The role of chromatin in the transcriptional regulation of Hoxd4 during hindbrain development and neural differentiation /

Mammalian Hox genes encode a highly conserved family of homeodomain-containing transcription factors that play a crucial role in specifying regional identity and embryonic patterning. Thirty-nine Hox genes are organized into four clusters on different chromosomes each containing up to eleven genes. Importantly, there is a correlation between the genomic location of a specific Hox gene within a cluster and its time and functional domain of expression during development, a phenomenon termed "colinearity". Altering normal Hox expression results in homeotic transformations and malformation, establishing the biological relevance of this tightly controlled expression pattern. Given the integral role of chromatin remodeling in gene regulation, colinear Hox gene activation could be explained in part by successive 3' to 5' conversion from closed to open chromatin along the length of a Hox cluster. We investigated the role of chromatin in regulating Hoxd4, a member of Hox paralog group 4, which is expressed in the CNS with an anterior boundary at r6/7. Previous studies in our laboratory mapped a 3' neural enhancer containing a critical retinoic acid response element absolutely necessary for Hoxd4 expression in the CNS. Moreover, transcripts originating from the P1 proximal promoter are more active in anterior domains of Hoxd4 expression and are responsive to the 3' neural enhancer. We used chromatin immunoprecipitation to detect chromatin changes at the Hoxd4 locus during neurogenesis in P19 cells and embryonic day (E) 8.0 and E 10.5 mouse embryos. In Chapter II, we show that during Hoxd4 induction in both systems, histone modifications typical of transcriptionally active chromatin occur first at the 3' neural enhancer, followed by the promoter. Moreover, the sequential distribution of histone modifications between E8.0 and E10.5 is consistent with a spreading of open chromatin starting with the enhancer, followed by successively more 5' intervening sequences, and culminating at the promoter. In Chapter III, we identify components of the Hoxd4 P1 promoter directing expression in neurally differentiating P19 cells. Our results show that three nucleosomes are positioned at the P1 promoter and are subsequently remodeled into an open chromatin state upon RA-induced Hoxd4 transcription. Moreover, an autoregulatory element was shown to recruit HOXD4 and its cofactor P13X1, and to positively regulate Hoxd4 expression. Conversely, YY1 binds to an inter-nucleosomel linker and represses Hoxd4 transcription before and during transcriptional activation, while the Polycomb Group protein member MEL 18 is co-recruited with YY1 only in undifferentiated P19 cells. In Chapter IV, we discuss the role of sequential chromatin opening in regulating Hox gene expression, and more specifically in controlling transcriptional initiation from P1. Finally, we discuss the consequences of transcription factor binding to P1, and the transcriptional characteristics of P1 that are crucial for regulating Hoxd4 transcription

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

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