Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: Evidence for the role of Sp1 and not of c- myb or PU.1 in myelomonocytic lineage-specific expression

Woei Yau Kao, Judith A. Briggs, Marsha C Kinney, Roy A. Jensen, Robert C. Briggs

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20 Citations (Scopus)

Abstract

The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelonloncytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in hone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to - 16) that did not include the cluster of c-Myb sites. A 4-hp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22)including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4- bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.

Original languageEnglish (US)
Pages (from-to)231-244
Number of pages14
JournalJournal of Cellular Biochemistry
Volume65
Issue number2
DOIs
StatePublished - May 1997
Externally publishedYes

Fingerprint

Differentiation Antigens
Nuclear Antigens
Genes
Transcription Initiation Site
Mutation
Transcriptional Activation
5' Flanking Region
Deoxyribonuclease I
Gene expression
Null Lymphocytes
Paraffin
Chromatin
Epitopes
Consensus Sequence
Cell Lineage
DNA Methylation
Reporter Genes
Granulocytes
Tissue
Monocytes

Keywords

  • cell maturation
  • granulocytes
  • monocytes
  • promoter
  • transcription

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

@article{5728cfcb9def47168878f9290c28d797,
title = "Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: Evidence for the role of Sp1 and not of c- myb or PU.1 in myelomonocytic lineage-specific expression",
abstract = "The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelonloncytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in hone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to - 16) that did not include the cluster of c-Myb sites. A 4-hp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22)including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4- bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.",
keywords = "cell maturation, granulocytes, monocytes, promoter, transcription",
author = "Kao, {Woei Yau} and Briggs, {Judith A.} and Kinney, {Marsha C} and Jensen, {Roy A.} and Briggs, {Robert C.}",
year = "1997",
month = "5",
doi = "10.1002/(SICI)1097-4644(199705)65:2<231::AID-JCB8>3.0.CO;2-V",
language = "English (US)",
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T1 - Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter

T2 - Evidence for the role of Sp1 and not of c- myb or PU.1 in myelomonocytic lineage-specific expression

AU - Kao, Woei Yau

AU - Briggs, Judith A.

AU - Kinney, Marsha C

AU - Jensen, Roy A.

AU - Briggs, Robert C.

PY - 1997/5

Y1 - 1997/5

N2 - The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelonloncytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in hone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to - 16) that did not include the cluster of c-Myb sites. A 4-hp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22)including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4- bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.

AB - The human myeloid nuclear differentiation antigen (MNDA) is expressed specifically in maturing cells of the myelonloncytic lineage and in monocytes and granulocytes. Epitope enhancement was used to confirm the strict lineage- and stage-specific expression of MNDA in hone marrow as well as in other paraffin-embedded fixed tissues. A 1-kb region of the gene that includes 5' flanking sequence was reported earlier to contain functional promoter activity and was specifically demethylated in expressing cells in contrast to null cells. Further analysis has revealed that this 1-kb fragment promotes higher reporter gene activity in MNDA-expressing cells than non-expressing cells, indicating cell-specific differences in transactivation. This sequence contains consensus elements consistent with myeloid-specific gene expression, including a PU.1 consensus site near the major transcription start site and a cluster of c-Myb sites located several hundred bases upstream of this region. However, analysis of deletion mutants localized nearly all of the promoter activity to a short region (-73 to - 16) that did not include the cluster of c-Myb sites. A 4-hp mutation of the core Sp1 consensus element (GC box) (-20) reduced overall promoter activity of the 1-kb fragment. Mutation of the PU.1 site did not significantly affect promoter activity. Only a small region (-35 to +22)including the Sp1 element and transcription start site, but not the PU.1 site was footprinted. The 4- bp mutation of the core Sp1 consensus element abolished footprinting at the site and an antibody super shift reaction showed that Sp1 is one of the factors binding the consensus site. The Sp1 site also co-localizes with a DNase I hypersensitive site. The results indicate that DNA methylation, chromatin structure, and transactivation at an Sp1 site contribute to the highly restricted expression of this myelomonocytic lineage specific gene.

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