The role of chromatin structure in long-range eukaryotic gene regulation.
chromatin structure, histone modifications, growth hormone gene, chorionic somatomammotropin gene, Pit-1, pituitary, placenta, transcriptional controls, mRNA expression.
Description of Research
Long-Range, Epigenetic Gene Activation by the Growth Hormone Locus Control Region.
The human growth hormone (hGH) gene cluster includes 5 genes: the pituitary hGH-N gene, and hCS-A, hCS-L, hGH-V, and hCS-B expressed in placenta. These 5 genes are regulated by a locus control region (LCR) located in the far 5’ region of this multigene locus. The B-cell specific CD79b/Igß gene is interposed between the hGH LCR and the hGH-N gene. The LCR is made up of 5 DNaseI hypersensitive sites (HS) located between -14.5 kb (HSI) and -32 kb (HSV) 5' to hGH-N. These HS are only present in placenta and/or pituitary. When this 32 kb domain plus the linked GH genes are present in a transgenic construct, transgenic mice reproducibly and robustly express the hGH-N gene specifically in pituitary somatotropes and the hCS genes specifically in the placenta. Expression in both tissues is copy-number-dependent and site-of-integration independent. This expression is paralleled by the establishment of a pituitary-specific 32 kb acetylated chromatin domain encompassing the hGH LCR and the contiguous hGH-N promoter. Levels of histone acetylation peak at the centrally located HSI,II. Determinants critical to hGH-N activation map to an array of three Pit-1 sites at HSI. Remarkably, deletion of HSI (99 bp) leads to a loss of histone acetylation throughout the 32 kb domain and >20-fold decrease in hGH-N expression. The acetylated domain encompassing the hGH LCR and hGH-N is organized into two pituitary-specific sub-domains defined by histone H3K4 tri-methylation (H3K4 tri-me); one sub-domain encompasses the LCR region including the CD79b/Igß locus while a second sub-domain encompasses hGH-N. Of note, the epigenetic modifications within the LCR lead to “bystander transcriptional activation” of the adjacent B-cell specific CD79b/Igß gene. These two sub-domains are separated by an intervening segment of minimally modified chromatin. By ChIP, RNA PolII is present throughout the hGH LCR as well as at the active hGH-N gene, and this pituitary-specific distribution of PolII is bimodal, closely paralleling the distribution of H3K4 tri-me. RT-PCR reveals non-coding, bi-directional RNA transcripts in this same region, confirming the presence of actively transcribing PolII within the hGH LCR. Deletion of HSI (99 bp) results in the marked and coordinate loss of the H3K4 tri-me, diminution of PolII recruitment within the LCR, and loss of the non-coding RNA transcripts. Insertion of a PolII terminator downstream of HSI,II blocks CD79b transcription and represses hGH-N expression, documenting a regulatory role for the noncoding transcripts and linking the “bystander” CD79b transcription to hGH-N regulation. Ongoing experiments will attempt to further understand the role of noncoding and CD79b transcripts in hGH-N gene regulation; to determine whether DNA looping between the LCR and GH genes is essential; to understand how the LCR activates the four placental GH genes.
The significance of these lines of investigation will be the detailed understanding of the regulation of the hGH/hCS gene cluster by its LCR via epigenetic changes in these tissues. These findings will serve as the foundation for a sophisticated understanding of the pathophysiology of human growth and development, and the role of epigenetic regulation of CD79b/Igß.
general adult endocrinology
genetically modified mouse models, mouse embryo cryopreservation
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Ho Y, Elefant F., Cooke NE, Liebhaber SA: A defined locus control region determinant links chromatin domain acetylation with long-range gene activation. Molecular Cell 9: 291-302, 2002.
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Last updated: 06/22/2012
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