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An extended regulatory landscape drives Tbx18 activity in a variety of prostate-associated cell lineages

ABSTRACT: The evolutionarily conserved transcription factor, Tbx18, is expressed in a dynamic pattern throughout embryonic and early postnatal life and plays crucial roles in the development of multiple organ systems. Previous studies have indicated that this dynamic function is controlled by an expansive regulatory architecture, extending far upstream and downstream of the gene. With the goal of identifying elements that interact with the Tbx18 promoter in developing prostate, we coupled chromatin conformation capture (4C) and ATAC-seq from embryonic day 18.5 (E18.5) mouse urogenital sinus (UGS), where Tbx18 is highly expressed. The data revealed dozens of active chromatin elements distributed throughout a 1.5 million base pair topologically associating domain (TAD). To identify cell types contributing to this chromatin signal, we used lineage tracing methods with a Tbx18 Cre “knock-in” allele; these data show clearly that Tbx18-expressing precursors differentiate into wide array of cell types in multiple tissue compartments, most of which have not been previously reported. We also used a 209 kb Cre-expressing Tbx18 transgene, to partition enhancers for specific precursor types into two rough spatial domains. Within this central 209 kb compartment, we identified ECR1, previously described to regulate Tbx18 expression in ureter, as an active regulator of UGS expression. Together these data define the diverse fates of Tbx18+ precursors in prostate-associated tissues for the first time, and identify a highly active TAD controlling the gene’s essential function in this tissue.

ORGANISM(S): Mus musculus

PROVIDER: GSE123102 | GEO | 2018/12/29

REPOSITORIES: GEO

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Project description:Studies centered at the intersection of embryogenesis and carcinogenesis have identified striking parallels involving signaling pathways that modulate both developmental and neoplastic processes. In the prostate, reciprocal interactions between epithelium and stroma are known to influence neoplasia and also exert morphogenic effects via the urogenital sinus mesenchyme. In this study, we sought to determine molecular relationships between aspects of normal prostate development and prostate carcinogenesis. We first characterized the gene expression program associated with key points of murine prostate organogenesis spanning the initial in utero induction of prostate budding through maturity. We identified a highly reproducible temporal program of gene expression that partitioned according to the broad developmental stages of prostate induction, branching morphogenesis, and secretory differentiation. Comparisons of gene expression profiles of murine prostate cancers arising in the context of genetically engineered alterations in the Pten tumor suppressor and Myc oncogene identified significant associations between the profile of branching morphogenesis and both cancer models. Further, the expression of genes comprising the branching morphogenesis program, such as PRDX4, SLC43A1, and DNMT3A, was significantly altered in human neoplastic prostate epithelium. These results indicate that components of normal developmental processes are active in prostate neoplasia and provide further rationale for exploiting molecular features of organogenesis to understand cancer phenotypes. Whole male UGS (E14.5, E15.5, E16.5, and E17.5) or separated prostate lobes (P7, P30, and DP90) were dissected from C57BL6/J mice and snap frozen in liquid nitrogen. For each biological replication, we pooled 3 to 10 mice representing one or two litters. RNA from pools of UGS or specific prostate lobes (vp, ap, and dlp) was prepared using the Qiagen RNeasy Mini kit. We included an on-column DNaseI treatment to remove contaminating DNA. Before RNA amplification, we combined equal quantities of RNA from vp, ap, and dlp for the postnatal prostate samples. We amplified 1 ug of total RNA from each sample through one round using the Arcturus RiboAmp kit. For the E14.5 UGS reference sample, a second round of amplification was done to provide enough RNA for all microarrays. Each developmental sample was hybridized against the E14.5 UGS reference sample with a dye swap, for a total of 42 arrays. Warning: the normalized data for this study which was made public on December 1, 2009, was swapped for some samples. All normalized data has now been corrected as of February 25, 2010. Raw data was not affected.

Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation. We investigated a 2.8 Mb region on Chromosome 7 (hg18 chr7: 115797757-118405450) containing the ENCODE region ENm001 42. The 5C experiment was designed to interrogate looping interactions between HindIII fragments containing transcription start sites (TSSs) and any other HindIII restriction fragment (distal fragments) in the target region. Libraries were generated for five cell lines: Caco2, Calu3, Capan1, GM12878 and HepG2, with two biological replicates for each line. 5C probes were designed at HindIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made publicly available online at our My5C website (http://my5C.umassmed.edu). Probes were designed based on the ENCODE manual region 1 (ENM001) design 25 with additional probes placed throughout the region when appropriate. We also added probes to extend the analysis to include a 700 Kb gene desert region located directly adjacent to ENM001. Probe settings were: U-BLAST, 3; S-BLAST, 100; 15-MER, 3,000; MIN_FSIZE, 250; MAX_FSIZE, 20,000; OPT_TM, 65; OPT_PSIZE, 40. We designed 74 reverse 5C probes, and 605 forward 5C probes.

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An extended regulatory landscape drives Tbx18 activity in a variety of prostate-associated cell lineages

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