Progress 01/01/16 to 12/31/19
Outputs
Target Audience:Animal scientists, in particular, animal geneticists and animal physiologists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training opportunities for two PhD students. How have the results been disseminated to communities of interest?Through publication in peer-reviewed journals and presentations at relevant conferences such as PAG. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the last reporting period, we conducted RNA-seq and ChIP-seq experiments in bovine preadipocytes and adipocytes. We isolated the stromal vascular fraction from the subcutaneous fat of Angus-crossbred steers using collagenase digestion. We expanded the isolated stromal vascular fraction as preadipocytes in growth medium consisting of medium DMEM and 10% FBS (fetal bovine serum). To differentiate bovine preadipocytes into adipocytes, we cultured them in growth medium until confluency and then cultured them for 2 days in differentiation induction medium (DMEM/F12, insulin, dexamethasone, 3-isobutyl-1-methylxanthine, rosiglitazone) followed by 4 days in differentiation maintenance medium (DMEM/F12, insulin, and rosiglitazone). We confirmed adipocyte differentiation by significantly increased mRNA expression of selected adipocyte markers including PPAR gamma, C/EBP alpha, fatty acid binding protein 4, and adiponectin. We constructed 10 RNA-seq libraries using total RNA from preadipocytes and adipocytes from five different steers. For ChIP-seq experiments, we immunoprecipitated crosslinked and sonicated chromatin from preadipocytes and adipocytes from two steers with an H3K27ac antibody and constructed four ChIP-seq libraries using co-immunoprecipitated DNA from these CHIP assays. We also made 4 Input-seq libraries using chromatin prior to immunoprecipitation from the same preadipocytes and adipocytes. We marked each of these RNA-seq and ChIP-seq libraries with a unique index. We had these libraries sequenced on an Illumina Highseq 2000. We have received ~400 Gb sequencing data and are in the process of analyzing these data. We expect to identify genes differentially expressed between bovine preadipocytes and adipocytes, and H3K27ac-marked genomic regions differentially associated with preadipocytes and adipocytes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Leng, X., Ji, X., Hou, Y., Settlage, R., and Jiang, H. 2019. Roles of the proteasome and inhibitor of DNA binding 1 protein in myoblast differentiation. FASEB J. 33(6):7403-7416.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Jiang, H., Settlage, R., & Leng, X. (2019). Identification of Active Enhancers Associated with Bovine Myoblast Differentiation. Poster session presented at the meeting of The Plant and Animal Genome ASIA 2019 Conference in Shenzhen, China.
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Progress 01/01/18 to 12/31/18
Outputs
Target Audience:Scientists that work in the areas of animal genomics and animal growth. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Provided a training opportunity for a PhD student How have the results been disseminated to communities of interest?Publications in peer-reviewed journals What do you plan to do during the next reporting period to accomplish the goals?Continute and complete the studies that are aimed to identify active enhancers in bovine preadipocytes and adipocytes.
Impacts
What was accomplished under these goals?
During this reporting period, we conducted additional ChIP-seq experiments to identify genomic regions with H3K27ac modification (i.e., active enhancers) in undifferentiated and differentiating bovine myoblasts. Satellite cells (i.e., myogenic progenitor cells in adult animals) isolated from Angus crossbred steers were cultured in growth medium containing 10% fetal bovine serum to proliferate as myoblasts or in differentiation medium containing 2% horse serum to differentiate and fuse into myotubes. ChIP-seq libraries were constructed from DNA precipitated with an H3K27ac antibody from myoblasts cultured in growth medium for 10 days (i.e., undifferentiated myoblasts) and myoblasts subsequently cultured in differentiation medium for 2 days (i.e., differentiating myoblasts). A ChIP-seq library was also constructed from Input DNA combined from undifferentiated and differentiating myoblasts. Sequencing the libraries and mapping the sequence reads to the bovine genome generated approximately 5, 6, and 10 million uniquely mapped sequence reads from the Input DNA, H3K27ac antibody-precipitated DNA from undifferentiated myoblasts, and H3K27ac antibody-precipitated DNA from differentiating myoblasts, respectively. Analyzing the uniquely mapped sequences using MACS revealed 57,703 and 69,039 H3K27ac-marked genomic regions in undifferentiated and differentiating myoblasts, respectively. Of these regions, 15,223 were unique to undifferentiated myoblasts, 26,559 to differentiating myoblasts, and 42,480 common to both undifferentiated and differentiating myoblasts. In both undifferentiated and differentiating myoblasts, H3K27ac-marked genomic regions were associated with greater gene expression, consistent with their role as enhancers in gene transcription. Approximately, 44%, 35%, and 14% of the H3K27ac-marked genomic regions were located in the intergenic, intron, and promoter regions, respectively. Less than 5% of these regions were located in exons or transcription termination regions. A HOMER analysis indicated that H3K27ac-marked regions were enriched with binding sites for transcription factors RXR, MyoD, and PPAR in undifferentiated myoblasts and that they were enriched with binding sites for transcription factors including MyoD, Myf5, MyoG, and NFkB in differentiating myoblasts. We are in the process of determining the role of some of these transcription factors in myoblast differentiation.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Leng X., and Jiang, H. Effects of arachidonic acid and its major prostaglandin derivatives on bovine myoblast proliferation, differentiation, and fusion. Domestic Animal Endocrinology. DOI information: 10.1016/j.domaniend.2018.12.006
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Investigators who are interested in animal genomics and growth biology Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research provided training opportunities to one PhD student. How have the results been disseminated to communities of interest?Some of the results from this project have been reported in the 2017 ASAS meeting. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we willconduct ChIP-seq and RNA-seq experiments in bovine preadipocytes and adipocytes.
Impacts
What was accomplished under these goals?
During this reporting period, we conducted a study to determine the role of proteasome-mediated protein degradation in the differentiation and fusion of bovine myoblasts into myotubes. This study was based on our earlier discovery that many genes encoding the ubiquitin-proteasome pathway in bovine myoblasts were upregulated during their differentiation and fusion into myotubes. We isolated satellite cells from 5 Angus or Angus crossbred steers, expanded them as myoblasts in growth medium for up to 10 days, and then induced them to differentiate and fuse into myotubes in differentiation medium. An assay of the 20S proteasome activity in myoblasts at 0, 24, 48, and 72 h of differentiation confirmed that the proteasome activity increased during myoblast differentiation and fusion. Myoblast differentiation and fusion into myotubes was nearly completely blocked by adding 5 μM lactacystin, a specific inhibitor of the 20S proteasome, to the differentiation medium, as indicated by Giemsa staining of cells, calculation of fusion index, and quantification of the mRNA expression levels of markers for differentiated myoblasts and fused myotubes. A western blot analysis of poly-ubiquitinated protein confirmed the inhibition of lactacystin on the proteasome in bovine myoblasts. Inhibitor of DNA-binding 1 (ID1) is one of the proteins that inhibit myoblast differentiation and fusion and that is known to be degraded by the proteasome in several cell types. Both ID1 protein and mRNA expression in bovine myoblasts decreased during differentiation and fusion into myotubes, and the decrease in ID1 protein, not in ID1 mRNA, was reversed by including lactacystin in the differentiation medium. Collectively, these results suggest that the proteasome-mediated protein degradation is beneficial to bovine myoblast differentiation and fusion into myotubes, and that this benefit may be mediated in part by the ID1 protein.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Xinyan Leng and Honglin Jiang. Identification of a beneficial role of proteasome-mediated protein degradation in the differentiation of bovine myoblasts into myotubes. 2017 ASAS-CSAS Annual Meeting and Trade Show.
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Animal physiologists, animal genomicists, and animal geneticists and their students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research providedtraining opportunities to two graduate students (one PhD, one MS). How have the results been disseminated to communities of interest?Some of the results from this project have been reported in 2016 ASDS/ASAS meeting. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will complete analyzing the ChIP-seq and RNA-seq data from bovine satellite cells and will conduct experiments to identify CBP/p300-binding genomic regions in undifferentiated and differentiated bovine satellite cells.
Impacts
What was accomplished under these goals?
Two ChIP-seq libraries were made from DNA immunoprecipitated by anti-H3K27ac antibody from undifferentiated and differentiated bovine satellite cells. A third library was made from input DNA pooled from undifferentiated and differentiated satellite cells. Hiseq 2500 sequencing generated approximately 50 million of high-quality sequencing reads from each library. Analyzing the uniquely mapped reads (7 to 13 million) to the bovine genome (Bos_taurus UMD3.1) using MACS1.4 revealed 58,878 and 52,452 genomic regions enriched with H3K27ac modification in differentiated and undifferentiated bovine satellite cells, respectively.Examples of genes associated with a greater number of H3K27ac peaks in differentiated than undifferentiated bovine satellite cells included MYH3, MYOG, and CKM, which were expressed at greater levels in differentiated than undifferentiated bovine satellite cells.Eight RNA-seq libraries were made from undifferentiated and differentiated bovine satellite cells. A total of 5,538 transcripts differentially expressed (counts > 20; the FDR-adjusted P < 0.05) between undifferentiated and differentiated satellite cells, of which 2,937 were upregulated and 2,601 downregulated in differentiated compared to undifferentiated cells. Genes upregulated in differentiated satellite cells included MYOG and MYF6, which are known stimulators of myogenic differentiation. Genes downregulated in differentiated satellite cells included MSTN and MDFI, which are known inhibitors of myogenic differentiation. Functional clustering of the upregulated genes revealed enrichment in contractile fiber, sarcoplasmic reticulum, calcium signaling, muscle contraction, cell adhesion, and lipid synthesis. Functional clustering of the downregulated genes revealed enrichment in cell cycle, microtubule cytoskeleton, growth factor binding, and ATP binding.
Publications
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
Honglin Jiang, Robert Settlage, Xinyan Leng1, and Yuguo Hou. Identification of Novel Genes and Mechanisms Involved in Bovine Myogenic Differentiation. 2016 JAM.
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
Honglin Jiang, Robert Settlage, Xinyan Leng1, and Yuguo Hou. Identification of Novel Genes and Mechanisms Involved in Bovine Myogenic Differentiation. 2016 JAM.
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