Article

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: 5.04). 10/2007; 27(18):6555-68. DOI: 10.1128/MCB.00273-07
Source: PubMed

ABSTRACT 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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    • "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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    • "The four placental genes are expressed at the highest levels, whereas hGH-N, lacking the epigenetic modifications, remains inactive in STBs. From Kimura et al., 2007 "
    Molecular and Cellular Endocrinology 07/2010; 323(1):94-104. DOI:10.1016/j.mce.2009.12.015 · 4.24 Impact Factor
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    • "An explanation may be provided by the work of Kimura and colleagues, who used chromatin immunoprecipitation to examine histone modifications at the human growth hormone (hGH) gene cluster in fusing primary CTBs isolated from human term placentas [23] [24]. Activation of these genes during fusion is associated with robust acetylation of histones H3 and H4, but the lower quantity and temporal pattern of H3 lysine 4 methylation suggest it plays a role in 'priming' the genes for activation [24]. These data are consistent with ultrastructural observations of the STB nuclei. "
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    ABSTRACT: The aim was to test for evidence of transcriptional activity within the nuclei of the syncytiotrophoblast of the human placenta. The syncytiotrophoblast forms the epithelial covering of the villous tree, and is a multinucleated, terminally-differentiated syncytium generated through fusion of the underlying progenitor cytotrophoblast cells. Its nuclei are heterogeneous with respect to chromatin condensation, and previous functional studies of 3H-uridine uptake in vitro have indicated that they are transcriptionally inactive. This observation is surprising given the key roles this tissue plays in active transport, hormone synthesis and metabolic regulation, and has widespread implications for trophoblast physiology and pathophysiology. We used three different approaches to look for evidence of transcriptional activity. First, immunofluorescence staining was performed on paraffin-embedded early pregnancy and term placental villi, using an antibody directed specifically against the actively transcribing form of RNA polymerase II. Second, a nucleoside incorporation assay was applied to placental villi maintained in short-term culture, with and without the transcription blocker alpha-amanitin. Third, histone modifications associated with active chromatin were identified by immunohistochemistry and immunofluorescence. Each of these methods showed transcription to be occurring in a proportion of syncytiotrophoblast nuclei, with qualitative evidence for transcription being more abundant in the first trimester than at term. These findings correlated with electron microscopical observations of prominent nucleoli within the nuclei, particularly during early pregnancy, signifying transcription of ribosomal RNA. Contrary to previous findings, these results confirm that a proportion of syncytiotrophoblast nuclei actively produce mRNA transcripts.
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