Identification of pituitary thyrotrope signature genes and regulatory elements

2020 ◽  
Author(s):  
Alexandre Z. Daly ◽  
Lindsey A. Dudley ◽  
Michael T. Peel ◽  
Stephen A. Liebhaber ◽  
Stephen C. J. Parker ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Lines ◽  
Binding Sites ◽  
Critical Factor ◽  
Regulatory Elements ◽  
Thyroid Stimulating Hormone ◽  
Bzip Transcription Factor ◽  
Enhancer Element ◽  
Transcriptional Regulatory Elements

AbstractPituitary thyrotropes are specialized cells that produce thyroid stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate and transcriptional regulation of the beta subunit of TSH, Tshb, but no transcriptomic or epigenomic analyses of these cells has been undertaken. The goal of this work was to discover key transcriptional regulatory elements that drive thyrotrope fate. We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes, a process modeled by two cell lines: one that represents an early, undifferentiated Pou1f1 lineage progenitor (GHF-T1) and one that is a committed thyrotrope that produces TSH (TαT1). We generated and compared RNA-seq, ATAC-seq, histone modification (including H3K27Ac, H3K4Me1, and H3K27Me3), and transcription factor (POU1F1) binding in these two cell lines to identify regulatory elements and candidate transcriptional regulators. We identified POU1F1 binding sites that were unique to each cell line. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding to unique sites. We validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element. These results extend the ENCODE multi-omic profiling approach to an organ that is critical for growth and metabolism, which should be valuable for understanding pituitary development and disease pathogenesis.

scholarly journals Multi-omic profiling of pituitary thyrotropic cells and progenitors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Alexandre Z. Daly ◽  
Lindsey A. Dudley ◽  
Michael T. Peel ◽  
Stephen A. Liebhaber ◽  
Stephen C. J. Parker ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Pituitary Gland ◽  
Cell Lines ◽  
Binding Sites ◽  
Critical Factor ◽  
Regulatory Elements ◽  
Thyroid Stimulating Hormone ◽  
Neuroendocrine Cells ◽  
Bzip Transcription Factor ◽  
Enhancer Element

Abstract Background The pituitary gland is a neuroendocrine organ containing diverse cell types specialized in secreting hormones that regulate physiology. Pituitary thyrotropes produce thyroid-stimulating hormone (TSH), a critical factor for growth and maintenance of metabolism. The transcription factors POU1F1 and GATA2 have been implicated in thyrotrope fate, but the transcriptomic and epigenomic landscapes of these neuroendocrine cells have not been characterized. The goal of this work was to discover transcriptional regulatory elements that drive thyrotrope fate. Results We identified the transcription factors and epigenomic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an undifferentiated Pou1f1 lineage progenitor (GHF-T1) and a committed thyrotrope line that produces TSH (TαT1). We compared RNA-seq, ATAC-seq, histone modification (H3K27Ac, H3K4Me1, and H3K27Me3), and POU1F1 binding in these cell lines. POU1F1 binding sites are commonly associated with bZIP transcription factor consensus binding sites in GHF-T1 cells and Helix-Turn-Helix (HTH) or basic Helix-Loop-Helix (bHLH) factors in TαT1 cells, suggesting that these classes of transcription factors may recruit or cooperate with POU1F1 binding at unique sites. We validated enhancer function of novel elements we mapped near Cga, Pitx1, Gata2, and Tshb by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice, and we demonstrate that GATA2 enhances Tshb expression through this element. Conclusion These results extend the ENCODE multi-omic profiling approach to the pituitary gland, which should be valuable for understanding pituitary development and disease pathogenesis. Graphical abstract

scholarly journals MON-723 Identification of Thyrotrope Signature Genes and Regulatory Elements

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Alexandre Daly ◽  
Leonard Cheung ◽  
Michelle Brinkmeier ◽  
Sally Ann Camper
Keyword(s):  
Gene Expression ◽  
Risk Factors ◽  
Transcription Factors ◽  
Cell Line ◽  
Cell Lines ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Genome Wide Association Studies ◽  
Expression Data ◽  
Enhancer Element

Abstract Recent genome wide association studies have begun to identify loci that are risk factors for sporadic pituitary adenomas, but the genes associated with these loci are unknown. In general, ~90% of GWAS hits are in noncoding regions, making it difficult to transition from genetic mapping to a biological understanding of risk factors. Recent studies that identify enhancer regions by undertaking large scale functional genomic annotation of non-coding elements like Encyclopedia of DNA Elements (ENCODE) have begun to yield a better understanding of some complex diseases. Dense molecular profiling maps of the transcriptome and epigenome have been generated for more than 250 cell lines and 150 tissues, but pituitary cell lines or tissues were not included. Epigenetic and gene expression data are emerging for somatotropes, gonadotropes and corticotropes, but there is very little available data on thyrotropes. We identified the transcription factors and epigenetic changes in chromatin that are associated with differentiation of POU1F1-expressing progenitors into thyrotropes using cell lines that represent an early, undifferentiated Pou1f1 lineage progenitor (GHF-T1) and a committed thyrotrope (TαT1). TαT1 is an excellent cell line for this purpose because it responds to TRH, retinoids, and secretes TSH in response to diurnal cues. We have also used genetic labeling and fluorescence activated cell sorting to purify thyrotropes from pituitaries of young mice and analyzed gene expression using single cell transcriptomics. We used the Assay for TransposaseAccessible Chromatin with sequencing (ATACseq) and Cleavage Under Target and Release Using Nuclease (CUT&RUN) to identify POU1F1 binding sites and histone marks associated with active enhancers, H3K27Ac and H3K4Me1, or inactive regions, H3K27Me3, in GHF-T1 and TαT1 cells. We integrated DNA accessibility, histone modification patterns, transcription factor binding and RNA expression data to identify regulatory elements and candidate transcriptional regulators. We identified POU1F1 binding sites that were unique to each cell line. For example, POU1F1 binds sites in and around Cga and Tshb only in TαT1 cells and Twist1 and Gli3 only in GHFT1 cells. POU1F1 binding sites are commonly associated with bZIP factor consensus binding sites in GHFT1 cells and Helix-Turn-Helix or basic Helix-Loop-Helix in TαT1 cells, suggesting classes of transcription factors that may recruit POU1F1 to unique sites. We validated enhancer function of novel elements we mapped near Tshb, Gata2, and Pitx1 by transfection in TαT1 cells. Finally, we confirmed that an enhancer element near Tshb can drive expression in thyrotropes of transgenic mice. These data extend the ENCODE analysis to an organ that is critical for growth and metabolism. This information could be valuable for understanding pituitary development and disease pathogenesis.

scholarly journals Evolutionarily conserved transcription factors drive the oxidative stress response in Drosophila

2020 ◽  
Vol 223 (14) ◽  
pp. jeb221622
Author(s):  
Sarah M. Ryan ◽  
Kaitie Wildman ◽  
Briseida Oceguera-Perez ◽  
Scott Barbee ◽  
Nathan T. Mortimer ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Stress Responses ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Genomic Locus ◽  
Biologically Relevant ◽  
Protein Levels ◽  
Putative Transcription Factor

ABSTRACTAs organisms are constantly exposed to the damaging effects of oxidative stress through both environmental exposure and internal metabolic processes, they have evolved a variety of mechanisms to cope with this stress. One such mechanism is the highly conserved p38 MAPK (p38K) pathway, which is known to be post-translationally activated in response to oxidative stress, resulting in the activation of downstream antioxidant targets. However, little is known about the role of p38K transcriptional regulation in response to oxidative stress. Therefore, we analyzed the p38K gene family across the genus Drosophila to identify conserved regulatory elements. We found that oxidative stress exposure results in increased p38K protein levels in multiple Drosophila species and is associated with increased oxidative stress resistance. We also found that the p38Kb genomic locus includes conserved AP-1 and lola-PT transcription factor consensus binding sites. Accordingly, over-expression of these transcription factors in D. melanogaster is sufficient to induce transcription of p38Kb and enhances resistance to oxidative stress. We further found that the presence of a putative lola-PT binding site in the p38Kb locus of a given species is predictive of the species' survival in response to oxidative stress. Through our comparative genomics approach, we have identified biologically relevant putative transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38K genes and suggest that transcription may play as important a role in p38K-mediated stress responses as post-translational modifications.

Can Fibroblasts Be Reprogrammed into Macrophages?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 443-443
Author(s):  
Ru Feng ◽  
Thomas Graf
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Bzip Transcription Factor ◽  
Nih3t3 Cells ◽  
Activation Domains ◽  
Gm Csf ◽  
Infected Cells ◽  
Factor C

Abstract Previous work showed that B cell precursors can be reprogrammed into functional macrophages by the enforced expression of the bZip transcription factor C/EBPalpha. The efficient activation of myelomonocytic genes, such as Mac-1, required the co-operation with endogenous PU.1 (Xie et al. 2004), reflecting the fact that many myelomonocytic genes are regulated by a combination of the two transcription factors. We therefore asked: Is C/EBPa and PU.1 sufficient to convert non-hematopoietic cells into macrophages? To test this, NIH-3T3 cells were co-infected with PU.1-GFP and C/EBPa-hCD4 retrovirusesor control vectors encoding the indicators GFP and hCD4 only. Uninfected cells in the retrovirus treated cultures served as additional controls. Our results showed that ~25% of the PU.1 only infected cells express Mac-1 and that this percentage could be increased ~3 fold by co-expression with C/EBPa. In addition, most cells also expressed CD45 and some expressed F4/80 antigen. The PU.1 infected and the double infected cells, but not the C/EBPa only infected cells, also expressed a number of other myelomonocytic genes as detected by RT-PCR. These included CSF-1R (M-CSFR), GM-CSF Ralpha, Lysozyme, CD32, PYK2 as well as endogenous PU.1. The PU.1 induced reprogramming of fibroblasts required the DNA binding and transcription activation domains, but not the PEST domain of the transcription factor. To test whether the reprogrammed cells have functional macrophage properties, we generated two stable cell lines co-expressing C/EBPa and PU.1 delta PEST (wild type PU.1 is toxic in long-term cultures). These cells were morphologically altered, ingested carboxylated particles, and expressed functional Fc-gamma receptors but were unable to phagocytize antibody coated red blood cells. Remarkably, the two cells lines acquired CSF-1 dependence for growth. In accordance with this finding they exhibited a 10–15 fold reduction of CSF-1 production compared to NIH3T3 cells. The response observed was not restricted to fibroblast cell lines since both embryonic and adult fibroblasts could also be partially reprogrammed by co-infection with PU.1 and C/EBPa in that they expressed Mac-1, CD45, F4/80 and IA MHC antigens. In conclusion, enforced expression of PU.1 and C/EBPa converts fibroblasts into macrophage like cells, indicating that the combination of these two transcription factors is sufficient to regulate the majority of genes that define the myelomonocytic phenotype.

scholarly journals Identifying Transcription Regulatory Elements in the Human and Mouse Genomes Using Tissue-specific Gene Expression Profiles

2007 ◽  
Vol 4 (2) ◽  
pp. 1-23
Author(s):  
Amitava Karmaker ◽  
Kihoon Yoon ◽  
Mark Doderer ◽  
Russell Kruzelock ◽  
Stephen Kwek
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Tissue Specific Gene ◽  
Human And Mouse

Summary Revealing the complex interaction between trans- and cis-regulatory elements and identifying these potential binding sites are fundamental problems in understanding gene expression. The progresses in ChIP-chip technology facilitate identifying DNA sequences that are recognized by a specific transcription factor. However, protein-DNA binding is a necessary, but not sufficient, condition for transcription regulation. We need to demonstrate that their gene expression levels are correlated to further confirm regulatory relationship. Here, instead of using a linear correlation coefficient, we used a non-linear function that seems to better capture possible regulatory relationships. By analyzing tissue-specific gene expression profiles of human and mouse, we delineate a list of pairs of transcription factor and gene with highly correlated expression levels, which may have regulatory relationships. Using two closely-related species (human and mouse), we perform comparative genome analysis to cross-validate the quality of our prediction. Our findings are confirmed by matching publicly available TFBS databases (like TRANFAC and ConSite) and by reviewing biological literature. For example, according to our analysis, 80% and 85.71% of the targets genes associated with E2F5 and RELB transcription factors have the corresponding known binding sites. We also substantiated our results on some oncogenes with the biomedical literature. Moreover, we performed further analysis on them and found that BCR and DEK may be regulated by some common transcription factors. Similar results for BTG1, FCGR2B and LCK genes were also reported.

Deep Mining of Natural Genetic Variation in Erythroid Cells Reveals New Insights about In Vivo Transcription Factor Binding and Chromatin Accessibility

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3879-3879
Author(s):  
Vivek Behera ◽  
Perry Evans ◽  
Carolyne J Face ◽  
Laavanya Sankaranarayanan ◽  
Gerd A. Blobel
Keyword(s):  
Transcription Factor ◽  
Genetic Variation ◽  
Transcription Factors ◽  
Cell Line ◽  
Cell Lines ◽  
Genetic Variants ◽  
Binding Sites ◽  
Chromatin Accessibility ◽  
Erythroid Cell

Abstract Erythroid transcription factors (TFs) control gene expression programs, lineage decisions, and disease outcomes. How transcription factors contact DNA has been studied extensively in vitro, but in vivo binding characteristics are less well understood as they are influenced in a reciprocal manner by chromatin accessibility and neighboring transcription factors. Here, we present a comparative analysis approach that takes advantage of non-coding sequence variation between functionally equivalent erythroid cell lines to conduct an in-depth analysis of erythroid TF binding profiles and chromatin features. Specifically, we analyzed ChIP-seq datasets to identify millions of genetic non-coding variants between the mouse erythroleukemia cell line (MEL), a GATA1-inducible erythroid progenitor cell line (G1E-ER4), and primary murine erythroblast cells. We found that while these cell lines are highly positively correlated in chromatin features, larger differences in TF binding intensity are correlated with higher degrees of genetic variation between cell lines. We next examined discriminatory genetic variants between the cell lines that are located in ChIP-seq peaks of the erythroid transcription factor GATA1. Hundreds of such variants fall within GATA1 motifs. Differential GATA1 binding intensities associated with the variants revealed nucleotide positions that contribute most to in vivo GATA1 chromatin occupancy and identified which alternative nucleotides are most likely to disrupt binding. Notably, this additional information about GATA1's in vivo nucleotide binding preferences improved prediction of GATA1 binding sites genome-wide. We applied similar approaches to determine the bp-resolution in vivo binding preferences of TAL1/SCL and CTCF. We additionally identified thousands of discriminatory genetic variants within GATA1 sites that fall outside canonical GATA elements but within binding sites of other known TFs. Association of these variants with differential GATA1 binding intensities revealed that the hematopoietic transcription factors TAL1/SCL and KLF1 positively regulate GATA1 chromatin occupancy. Strikingly, we identified a number of motifs not previously implicated in cooperating with GATA1 that positively impact GATA1 chromatin binding. Notably, we also defined motifs associated with negative regulation of GATA1 chromatin occupancy. Applying a similar analysis to TAL1/SCL and CTCF revealed additional motifs involved in regulating the chromatin occupancy of these TFs. Finally, we associated discriminatory genetic variation between erythroid cell lines with large changes in sub-kb-scale DNase hypersensitivity. We found that single base pair substitutions within or near a number of erythroid TF motifs, including that for the RUNX family of nuclear factors, are strongly associated with changes in chromatin accessibility. Our findings use novel methods in comparative ChIP-seq and DNase-seq analysis to reveal new insights about the genetic basis for erythroid TF chromatin occupancy and chromatin accessibility. Disclosures No relevant conflicts of interest to declare.

Role of SCL/Tal-1, GATA, and Ets transcription factor binding sites for the regulation of Flk-1 expression during murine vascular development

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3078-3085 ◽  
Author(s):  
Andreas Kappel ◽  
Thorsten M. Schlaeger ◽  
Ingo Flamme ◽  
Stuart H. Orkin ◽  
Werner Risau ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Blood Vessel ◽  
Tumor Angiogenesis ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Vascular Development ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Ets Transcription Factors

The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in theFlk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed withFlk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream ofFlk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.

Role of SCL/Tal-1, GATA, and Ets transcription factor binding sites for the regulation of Flk-1 expression during murine vascular development

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3078-3085 ◽  
Author(s):  
Andreas Kappel ◽  
Thorsten M. Schlaeger ◽  
Ingo Flamme ◽  
Stuart H. Orkin ◽  
Werner Risau ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Blood Vessel ◽  
Tumor Angiogenesis ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Vascular Development ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Ets Transcription Factors

Abstract The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in theFlk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed withFlk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream ofFlk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.

scholarly journals Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

1998 ◽  
Vol 18 (11) ◽  
pp. 6293-6304 ◽  
Author(s):  
Vesco Mutskov ◽  
Delphine Gerber ◽  
Dimitri Angelov ◽  
Juan Ausio ◽  
Jerry Workman ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Molecular Biology ◽  
Binding Sites ◽  
Cross Linking ◽  
Uv Laser ◽  
Histone Tail ◽  
Histone Tails ◽  
Nucleosomal Dna ◽  
Core Histones

ABSTRACT In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

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