Abstract
Two functional genes encoding phosphoglycerate kinase are differentially expressed during spermatogenesis in the mouse. Expression of the X-linked Pgk-1 gene is repressed coincident with X chromosome inactivation during prophase of meiosis I. At this same stage, expression of the autosomal Pgk-2 gene is initiated by tissue-specific mechanisms. To investigate the role of chromatin structure in these processes, we have examined the appearance and disappearance of DNase I-hypersensitive (DH) sites in each gene, and correlated this with transcriptional activity as measured by nuclear run-off analysis at specific stages of spermatogenesis. Our results demonstrate that the occurrence of DH sites is related to periods of active transcription. Results with the Pgk-1 gene indicate that transcriptional inactivation of the X chromosome in spermatogenic cells may not be as complete as that in somatic cells, and that maximum repression may be limited to a very transient period during the pachytene stage of first meiotic prophase. Results with the Pgk-2 gene indicate that DH sites appear coincident with, or just prior to, transcriptional activation of this gene. The implications of these results are discussed with respect to the role of X chromosome inactivation in spermatogenic cells and the developmental order of molecular events that regulate differential gene expression during spermatogenesis.
Highlights
Gene is initiated [2, 3]
The absence of nucleosomes results in these regions becoming hypersensitive to digestion with endonucleases such as DNase I; the presence of bound regulatory proteins protects these same regions from digestion with exonucleases such as Bal-31. To identify such regions in the murine Pgk genes, we first investigated the presence of DNase I-hypersensitive (DH) sites in the 5Ј-flanking region, exon 1, and part of intron I of the Pgk-1 gene, and in the 5Ј-flanking region, coding region, and 3Ј-flanking region of the intronless Pgk-2 gene, in tissues known to express each gene
DH sites found within 1 kb of the 5Ј-flanking end of an active gene are typically most relevant to the control of gene expression [27], mapping to core promoters (16, 28 –30), enhancers [30], and/or silencers of transcription [31,32,33]
Summary
Isolation of Nuclei from Liver and Germ Cells—Nuclei from liver tissue were isolated by Dounce homogenization in the presence of 0.5% Triton X-100 as described elsewhere [16]. The Pgk-2 gene was analyzed with a 1.6-kb EcoRI-HindIII probe fragment from the 5Ј-flanking region of the mouse Pgk-2 gene [21] These probe fragments were isolated by agarose gel electrophoresis and recovered either by digestion with beta agarase (New England Biolabs) or electroelution (IBI/Kodak) according to manufacturer’s instructions.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
