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Donald C Wilkerson

Publications and source records attributed to Donald C Wilkerson.

6 recordsLinked to original sources
PubMed2005

Mechanism of hsp70i gene bookmarking.

In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking.

Adenosine Triphosphatases↗
PubMed2004

Regulatory factor X2 (RFX2) binds to the H1t/TE1 promoter element and activates transcription of the testis-specific histone H1t gene.

Transcription of the mammalian testis-specific linker histone H1t gene occurs only in pachytene primary spermatocytes during spermatogenesis. Studies of the wild type (Wt) and mutant H1t promoters in transgenic mice show that transcription of the H1t gene is dependent upon the TE promoter element. We purified an 85 kDa protein from rat testis nuclear extracts using the TE1 subelement as an affinity chromatography probe and analysis revealed that the protein was RFX2. The TE1 element is essentially an X-box DNA consensus element and regulatory factor X (RFX) binds specifically to this element. Polyclonal antibodies directed against RFX2 supershift the low mobility testis nuclear protein complex formed in electrophoretic mobility shift assays (EMSA). RFX2 derived from primary spermatocytes, where the transcription factor is relatively abundant, binds with high affinity to the TE1 element. Coexpression of RFX2 together with an H1t promoter/reporter vector activates the H1t promoter in a cultured GC-2spd germinal cell line, but mutation of either the TE1 subelement or the TE2 subelements represses activity. These observations lead us to conclude that the TE1 and TE2 subelements of the testis-specific histone H1t promoter are targets of the transcription factor RFX2 and that this factor plays a key role in activating transcription of the H1t gene in primary spermatocytes. Published 2003 Wiley-Liss, Inc.

Animals↗
PubMed2003

TE2 and TE1 sub-elements of the testis-specific histone H1t promoter are functionally different.

The testis-specific linker histone H1t gene is transcribed exclusively in pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in part by transcriptional factors that bind elements located within the proximal and distal promoter. A 40 bp promoter element, designated H1t/TE, that is located within the proximal promoter between the CCAAT-box and AC-box, is known to be essential for H1t gene transcription in transgenic animals. In the present study, we show by SDS-PAGE analysis of UV crosslinked protein and DNA and by electrophoretic mobility shift assays (EMSA) of testis nuclear proteins separated on a non-denaturing glycerol gradient that the TE1 sub-element is bound by a protein complex. Mutation of TE1 leads to a drop in H1t promoter activity in germinal GC-2spd cells as well as in nongerminal Leydig, NIH3T3, and C127I cell lines. Although TE1 and TE2 sub-elements have similar sequences, mutation of the TE2 sub-element causes an increase in promoter activity in C127I and Leydig cells. The rat TE1 but not TE2 contains a CpG dinucleotide and this cytosine is methylated in liver but not in primary spermatocytes. Methylation of the cytosine at this site almost eliminates nuclear protein binding. Thus, there are significant functional differences in the TE2 and TE1 sub-elements of the H1t promoter with TE1 serving as a transcriptional activator binding site and TE2 serving as a repressor binding site in some cell lines.

Animals↗
PubMed2002

Sp1 and Sp3 activate the testis-specific histone H1t promoter through the H1t/GC-box.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in large part through elements located within the proximal promoter. Previous work in transgenic animals showed that a unique 40 bp promoter element designated H1t/TE is essential for spermatocyte-specific expression. The H1t/TE element contains a GC-box, which is a perfect consensus binding site for members of the Sp family of transcription factors. We have shown that Sp1 and Sp3 are present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids and that they can bind to the H1t/GC-box. Mutagenesis of the GC-box reduced H1t promoter activity. Furthermore, a CpG dinucleotide within the GC-box was totally unmethylated in rat testis primary spermatocytes where the gene is transcribed but it was methylated in liver where the gene is silenced. These previous studies supported the importance of the GC-box and suggested that Sp transcription factors contribute to expression of the H1t gene. In this study, we show that co-transfection of Sp1 and Sp3 expression vectors leads to an upregulation of histone H1t promoter activity in several cell lines including testis GC-2spd cells. However, very low H1t promoter activity is seen in GC-2spd cells grown at 39 degrees C, which correlates with lower levels of Sp1 and Sp3 in these cells grown at this elevated temperature. Upregulation of the H1t promoter by Sp1 and Sp3 was also seen in cotransfected NIH3T3 and C127I cell lines. On the other hand, co-transfection of the Sp1 and Sp3 expression vectors does not lead to upregulation of activity of the cell-cycle dependent histone H1d promoter.

Animals↗
PubMed2002

H1t/GC-box and H1t/TE1 element are essential for promoter activity of the testis-specific histone H1t gene.

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue-specific expression of the gene is mediated primarily through elements located within the proximal promoter. Previous work in transgenic animals identified a unique 40-base pair promoter element designated H1t/TE that is essential for spermatocyte-specific expression. The H1t/TE element contains three subelements designated TE2, GC-box, and TE1 based on in vitro footprinting and electrophoretic mobility shift assays. Because GC-box is a consensus site for binding of Sp transcription-factor family members, experiments were performed demonstrating that two Sp family members, Sp1 and Sp3, were present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids. A 95- to 105-kDa form of Sp1 is most abundant in the tissues and cell lines examined, but a 60-kDa form of Sp1 is the most abundant species in spermatocytes and early spermatids. Further examination of Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids showed that they can bind to the H1t/TE element. In order to determine the contributions of the subelements to H1t transcription, we mutated each of them in H1t promoter luciferase reporter vectors. Mutation of the GC-box and TE1 subelement reduced expression 77% and 49%, respectively, compared with the wild-type H1t promoter in transient expression assays in a testis GC-2spd cell line that was derived from germinal cells. These studies suggest that Sp transcription factors may be involved in transcription of the H1t gene and the GC-box and the TE1 subelement are required for activation of the H1t promoter.

Animals↗