Modulation of Cx43 and Gap Junctional Intercellular Communication by Androstenedione in Rat Polycystic Ovary and Granulosa Cells in vitro.

BACKGROUND: Gap-junctional intercellular communication (GJIC) is implicated in physicological processes and it is vitally important for granulosa cell (GC) differentiation and oocyte growth. We investigated the expression of connexin 43 (Cx43), a gap junctional protein, in normal and androstenedione-induced polycystic ovary (PCO), the effects of androstenedione on Cx43 expression, GJIC and progesterone production in granulosa cells in vitro. METHODS: Isolated GCs from rat ovary were supplemented with FSH and dripped with EHS-matrix (EHS-drip) in culture media, were treated with physiological (10(-7) M) or pathological (10(-5) M) androstenedione concentrations to induce differentiation. Cx43 protein levels were assessed by Western blotting. Immunohistochemistry was also used to determine the localization of Cx43 in GCs and corpus luteum (CL) of controls and PCOs. Differentiation of GCs was determined by progesterone assay and Lucifer yellow dye transfer for GJIC status. The degree of significance of variations between the results was analyzed by ANOVA using SPSS (version 11.5; 2002). RESULTS: Cx43 localized in the GC layer of both the control and PCOs. Its protein levels were upregulated in PCO rat ovaries. GCs in culture with or without androstenedione had a punctate membranous distribution of Cx43. However, androstenedione increased GJIC and upregulated progesterone and Cx43 protein levels. Inhibiting GJIC by 18-α GA in androstenedione-treated GCs caused partial inhibition of progesterone production, suggesting a possible role of GJIC in mediating the action of androstenedione on GC differentiation. CONCLUSION: This study presented a suitable culture model for polycystic ovary syndrome and showed that Cx43 and GJIC might contribute to the pathogenesis of polycystic ovary syndrome.

Interplay between chromatin and trans-acting factors regulating the Hoxd4 promoter during neural differentiation.

Correct patterning of the antero-posterior axis of the embryonic trunk is dependent on spatiotemporally restricted Hox gene expression. In this study, we identified components of the Hoxd4 P1 promoter directing expression in neurally differentiating retinoic acid-treated P19 cells. We mapped three nucleosomes that are subsequently remodeled into an open chromatin state upon retinoic acid-induced Hoxd4 transcription. These nucleosomes spanned the Hoxd4 transcriptional start site in addition to a GC-rich positive regulatory element located 3' to the initiation site. We further identified two major cis-acting regulatory elements. An autoregulatory element was shown to recruit HOXD4 and its cofactor PBX1 and to positively regulate Hoxd4 expression in differentiating P19 cells. Conversely, the Polycomb group (PcG) protein Ying-Yang 1 (YY1) binds to an internucleosomal linker and represses Hoxd4 transcription before and during transcriptional activation. Sequential chromatin immunoprecipitation studies revealed that the PcG protein MEL18 was co-recruited with YY1 only in undifferentiated P19 cells, suggesting a role for MEL18 in silencing Hoxd4 transcription in undifferentiated P19 cells. This study links for the first time local chromatin remodeling events that take place during transcriptional activation with the dynamics of transcription factor association and DNA accessibility at a Hox regulatory region.

Sequential histone modifications at Hoxd4 regulatory regions distinguish anterior from posterior embryonic compartments.

Hox genes are differentially expressed along the embryonic anteroposterior axis. We used chromatin immunoprecipitation to detect chromatin changes at the Hoxd4 locus during neurogenesis in P19 cells and embryonic day 8.0 (E8.0) and E10.5 mouse embryos. During Hoxd4 induction in both systems, we observed that histone modifications typical of transcriptionally active chromatin occurred first at the 3' neural enhancer and then at the promoter. Moreover, the sequential distribution of histone modifications between E8.0 and E10.5 was consistent with a spreading of open chromatin, starting with the enhancer, followed by successively more 5' intervening sequences, and culminating at the promoter. Neither RNA polymerase II (Pol II) nor CBP associated with the inactive gene. During Hoxd4 induction, CBP and RNA Pol II were recruited first to the enhancer and then to the promoter. Whereas the CBP association was transient, RNA Pol II remained associated with both regulatory regions. Histone modification and transcription factor recruitment occurred in posterior, Hox-expressing embryonic tissues, but never in anterior tissues, where such genes are inactive. Together, our observations demonstrate that the direction of histone modifications at Hoxd4 mirrors colinear gene activation across Hox clusters and that the establishment of anterior and posterior compartments is accompanied by the imposition of distinct chromatin states.