Subtypes of inositol 1,4,5-trisphosphate receptor in human hematopoietic cell lines: Dynamic aspects of their cell-type specific expression

Abstract The transcription factor PU.1 (also known as Spi-1) plays a critical role in the development of the myeloid lineages, and myeloid cells derived from PU.1−/− animals are blocked at the earliest stage of myeloid differentiation. Expression of the PU.1 gene is tightly regulated during normal hematopoietic development, and dysregulation of PU.1 expression can lead to erythroleukemia. However, relatively little is known about how the PU.1 gene is regulated in vivo. Here it is shown that myeloid cell type–specific expression of PU.1 in stable cell lines and transgenic animals is conferred by a 91-kilobase (kb) murine genomic DNA fragment that consists of the entire PU.1 gene (20 kb) plus approximately 35 kb of upstream and downstream sequences, respectively. To further map the important transcriptional regulatory elements, deoxyribonuclease I hypersensitive site mapping studies revealed at least 3 clusters in the PU.1 gene. A 3.5-kb fragment containing one of these deoxyribonuclease I hypersensitive sites, located −14 kb 5′ of the transcriptional start site, conferred myeloid cell type–specific expression in stably transfected cell lines, suggesting that within this region is an element important for myeloid specific expression of PU.1. Further analysis of this myeloid-specific regulatory element will provide insight into the regulation of this key transcriptional regulator and may be useful as a tool for targeting expression to the myeloid lineage.

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Development of two bone-derived cell lines from the marine teleost Sparus aurata ; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

Cell and Tissue Research ◽

10.1007/s00441-003-0830-1 ◽

2004 ◽

Vol 315 (3) ◽

pp. 393-406 ◽

Cited By ~ 71

Author(s):

Ant�nio R. Pombinho ◽

Vincent Laiz� ◽

Duarte M. Molha ◽

Sandra M. P. Marques ◽

M. Leonor Cancela

Keyword(s):

Extracellular Matrix ◽

Cell Lines ◽

Sparus Aurata ◽

Matrix Gla Protein ◽

Marine Teleost ◽

Cell Type ◽

Specific Expression ◽

Matrix Mineralization ◽

Cell Type Specific Expression ◽

Cell Type Specific

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Structure, organization, and regulation of human metallothionein IF gene: differential and cell-type-specific expression in response to heavy metals and glucocorticoids.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.26 ◽

1986 ◽

Vol 6 (1) ◽

pp. 26-37 ◽

Cited By ~ 58

Author(s):

U Varshney ◽

N Jahroudi ◽

R Foster ◽

L Gedamu

Keyword(s):

Heavy Metals ◽

Cell Lines ◽

Regulatory Element ◽

Hepatoma Cell ◽

Cell Type ◽

Kinase Gene ◽

Specific Expression ◽

Cell Type Specific Expression ◽

Cell Type Specific ◽

Flanking Region

We describe a human genomic clone containing the metallothionein (MT) IF and MT IG genes. Southern blot analysis and partial DNA sequence determinations show that these genes are organized in a head-to-head fashion and are located approximately 7.0 kilobases apart from each other. Sequence analysis shows that the MT IF gene contains three exons separated by two introns. All of the intron-exon junctions are defined by the GT-AG rule. The 5' flanking region shows the presence of a duplicated metal regulatory element (TGCGC CCGGCCC) important in heavy-metal induction of this gene and a sequence for its basal level expression (GCGGGGCGGGTGCAAAG). The 5' flanking region is also highly G + C rich (approximately 75%) and contains several GC boxes (GGGCGG), probably important in the binding of transcription factors. The TATAA box and the AATAAA sequence are represented by their variants, the TATCAA box and the AATTAA sequence, respectively. This gene is functional and inducible by heavy metals but not by dexamethasone in mouse LMTK- cells after its transfer on a plasmid containing the herpes simplex virus thymidine kinase gene. Further studies on various human cell lines show that this gene is not expressed in a splenic lymphoblastoid cell line (WI-L2) but is expressed in two hepatoma cell lines (Hep 3B2 and Hep G2) in response to cadmium, zinc, and copper. Dexamethasone appears to have no significant effect on its expression. The studies suggest that the MT IF gene shows cell-type-specific expression and is differentially regulated by heavy metals and glucocorticoids.

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Structure, organization, and regulation of human metallothionein IF gene: differential and cell-type-specific expression in response to heavy metals and glucocorticoids

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.26-37.1986 ◽

1986 ◽

Vol 6 (1) ◽

pp. 26-37

Author(s):

U Varshney ◽

N Jahroudi ◽

R Foster ◽

L Gedamu

Keyword(s):

Heavy Metals ◽

Cell Lines ◽

Regulatory Element ◽

Hepatoma Cell ◽

Cell Type ◽

Kinase Gene ◽

Specific Expression ◽

Cell Type Specific Expression ◽

Cell Type Specific ◽

Flanking Region

We describe a human genomic clone containing the metallothionein (MT) IF and MT IG genes. Southern blot analysis and partial DNA sequence determinations show that these genes are organized in a head-to-head fashion and are located approximately 7.0 kilobases apart from each other. Sequence analysis shows that the MT IF gene contains three exons separated by two introns. All of the intron-exon junctions are defined by the GT-AG rule. The 5' flanking region shows the presence of a duplicated metal regulatory element (TGCGC CCGGCCC) important in heavy-metal induction of this gene and a sequence for its basal level expression (GCGGGGCGGGTGCAAAG). The 5' flanking region is also highly G + C rich (approximately 75%) and contains several GC boxes (GGGCGG), probably important in the binding of transcription factors. The TATAA box and the AATAAA sequence are represented by their variants, the TATCAA box and the AATTAA sequence, respectively. This gene is functional and inducible by heavy metals but not by dexamethasone in mouse LMTK- cells after its transfer on a plasmid containing the herpes simplex virus thymidine kinase gene. Further studies on various human cell lines show that this gene is not expressed in a splenic lymphoblastoid cell line (WI-L2) but is expressed in two hepatoma cell lines (Hep 3B2 and Hep G2) in response to cadmium, zinc, and copper. Dexamethasone appears to have no significant effect on its expression. The studies suggest that the MT IF gene shows cell-type-specific expression and is differentially regulated by heavy metals and glucocorticoids.

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CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein

10.1101/450320 ◽

2018 ◽

Cited By ~ 1

Author(s):

Milad Babaei ◽

Yuekun Liu ◽

Shelly M. Wuerzberger-Davis ◽

Alan T. Yeo ◽

Larisa Kagermazova ◽

...

Keyword(s):

Cell Lines ◽

Cell Clone ◽

293T Cell ◽

Scaffold Protein ◽

Response To Treatment ◽

Cell Type ◽

Specific Expression ◽

Sequence Element ◽

Cell Type Specific Expression ◽

Cell Type Specific

ABSTRACTThe use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-κB signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-κB signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.

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