In Vivo Footprinting and Genomic Sequencing by Ligation-Mediated PCR

Hornstra, Ian; Yang, Tianbo

doi:10.1006/abio.1993.1407

Cited by 57 publications

(58 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: -Azadeoxycytidine Reactivation Of the Hprt Gene 14922mentioning

confidence: 99%

“…To visualize the final DNA sequencing ladder, strand-specific hybridization probes were synthesized from M13 clones containing the human HPRT promoter region. Probe synthesis, hybridization, washing, and autoradiography were performed as described previously (24,26,29).Quantification of Footprints by Autoradiogram Densitometry-Quantification of transcription factor binding at position Ϫ91 was carried out by densitometry of the DNA sequencing autoradiogram. The densitometric value for the band intensity at position Ϫ91 in each sample was first normalized for loading differences using the average band intensity of eight nonfootprinted bands flanking both sides of the band at position Ϫ91 in each lane.…”

mentioning

confidence: 99%

See 1 more Smart Citation

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

Litt¹,

Hansen²,

Hornstra³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

During the process of 5-aza-2-deoxycytidine (5aCdr)-induced reactivation of the X-linked human hypoxanthine phosphoribosyltransferase (HPRT) gene on the inactive X chromosome, acquisition of a nucleasesensitive chromatin conformation in the 5 region occurs before the appearance of HPRT mRNA. In vivo footprinting experiments reported here show that the 5aCdr-induced change in HPRT chromatin structure precedes the appearance of three footprints in the immediate 5 flanking region that are characteristic of the active HPRT allele. These and other data suggest the following sequence of events that lead to the reactivation of the HPRT gene after 5aCdr treatment: (a) hemidemethylation of the promoter, (b) an "opening" of chromatin structure detectable as increased nuclease sensitivity, (c) transcription factor binding to the promoter, (d) assembly of the transcription complex, and (e) synthesis of HPRT RNA. This sequence of events supports the view that inactive X-linked genes are silenced by a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter.A unique system of differential gene expression in mammals is established during female embryogenesis by X chromosome inactivation (1, 2). The inactivation of one X chromosome within each female somatic nucleus generates a transcriptionally active and inactive allele of most X-linked genes and results in dosage compensation for X-linked genes between males and females. A variety of molecular mechanisms have been implicated in regulating the initiation, spreading, and maintenance of X inactivation (1-7). The involvement of DNA methylation in this process has been established by studies using methyl-sensitive restriction enzymes (8 -10), DNA-mediated transformation (11-13), genomic sequencing (14 -16), and the DNA demethylating agent 5-azacytidine (6,13,(17)(18)(19). All of these studies support the notion that hypermethylation of the 5Ј CpG island associated with many X-linked housekeeping genes is involved in the transcriptional silencing of these genes on the inactive X chromosome.The ability to demethylate and reactivate individual genes on the human inactive X chromosome in rodent-human somatic cell hybrids by treatment with 5-azacytidine or 5-aza-2Ј-deoxycytidine (5aCdr) 1 (6, 20) suggests that transcriptional regulation of X-linked genes by X chromosome inactivation involves some measure of local control either at the level of individual genes or at the level of chromatin domains. Reactivation of inactive X-linked genes such as the hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase (PGK-1) genes after 5-azacytidine or 5aCdr treatment is associated with both a change in chromatin structure from a nuclease-inaccessible to a nuclease-accessible conformation and a reduction in DNA methylation levels in the 5Ј CpG island (17,21).In previous studies, Sasaki et al. (22) assayed four parameters during 5aCdr reactivation of the human HPRT gene in a hamster-human somatic cell hybrid cell line (X8 -6T2) conta...

show abstract

Section: -Azadeoxycytidine Reactivation Of the Hprt Gene 14922mentioning

confidence: 99%

mentioning

confidence: 99%

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

Litt¹,

Hansen²,

Hornstra³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In vivo footprinting was performed by using Arabidopsis suspension cells (24) or mature Arabidopsis leaves as described (25)(26)(27), using separate primer sets for upper and lower strands (see Supporting Text).…”

mentioning

confidence: 99%

Arabidopsis TCP20 links regulation of growth and cell division control pathways

Potuschak²,

Colón‐Carmona³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

305

287

View full text Add to dashboard Cite

During postembryonic plant development, cell division is coupled to cell growth. There is a stringent requirement to couple these processes in shoot and root meristems. As cells pass through meristems, they transit through zones with high rates of cell growth and proliferation during organogenesis. This transition implies a need for coordinate regulation of genes underpinning these two fundamental cell functions. Here, we report a mechanism for coregulation of cell division control genes and cell growth effectors. We identified a GCCCR motif necessary and sufficient for high-level cyclin CYCB1;1 expression at G 2/M. This motif is overrepresented in many ribosomal protein gene promoters and is required for high-level expression of the S27 and L24 ribosomal subunit genes we examined. p33 TCP20 , encoded by the Arabidopsis TCP20 gene, binds to the GCCCR element in the promoters of cyclin CYCB1;1 and ribosomal protein genes in vitro and in vivo. We propose a model in which organ growth rates, and possibly shape in aerial organs, are regulated by the balance of positively and negatively acting teosinte-branched, cycloidea, PCNA factor (TCP) genes in the distal meristem boundary zone where cells become mitotically quiescent before expansion and differentiation.

show abstract

“…Although signals around Ϫ135 are weak, especially 0.05% DMS-treated DAB-1 specimen, signal intensities of Ϫ130 guanine on the sense strand of both cancer cells are almost equal to that of G ladder derived from naked genomic DNA, whereas that of SV-HUC cells is clearly low (protected). Sometimes a hypersensitive signal is observed in adjacent position to the protected region, 25,26 and we detected such a type of hypersensitive signal at Ϫ126 cytosine in SV-HUC cells but not in DAB-1 and KU-1 cells. Thus, low signal intensities (protection) in guanine cluster (Ϫ135 to Ϫ138), protection of Ϫ130 guanine and appearance of hypersensitive signal at Ϫ126 cytosine were observed in only SV-HUC-1 cells.…”

Section: In Vivo Dms Footprintingmentioning

confidence: 61%

Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO₄in vivo footprinting on its promoter region

Haga

Fujita

Nomoto

et al. 2004

Intl Journal of Cancer

View full text Add to dashboard Cite

We previously reported that gelsolin gene expression is reduced in various tumors. In an effort to gain further insights into the mechanism of gelsolin downregulation in tumors, we examined the in vivo properties of the gelsolin promoter in urinary bladder cancer cell lines. Neither mutation nor hypermethylation was responsible for gene silencing at the promoter. After exposure to trichostatin A (TSA), a histone deacetylase inhibitor, gelsolin promoter activity was markedly enhanced in the cancer cells, not in cells derived from normal tissue. Chromatin immunoprecipitation assays revealed that both histones H3 and H4 were hypoacetylated in the promoter region of the cancer cells, and the accumulation of acetylated histones was detected by TSA treatment. Key words: gelsolin; gene silencing; dimethylsulfate hypersensitivity; bladder cancer; in vivo footprintingCancers occur due to the accumulation of abnormalities in gene function. While many of them are genetic (mutation and deletion), epigenetically mediated changes in gene expression have been increasingly investigated. Hypermethylation of CpG islands, which are GC-rich regions usually located at the 5Ј end of genes, results in alterations in the histone acetylation and methylation status around gene promoter regions. Aberrant cytosine methylation and concomitant histone modifications play important roles in the repression of over half of the tumor suppressor genes during malignant transformation. 1,2Previously, we reported that the expression of gelsolin, an actin-regulatory protein, is frequently silenced in various cancers, including stomach, colon, urinary bladder and lung, and that gelsolin functions as a tumor suppressor. [3][4][5][6][7] These findings were also demonstrated in breast and prostate cancers. 8,9 On the other hand, a subset of non small cell lung cancers is characterized by high expression levels of gelsolin correlated with lymphatic invasion, 10 and there was a gradual increase in gelsolin with an increase in tumor grade and stage in uroepithelial carcinoma. 11 Gelsolin induced invasion of epithelial cells as well as colon adenocarcinoma cells. 12 It is therefore possible that a decrease in expression during a particular stage is followed by an increase during another stage. To understand roles of gelsolin in the whole process of tumorigenesis, mechanisms for both down-and upregulations should be analyzed. In breast cancers, gelsolin genes were not mutated; however, epigenetic changes in the chromatin structure were responsible for the deficiency. 13 Histone deacetylase (HDAC) inhibitors, such as trichostatin A (TSA) and apicidin, selectively induced the expression of gelsolin. 14,15 Recent studies have suggested a new mechanism for cell-specific gene regulation linking nuclear architecture, chromatin structure and functional organization of DNA sequences. 16 In an effort to determine the molecular basis of gelsolin downregulation in urinary bladder cancer and to better understand the carcinogenic process, we investigated the gelsolin prom...

show abstract

In Vivo Footprinting and Genomic Sequencing by Ligation-Mediated PCR

Cited by 57 publications

References 0 publications

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

Arabidopsis TCP20 links regulation of growth and cell division control pathways

Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO₄in vivo footprinting on its promoter region

Contact Info

Product

Resources

About

In Vivo Footprinting and Genomic Sequencing by Ligation-Mediated PCR

Cited by 57 publications

References 0 publications

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

Arabidopsis TCP20 links regulation of growth and cell division control pathways

Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO4in vivo footprinting on its promoter region

Contact Info

Product

Resources

About

Gelsolin gene silencing involving unusual hypersensitivities to dimethylsulfate and KMnO₄in vivo footprinting on its promoter region