Purα Is Essential for Postnatal Brain Development and Developmentally Coupled Cellular Proliferation As Revealed by Genetic Inactivation in the Mouse

ABSTRACT The single-stranded DNA- and RNA-binding protein, Purα, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA−/− mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA−/− mice. Levels of proliferation were also absent or low in several other tissues of the PURA−/− mice, including those of myeloid lineage, whereas those of PURA+/− mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Purα and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA+/+ mice, but both were absent in dendrites of PURA−/− mice. Purα and Cdk5 can be coimmunoprecipitated from brain lysates of PURA+/+ mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Purα in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

Download Full-text

Cellular Functions of OCT-3/4 Regulated by Ubiquitination in Proliferating Cells

Cancers ◽

10.3390/cancers12030663 ◽

2020 ◽

Vol 12 (3) ◽

pp. 663

Author(s):

Kwang-Hyun Baek ◽

Jihye Choi ◽

Chang-Zhu Pei

Keyword(s):

Stem Cells ◽

Cellular Proliferation ◽

Critical Role ◽

Embryonic Stem ◽

Cell Types ◽

Ubiquitin Proteasome System ◽

Specific Cell ◽

Cellular Mechanisms ◽

Proliferating Cells ◽

Proliferation And Differentiation

Octamer-binding transcription factor 3/4 (OCT-3/4), which is involved in the tumorigenesis of somatic cancers, has diverse functions during cancer development. Overexpression of OCT-3/4 has been detected in various human somatic tumors, indicating that OCT-3/4 activation may contribute to the development and progression of cancers. Stem cells can undergo self-renewal, pluripotency, and reprogramming with the help of at least four transcription factors, OCT-3/4, SRY box-containing gene 2 (SOX2), Krüppel-like factor 4 (KLF4), and c-MYC. Of these, OCT-3/4 plays a critical role in maintenance of undifferentiated state of embryonic stem cells (ESCs) and in production of induced pluripotent stem cells (iPSCs). Stem cells can undergo partitioning through mitosis and separate into specific cell types, three embryonic germ layers: the endoderm, the mesoderm, and the trophectoderm. It has been demonstrated that the stability of OCT-3/4 is mediated by the ubiquitin-proteasome system (UPS), which is one of the key cellular mechanisms for cellular homeostasis. The framework of the mechanism is simple, but the proteolytic machinery is complicated. Ubiquitination promotes protein degradation, and ubiquitination of OCT-3/4 leads to regulation of cellular proliferation and differentiation. Therefore, it is expected that OCT-3/4 may play a key role in proliferation and differentiation of proliferating cells.

Download Full-text

cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21Cip1, and PCNA

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0501 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1527-1542 ◽

Cited By ~ 34

Author(s):

Soheil Naderi ◽

Jean Y.J. Wang ◽

Tung-Ti Chen ◽

Kristine B. Gutzkow ◽

Heidi K. Blomhoff

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Cellular Proliferation ◽

Kinase Activity ◽

Antitumor Agents ◽

Cell Types ◽

S Phase ◽

3T3 Fibroblasts ◽

Phase Progression ◽

Inhibition Of Dna Synthesis

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1proteins, since both processes were only marginally affected by increased levels of cAMP in Rb-/-and p21Cip1-/-3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase–specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase–specific antitumor agents.

Download Full-text

CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses

10.1101/2020.03.13.991224 ◽

2020 ◽

Author(s):

Kristina Sakers ◽

Yating Liu ◽

Lorida Llaci ◽

Michael J. Vasek ◽

Michael A. Rieger ◽

...

Keyword(s):

Rna Binding ◽

Gene Knockout ◽

Binding Protein ◽

Rna Binding Protein ◽

Cell Types ◽

Specific Gene ◽

Specific Cell ◽

Normal Brain ◽

Oligodendrocyte Development

AbstractQuaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’ UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte’s territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

Download Full-text

Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

10.1101/244905 ◽

2018 ◽

Cited By ~ 1

Author(s):

Yuhki Saito ◽

Yuan Yuan ◽

Ilana Zucker-Scharff ◽

John J. Fak ◽

Yoko Tajima ◽

...

Keyword(s):

Motor Coordination ◽

Rna Binding ◽

Synapse Formation ◽

Rna Binding Proteins ◽

Cell Types ◽

Knock Out ◽

Knock Out Mice ◽

Cerebellar Purkinje Cells ◽

The Brain

SUMMARYRNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell-types in vivo is unknown. We employed NOVA2 cTag-CLIP to generate functional RBP-RNA maps from single neuronal populations in the mouse brain. Combining cell-type specific data from Nova2-cTag and Nova2 conditional knock-out mice revealed differential NOVA2 regulatory actions (e.g. alternative splicing) on the same transcripts in different neurons, including in cerebellar Purkinje cells, where NOVA2 acts as an essential factor for proper motor coordination and synapse formation. This also led to the discovery of a mechanism by which NOVA2 action leads to different outcomes in different cells on the same transcripts: NOVA2 is able to regulate retained introns, which subsequently serve as scaffolds for another trans-acting splicing factor, PTBP2. Our results describe differential roles and mechanisms by which RBPs mediate RNA diversity in different neurons and consequent functional outcomes within the brain.

Download Full-text

Comprehensive analysis of DNA replication timing in genetic diseases and gene knockouts identifies MCM10 as a novel regulator of the replication program

10.1101/2021.09.08.459433 ◽

2021 ◽

Author(s):

Madison Caballero ◽

Tiffany Ge ◽

Ana Rita Rebelo ◽

Seungmae Seo ◽

Sean Kim ◽

...

Keyword(s):

Dna Replication ◽

Cellular Proliferation ◽

Genetic Diseases ◽

Replication Timing ◽

Cell Types ◽

Replication Initiation ◽

Timing Variability ◽

Gene Knockouts ◽

Dna Replication Timing ◽

Mutant Cells

AbstractCellular proliferation depends on the accurate and timely replication of the genome. Several genetic diseases are caused by mutations in key DNA replication genes; however, it remains unclear whether these genes influence the normal program of DNA replication timing. Similarly, the factors that regulate DNA replication dynamics are poorly understood. To systematically identify trans-acting modulators of replication timing, we profiled replication in 184 cell lines from three cell types, encompassing 60 different gene knockouts or genetic diseases. Through a rigorous approach that considers the background variability of replication timing, we concluded that most samples displayed normal replication timing. However, mutations in two genes showed consistently abnormal replication timing. The first gene was RIF1, a known modulator of replication timing. The second was MCM10, a highly conserved member of the pre-replication complex. MCM10 mutant cells demonstrated replication timing variability comprising 46% of the genome and at different locations than RIF1 knockouts. Replication timing alterations in MCM10-mutant cells was predominantly comprised of replication initiation defects. Taken together, this study demonstrates the remarkable robustness of the human replication timing program and reveals MCM10 as a novel modulator of DNA replication timing.

Download Full-text

Control of synaptic specificity by limiting promiscuous synapse formation

10.1101/415695 ◽

2018 ◽

Cited By ~ 2

Author(s):

Chundi Xu ◽

Emma Theisen ◽

Elijah Rumbaut ◽

Bryan Shum ◽

Jing Peng ◽

...

Keyword(s):

Visual System ◽

Synapse Formation ◽

Neural Circuits ◽

Local Environment ◽

Cell Types ◽

Partner Choice ◽

Synaptic Connectivity ◽

Specific Cell ◽

Synaptic Specificity ◽

And Function

SUMMARYThe ability of neurons to distinguish appropriate from inappropriate synaptic partners in their local environment is fundamental to the proper assembly and function of neural circuits. How synaptic partner selection is regulated is a longstanding question in Neurobiology. A prevailing hypothesis is that appropriate partners express complementary molecules that match them together and promote synaptogenesis. Dpr and DIP IgSF proteins bind heterophilically and are expressed in a complementary manner between synaptic partners in the Drosophila visual system. Here, we show that in the lamina, DIP mis-expression is sufficient to promote synapse formation with Dpr-expressing neurons, and that DIP proteins are not necessary for synaptogenesis but rather function to prevent ectopic synapse formation. These findings indicate that Dpr-DIP interactions regulate synaptic specificity by biasing synapse formation towards specific cell-types. We propose that synaptogenesis occurs independent of synaptic partner choice, and that precise synaptic connectivity is established by limiting promiscuous synapse formation.

Download Full-text

Regulation of Pituitary Progenitor Differentiation by β-Catenin

Endocrinology ◽

10.1210/en.2018-00563 ◽

2018 ◽

Vol 159 (9) ◽

pp. 3287-3305 ◽

Cited By ~ 3

Author(s):

Julie L Youngblood ◽

Tanner F Coleman ◽

Shannon W Davis

Keyword(s):

Critical Time ◽

Cell Types ◽

Pituitary Hormones ◽

Pituitary Hormone ◽

Specific Cell ◽

Lineage Specification ◽

Gain Of Function ◽

Physiological Processes ◽

Critical Organ ◽

Time Point

Abstract The pituitary gland is a critical organ that is necessary for many physiological processes, including growth, reproduction, and stress response. The secretion of pituitary hormones from specific cell types regulates these essential processes. Pituitary hormone cell types arise from a common pool of pituitary progenitors, and mutations that disrupt the formation and differentiation of pituitary progenitors result in hypopituitarism. Canonical WNT signaling through CTNNB1 (β-catenin) is known to regulate the formation of the POU1F1 lineage of pituitary cell types. When β-catenin is deleted during the initial formation of the pituitary progenitors, Pou1f1 is not transcribed, which leads to the loss of the POU1F1 lineage. However, when β-catenin is deleted after lineage specification, there is no observable effect. Similarly, the generation of a β-catenin gain-of-function allele in early pituitary progenitors or stem cells results in the formation of craniopharyngiomas, whereas stimulating β-catenin in differentiated cell types has no effect. PROP1 is a pituitary-specific transcription factor, and the peak of PROP1 expression coincides with a critical time point in pituitary organogenesis—that is, after pituitary progenitor formation but before lineage specification. We used a Prop1-cre to conduct both loss- and gain-of-function studies on β-catenin during this critical time point. Our results demonstrate that pituitary progenitors remain sensitive to both loss and gain of β-catenin at this time point, and that either manipulation results in hypopituitarism.

Download Full-text

Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1681-1690.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1681-1690

Author(s):

W J Tang ◽

S L Berger ◽

S J Triezenberg ◽

W R Folk

Keyword(s):

Dna Replication ◽

Mouse Embryo ◽

Simian Virus 40 ◽

Cell Types ◽

Simian Virus ◽

Common Element ◽

Specific Cell ◽

Bovine Papillomavirus ◽

Embryo Cells ◽

Better Than

The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 E1A enhancer core AGGAAGTGACT; acquisition of an A----G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with immunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein.

Download Full-text