sikorski ◽  
Myra M. Hurt
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Transcription Factor ◽  
Protein Interaction ◽  
Histone Gene ◽  
Cycle Phase ◽  
Histone Gene Expression ◽  
Dna Protein Interaction

ABSTRACT Expression of the highly conserved replication-dependent histone gene family increases dramatically as a cell enters the S phase of the eukaryotic cell cycle. Requirements for normal histone gene expression in vivo include an element, designated α, located within the protein-encoding sequence of nucleosomal histone genes. Mutation of 5 of 7 nucleotides of the mouse H3.2 α element to yield the sequence found in an H3.3 replication-independent variant abolishes the DNA-protein interaction in vitro and reduces expression fourfold in vivo. A yeast one-hybrid screen of a HeLa cell cDNA library identified the protein responsible for recognition of the histone H3.2 α sequence as the transcription factor Yin Yang 1 (YY1). YY1 is a ubiquitous and highly conserved transcription factor reported to be involved in both activation and repression of gene expression. Here we report that the in vitro histone α DNA-protein interaction depends on YY1 and that mutation of the nucleotides required for the in vitro histone α DNA-YY1 interaction alters the cell cycle phase-specific up-regulation of the mouse H3.2 gene in vivo. Because all mutations or deletions of the histone α sequence both abolish interactions in vitro and cause an in vivo decrease in histone gene expression, the recognition of the histone α element by YY1 is implicated in the correct temporal regulation of replication-dependent histone gene expression in vivo.

scholarly journals Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis

2019 ◽  
Author(s):  
Chiaowen Joyce Hsiao ◽  
PoYuan Tung ◽  
John D. Blischak ◽  
Jonathan E. Burnett ◽  
Kenneth A. Barr ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Cell ◽  
Cell Cycle Progression ◽  
Cell Types ◽  
Cell Cycle Phase ◽  
Cellular Heterogeneity ◽  
Cycle Phase ◽  
Cycle Progression ◽  
Cellular Environment

AbstractCellular heterogeneity in gene expression is driven by cellular processes such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity, and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). Using these data, we developed a novel approach to characterize cell cycle progression. While standard methods assign cells to discrete cell cycle stages, our method goes beyond this, and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell’s position on the cell cycle continuum to within 14% of the entire cycle, and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell-cycle-related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.

scholarly journals Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis

2020 ◽  
Author(s):  
Jonathan Lenz ◽  
Robert Liefke ◽  
Julianne Funk ◽  
Samuel Shoup ◽  
Andrea Nist ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cell ◽  
Specific Gene ◽  
Cell Cycle Regulators ◽  
Cell Type ◽  
Nurd Complex ◽  
Nucleosome Remodeling ◽  
Specific Gene Expression ◽  
Cell Type Specific

AbstractThe generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

scholarly journals Inherited Chromosomally Integrated Human Herpesvirus 6 Demonstrates Tissue-Specific RNA Expression In Vivo That Correlates with an Increased Antibody Immune Response

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
Vikas Peddu ◽  
Isabelle Dubuc ◽  
Annie Gravel ◽  
Hong Xie ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Antibody ◽  
Human Herpesvirus ◽  
Specific Gene ◽  
Specific Antibody Response ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Specific Gene Expression ◽  
Human Herpesviruses

ABSTRACT Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carries one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response are still unclear. In this study, we screened DNA sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A- and 4 iciHHV-6B-positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for HHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A- and iciHHV-6B-positive individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B+ subjects were identified. Plasma samples from iciHHV-6A/B+ and age- and sex-matched controls were analyzed for antibodies to control antigens (cytomegalovirus [CMV], Epstein-Barr virus [EBV], and influenza virus [FLU]) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B+ subjects have significantly more antibodies against the U90 gene product (IE1) than do non-iciHHV-6-positive individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57, and U72, were identical between controls and iciHHV-6A/B+ subjects. CMV-seropositive individuals with iciHHV-6A/B+ have more antibodies against CMV pp150 than do CMV-seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B-specific antibody response. IMPORTANCE HHV-6A and -6B are human herpesviruses that have the unique property of being able to integrate into the telomeric regions of human chromosomes. Approximately 1% of the world’s population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression data set, we showed that the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate data sets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B+ subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks, and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production.

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