Epigenetic Activation of the Human Growth Hormone Gene Cluster during Placental Cytotrophoblast Differentiation

Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 10/2007; 27(18):6555-68. DOI: 10.1128/MCB.00273-07
Source: PubMed


The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.

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Available from: Nancy E Cooke, Oct 07, 2015
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    • "Although the primary structure of the five genes is well conserved, hGH-N is specifically expressed in the pituitary and the other four genes are placenta specific [21] [22]. The tissue-specific activation of the hGH cluster is dependent on the 5′-distal locus control region (LCR) [23] [24], and epigenetic regulation and noncoding transcription are known to play crucial roles in activation by the hGH LCR [25] [26] [27] [28] [29]. Interestingly , the hGH cluster is linked to two other tissuespecific genes: the B-cell-specific CD79b gene, which is located between the cluster and LCR, and the testis-specific testicular cell adhesion molecule (TCAM1P) gene in the 3′-region of the cluster. "
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    ABSTRACT: Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5'-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.
    Journal of Molecular Biology 07/2014; 426(17). DOI:10.1016/j.jmb.2014.06.018 · 4.33 Impact Factor
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    • "Kumar et al. detected an augmented H3K9/14 acetylation and decreased H3K9 methylation linked to the transcriptional induction of the human CYP19 gene during the differentiation of trophoblast cells [57]. Additionally, transcriptional activation of the hGH genes has been linked to unique roles not only for HAT, but also for histone methyltransferase co-activator complexes, and to an expansive histone H3 and H4 acetylation of the locus in syncytium, where the four placental hGH genes are expressed at the highest levels [58]. Collectively, these data support our findings suggesting that lysine acetylation may contribute to the remarkable increase of PSG gene expression in trophoblast cells. "
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    ABSTRACT: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs) up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140), was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA). This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT) function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.
    PLoS ONE 02/2013; 8(2):e55992. DOI:10.1371/journal.pone.0055992 · 3.23 Impact Factor
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    • "We have intensely studied a small subset of genes, particularly the casein genes, so there are limits to which these findings can apply to the whole genome. However, similar findings for other spatially, temporally and lineages regulated genes in different tissues [34], [71]–[73] show the general applicability of these studies and the mammary gland is an excellent model system for the understanding of the role of epigenetic regulation in development and functional differentiation of somatic tissue. In contrast to whole genome studies, this focused study of the casein region enabled us to finely characterize regions of interest such as the evolutionary conserved regions and learn more about the regulation of casein gene cluster, encoding major milk proteins, with major nutritional functions. "
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    ABSTRACT: Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation. Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization. A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland.
    PLoS ONE 01/2013; 8(1):e53270. DOI:10.1371/journal.pone.0053270 · 3.23 Impact Factor
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