scholarly journals MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies

2015 ◽  
Vol 84 (2) ◽  
pp. 443-450 ◽  
Author(s):  
On Sun Lau ◽  
Dominique C. Bergmann
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Large Scale ◽  
Cell Type ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Cell Type Specific

The microenvironment for erythropoiesis is regulated by HIF-2α through VCAM-1 in endothelial cells

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1482-1492 ◽  
Author(s):  
Toshiharu Yamashita ◽  
Osamu Ohneda ◽  
Ai Sakiyama ◽  
Fumiko Iwata ◽  
Kinuko Ohneda ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chromatin Immunoprecipitation ◽  
Normocytic Anemia ◽  
Reporter Assay ◽  
Cell Type ◽  
Erythroid Progenitors ◽  
Chromatin Immunoprecipitation Assay ◽  
Hematopoietic Microenvironment ◽  
Cell Type Specific ◽  
Vascular Adhesion

Abstract Erythropoiesis is a dynamic process regulated by oxygen in vertebrates. Recent evidence has indicated that erythropoietin (Epo) expression is regulated by hypoxia-inducible transcription factors (HIFs), HIF-2α in particular. In this study, we report that knockdown mutation of HIF-2α in mice (kd/kd) results in normocytic anemia, despite Epo induction in response to hypoxia not being severely affected. Transplantation analyses clearly demonstrated that the hematopoietic microenvironment, but not the hematopoietic cells, was altered in kd/kd. Furthermore, cell-type specific recovery of HIF-2α expression in endothelial cells (ECs) abrogated the anemic condition of the kd/kd mice, indicating that HIF-2α in EC plays an essential role in supporting erythropoiesis. In the absence of HIF-2α, the expression of vascular adhesion molecule-1 (VCAM-1) was reduced significantly and restoration of VCAM-1 expression in kd/kd ECs enhanced the development of erythroid progenitors. Finally, a chromatin immunoprecipitation assay and a reporter assay indicated that VCAM-1 gene transcription is directly regulated by HIF-2α. These data suggest that the hematopoietic microenvironment required for erythropoiesis is dynamically regulated by oxygen through the functions of HIF-2α in ECs.

scholarly journals Quantitative synapse analysis for cell-type specific connectomics

2018 ◽  
Author(s):  
Dika A. Kuljis ◽  
Khaled Zemoura ◽  
Cheryl A. Telmer ◽  
Jiseok Lee ◽  
Eunsol Park ◽  
...  
Keyword(s):  
Large Scale ◽  
Neural Circuit ◽  
Apical Dendrite ◽  
Automated Detection ◽  
Cell Type ◽  
Disease States ◽  
Specific Localization ◽  
Cell Type Specific ◽  
Dendritic Branches ◽  
Circuit Function

AbstractAnatomical methods for determining cell-type specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed new genetically-encoded reagents for fluorescence-synapse labeling and connectivity analysis in brain tissue, using a fluorogen-activating protein (FAP)-or YFP-coupled, postsynaptically-localized neuroligin-1 targeting sequence (FAP/YFPpost). Sparse viral expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific localization of synapses across diverse neuron types in mouse somatosensory cortex. High-resolution confocal image stacks of virally-transduced neurons were used for 3D reconstructions of postsynaptic cells and automated detection of synaptic puncta. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, synapses, and presynaptic neurites to assess subtype-specific inhibitory connectivity onto L2 neocortical pyramidal (Pyr) neurons. Quantitative and compartment-specific comparisons show that PV inputs are the dominant source of inhibition at both the soma and across all dendritic branches examined and were particularly concentrated at the primary apical dendrite, a previously unrecognized compartment of L2 Pyr neurons. Our fluorescence-based synapse labeling reagents will facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.

scholarly journals Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Yong Feng ◽  
Yunfei Liao ◽  
Jianming Zhang ◽  
Jacson Shen ◽  
Zengwu Shao ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Transcriptional Analysis ◽  
Luciferase Assay ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Gel Shift Assay ◽  
Gel Shift

Abstract Background Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. Methods In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFβ) were further accessed in osteosarcoma. Results We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFβ expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. Conclusions These data establish CBFβ as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Graphical Abstract

scholarly journals ASCOT identifies key regulators of neuronal subtype-specific splicing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonathan P. Ling ◽  
Christopher Wilks ◽  
Rone Charles ◽  
Patrick J. Leavey ◽  
Devlina Ghosh ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Large Scale ◽  
Splice Variants ◽  
Next Generation Sequencing Data ◽  
Data Sets ◽  
Cell Type ◽  
Sequencing Data ◽  
Large Scale Analysis ◽  
Cell Type Specific ◽  
Public Archives

AbstractPublic archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.

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