Progress 01/01/09 to 12/31/12
Outputs
OUTPUTS: Transcription factor binding sites are important regulatory DNA sequences. Signal transducer and activator of transcription 5 (STAT5) is a versatile transcription factor involved in multiple biological processes, including muscle growth, mammary gland development, lactation, and adipogenesis. STAT5 is involved in these processes by mediating the effects of growth hormone, prolactin, and interleukins on gene transcription. Identifying the binding sites for STAT5 in the bovine genome is essential to understanding how STAT5 controls various biological processes and to identifying single nucleotide polymorphisms, deletions, or insertions in STAT5 binding sites that contribute to variations in production traits in cattle. The overall goal of this project was to identify STAT5 target genes and STAT5 binding sites in the cattle genome. To achieve this goal, the following studied have been conducted: 1) Identifying STAT5 binding sites and target genes in bovine skeletal muscle, adipose tissue, and mammary gland; 2) Identifying growth hormone (GH)-induced STAT5 binding sites and target genes in bovine liver. PARTICIPANTS: Dr. Honglin Jiang from the Department of Animal and Poultry Sciences and Dr. Liqing Zhang from the Department of Computer Science of Virginia Tech served as PD and co-PD, respectively, on this project. Postdoctoral research associates Dr. Aihua Wang, Dr. Xiaomei Ge and graduate students Satyanarayana Eleswarapu, Jie Yu, Xiaomei Ge, Lidan Zhao (Animal science) and Dan Qiao (Computer Science), and Dr. Honglin Jiang carried out experiments and analyzed data for this project. TARGET AUDIENCES: The target audiences for this project include farm animal geneticists and physiologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We used chromatin immunoprecipitation (ChIP) coupled with next generation sequencing (ChIP-Seq) to identify STAT5-binding genomic regions. This led to the identification of 1,548, 3,330, and 3,387 potential STAT5-binding regions in bovine liver, cultured bovine skeletal muscle cells, and cultured bovine adipose cells, respectively. These numbers suggest that STAT5 may regulate the transcription of thousands of genes in each of these bovine tissues. Interestingly, only a small percentage of these STAT5-binding genomic regions were shared by the three types of tissues. This suggests that STAT5 is activated by extracellular signals or STAT5 regulates gene transcription in a tissue-specific manner in cattle. The majority of the identified STAT5-binding regions were located in the distal 5'- and 3'-flanking regions and introns of genes, suggesting that the major mechanism by which STAT5 regulates gene transcription involves chromatin looping and chromatin modification. Motif scans revealed that nearly 50% of the identified STAT5-binding genomic regions contained at least one sequence that resembled STAT5 binding consensus sequence (TTCNNNGAA, where N is A, C, G, or T). Motif scans also revealed several additional motifs enriched in the identified STAT5-binding genomic regions. The enrichment of non-STAT5 binding site motifs in STAT5-binding genomic regions raises the possibility that STAT5 might also regulate gene transcription by interacting with transcription factors that bind directly to these DNA motifs. Using a microarray analysis, we identified 392 and 25 genes that were upregulated and downregulated, respectively, by GH in bovine liver. Whereas 60% of the GH-upregulated genes contained at least one STAT5 binding site in their 2-kb promoters, only 30% of the GH-downregulated genes contained putative STAT5 binding sites in their promoters. This difference suggests that whereas GH upregulation of gene expression in bovine liver is probably mainly mediated by STAT5, GH downregulation of gene expression in bovine liver is perhaps mediated mainly by other transcription factors. Many of the identified GH-regulated genes were previously not known to be regulated by GH. We selected two of such genes, FOXA3, which is also called hepatocyte nuclear factor 3 gamma, and FGF21, which is a recently discovered metabolic regulator, and studied their regulation by GH in detail. Using real-time RT-PCR, we confirmed their upregulation by GH in bovine liver. Using EMSA and ChIP-PCR, we validated STAT5 binding to the STAT5 binding sites in the promoters of these genes. Using co-transfection and reporter gene assays, we demonstrated the importance of these STAT5 binding sites in mediating GH-induced gene transcription in cultured cells.
Publications
- Ge, X., Zhang, Y., and Jiang, H. Signaling pathways mediating the effects of insulin-like growth factor-I in bovine muscle satellite cells. Molecular and Cellular Endocrinology. Available online 27 March 2013.
- Zhao, L., Wang, H., and Jiang, H. 2012. Growth hormone inhibits differentiation of bovine preadipocytes into adipocytes partly through signal transducer and activator of transcription 5. The Endocrine Society's Annual Meeting, Houston, TX, June 23-26, 2012.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The overall goal of this project was to identify STAT5 target genes and STAT5 binding sites in bovine liver, skeletal muscle, adipose tissue, and mammary gland. Using chromatin immunoprecipitation (ChIP) coupled with next generation sequencing (ChIP-Seq), we identified 1548, 3330, and 3387 potential STAT5-binding regions from bovine liver tissue, cultured bovine myoblasts and myotubes, and cultured bovine preadipocytes and adipocytes, respectively. This data suggests that STAT5 may regulate the transcription of thousands of genes in each of these bovine tissues. Interestingly, only a small percentage of these STAT5-binding genomic regions were shared by the three types of tissues. This suggests that activation of STAT5 by extracellular signals or action of STAT5 on gene transcription is tissue specific. The majority of the identified STAT5-binding regions were located in the distal 5'- and 3'-flanking regions and introns of genes, suggesting that the major mechanism by which STAT5 regulates gene transcription involves chromatin looping and chromatin modification. Motif scans revealed that nearly 50% of the identified STAT5-binding genomic regions contain at least one sequence that resembles consensus STAT5 binding site (TTCNNNGAA). Motif scans also revealed several additional motifs enriched in the identified STAT5-binding genomic regions. The enrichment of non-STAT5 binding site motifs in STAT5-binding genomic regions raises the possibility that STAT5 might also regulate gene transcription by interacting with transcription factors that bind directly to these DNA motifs. Using microarray analysis, we identified 392 and 25 genes that were upregulated and downregulated, respectively, by GH in bovine liver. Whereas 60% of the GH-upregulated genes contain at least one STAT5 binding site in their 2-kb promoters, only 30% of the GH-downregulated genes do so. This difference suggests that whereas STAT5 is a major transcription factor mediating GH upregulation of gene expression in bovine liver, it is not in terms of GH downregulation of gene expression in bovine liver. Many of the identified GH-regulated genes were previously not known to be regulated by GH. We selected two of such genes, FOXA3, which is also called hepatocyte nuclear factor 3 gamma, and FGF21, which is a recently discovered metabolic regulator, and studied their regulation by GH in detail. Using real-time RT-PCR, we confirmed their upregulation by GH in bovine liver. Using EMSA and ChIP-PCR, we validated STAT5 binding to the STAT5 binding sites in the promoters of these genes. Using co-transfection and reporter gene assays, we demonstrated the function of these STAT5 binding sites in mediating GH-induced gene transcription in cultured cells. PARTICIPANTS: Graduate students: Yu, J., Eleswarapu, S., Zhao, L., Ge, X. (Animal Science); Qiao, D., Liu, M. (Computer Science). Postdoctoral research associate: Wang, A. (animal Science). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have identified thousands of STAT5-DNA interactions in bovine liver, skeletal muscle and adipose tissues. Further characterization of these interactions may lead to novel understanding of the molecular mechanisms by which liver metabolism, muscle growth, and fat deposition are controlled by STAT5 and the hormones that activate STAT5. The finding that only a small percentage of the identified STAT5-binding genomic regions contain consensus STAT5 binding sites is unanticipated, but it raises the possibility that STAT5 might regulate gene transcription by interacting with other transcription factors that directly bind to DNA.
Publications
- Yu J, Zhao L, Wang A, Eleswarapu S, Ge X, Chen D, Jiang H. 2012. Growth hormone stimulates transcription of the fibroblast growth factor 21 gene in the liver through the signal transducer and activator of transcription 5. Endocrinology. 153(2):750-8.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: During this reporting period, we focused on identifying the STAT5-binding genomic regions in bovine liver, skeletal muscle, and adipose tissue using the ChIP-Seq approach. We collected liver, skeletal muscle, and adipose tissue from adult cattle. We isolated satellite cells from skeletal muscle using Pronase digestion and stromal-vascular cells from adipose tissue using Collagenase D digestion. Satellite cells were induced to fuse into myotubes in medium containing 2% horse serum, and stromal-vascular cells to differentiate into adipocytes in medium supplemented with dexamethasone, isobutylmethylxanthine (IBMX), and insulin. Chromatin was isolated from bovine liver, bovine myoblasts and myotubes, and bovine preadipocytes and adipocytes, and was immunoprecipitated with an anti-STAT5 antibody. ChIP-Seq libraries were made from the STAT5 antibody-precipitated chromatin or untreated chromatin (i.e. Input chromatin) using the ChIP-Seq DNA Sample Prep Kit from Illumina. ChIP-Seq libraries were sequenced on a Genome Analyzer. PARTICIPANTS: Dr. Honglin Jiang from the Department of Animal and Poultry Sciences and Dr. Liqing Zhang from the Department of Computer Science of Virginia Tech served as PD and co-PD, respectively, during this reporting period. Postdoctoral research associate Dr. Aihua Wang (Animal Science), graduate students Lidan Zhao (Animal Science), Dan Qiao (Computer Science), and Mingming Liu (Computer Science), and Dr. Honglin Jiang carried out experiments and analyzed data for this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Sequencing the library prepared from the STAT5 antibody-precipitated bovine liver chromatin (i.e. liver ChIP-Seq library) and the library from Input bovine liver chromatin (i.e. liver Input library) generated 14,182,004 and 36,386,567 short sequence reads, respectively. Sequencing the muscle ChIP-Seq library and the muscle Input library generated 39,562,939 and 39,198,425 sequence reads, respectively. Sequencing the adipose ChIP-Seq library and the adipose Input library generated 33,833,850 and 21,371,312 sequence reads, respectively. Between 50% and 60% of these sequence reads uniquely mapped to the bovine genome. Comparing the genomic distribution of the mapped sequences between the ChIP-Seq libraries and their respective Input libraries identified 1548, 3330, and 3387 genomic regions enriched in bovine liver, muscle, and adipose tissue, respectively. These genomic regions were potential STAT5-binding DNA regions, and identification of thousands of them from bovine liver, skeletal muscle, and adipose tissue suggests that STAT5 may regulate, directly or indirectly, the transcription of thousands of genes in these tissues. However, only a small percentage of STAT5-binding genomic regions were identical between the three types of tissues, suggesting that activation of STAT5 or action of STAT5 on gene transcription may be tissue specific. The majority of the identified STAT5-binding regions were located in the distal 5'- and 3'-flanking regions and introns of genes, suggesting that the major mechanism by which STAT5 regulates gene transcription involves chromatin looping and modification. A motif scanning revealed that 5% of the identified STAT5-binding regions contained at least one consensus STAT5 binding site (TTCNNNGAA) and that 40% or 60% of them contained sequences having one or two mismatches, respectively, with the consensus STAT5 binding sequence. It remains to be determined whether these STAT5 binding motif-like sequences are true STAT5 binding sites in vivo. The motif scanning also revealed several additional motifs enriched in the identified STAT5-binding genomic regions. The possibility that STAT5 indirectly binds to these motifs to regulate gene transcription will be evaluated.
Publications
- Jiang, H., Eleswarapu, S., and Wang, Y., 2010. Mechanism of growth hormone regulation of insulin‐like growth factor (IGF)‐I gene expression. The Asia-Pacific Journal of Endocrinology. (online).
- Zhao, L., Corl, B. A., and Jiang, H., 2010. Both growth hormone and signal transducer and activator of transcription 5b inhibit glycerol-3-phosphate dehydrogenase activity and CCAAT/enhancer binding protein α mRNA expression in differentiating bovine preadipocytes. Journal of Animal Science 88 (E-Suppl. 2): 632.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The primary goal of the proposed project was to identify genome-wide STAT5 binding sites in bovine liver, skeletal muscle, adipose, and mammary gland tissues using the ChIP-Seq approach. Our work during this initial reporting period has been focused on identifying a STAT5 antibody that can efficiently immunoprecipitate STAT5 protein-chromatin complexes from bovine tissues because a key step in successful ChIP-Seq is to identify an antibody that can bind efficiently to the transcription factor of interest while the transcription factor is cross-linked to chromatin. In addition, we have constructed and sequenced two ChIP-Seq libraries from liver tissue. PARTICIPANTS: Dr. Honglin Jiang from the Department of Animal and Poultry Sciences and Dr. Liqing Zhang from the Department of Computer Science of Virginia Tech served as PD and co-PD, respectively, for this project during this reporting period. Postdoctoral research associate Dr. Aihua Wang and graduate students Satyanarayana Eleswarapu (Animal science) and Dan Qiao (Computer Science) as well as Dr. Honglin Jiang carried out experiments and analyzed data for this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The bovine HNF-3γ gene contains two candidate STAT5 binding sites in its promoter region, and the transcription of this gene in the liver is upregulated by growth hormone (GH). Electrophoretic mobility shift assays demonstrated that one of the two candidate STAT5 binding sites could form a DNA-protein complex with bovine liver STAT5 protein in vitro. Co-transfection analyses showed that GH-activated STAT5 stimulated reporter gene expression from the bovine HNF-3γ promoter and that this stimulation was dependent on the confirmed STAT5 binding site. These in vitro data indicated that GH might stimulate binding of STAT5 protein to the HNF-3γ promoter and thereby increase HNF-3γ transcription in bovine liver. Therefore, the HNF-3γ promoter is a good DNA region for testing the qualification of STAT5 antibody for ChIP-Seq. We tested the ability of several commercially available STAT5 antibodies to immunoprecipitate the protein-chromatin complexes formed between STAT5 protein and the HNF-3γ promoter region from GH-treated bovine liver. These tests revealed that a STAT5 antibody from Santa Cruz Biotechnology was able to efficiently enrich the HNF-3γ promoter DNA from the GH-injected bovine liver, which means this STAT5 antibody is qualified for ChIP-Seq work. We have constructed two ChIP-Seq libraries, one from DNA immunoprecipitated by this STAT5 antibody from GH-injected liver and one from input DNA. Solexa sequencing of these two libraries generated 21,251,886 and 2,114,928 short sequence reads (35 nt). Bioinformatics analyses that map these sequences to the bovine genome and that identify potential STAT5 binding sites are underway.
Publications
- Eleswarapu S, Ge X, Wang Y, Yu J, Jiang H. 2009. Growth Hormone-activated STAT5 may indirectly stimulate IGF-I gene transcription through HNF-3γ. Mol. Endocrinol. 23:2026-2037.
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