Principles for the regulation of multiple developmental pathways by a versatile transcriptional factor, BLIMP1.
ABSTRACT: Single transcription factors (TFs) regulate multiple developmental pathways, but the underlying mechanisms remain unclear. Here, we quantitatively characterized the genome-wide occupancy profiles of BLIMP1, a key transcriptional regulator for diverse developmental processes, during the development of three germ-layer derivatives (photoreceptor precursors, embryonic intestinal epithelium and plasmablasts) and the germ cell lineage (primordial germ cells). We identified BLIMP1-binding sites shared among multiple developmental processes, and such sites were highly occupied by BLIMP1 with a stringent recognition motif and were located predominantly in promoter proximities. A subset of bindings common to all the lineages exhibited a new, strong recognition sequence, a GGGAAA repeat. Paradoxically, however, the shared/common bindings had only a slight impact on the associated gene expression. In contrast, BLIMP1 occupied more distal sites in a cell type-specific manner; despite lower occupancy and flexible sequence recognitions, such bindings contributed effectively to the repression of the associated genes. Recognition motifs of other key TFs in BLIMP1-binding sites had little impact on the expression-level changes. These findings suggest that the shared/common sites might serve as potential reservoirs of BLIMP1 that functions at the specific sites, providing the foundation for a unified understanding of the genome regulation by BLIMP1, and, possibly, TFs in general.
Project description:Transcription factors (TFs) regulate biological events depending on cellular contexts, precise mechanisms for which are elusive. BLIMP1 has been shown to play key roles in many developmental processes, canonically as a transcriptional repressor that targets to proximities of promoters. Here, we systematically and quantitatively characterized genomic binding patterns of BLIMP1 across four distinct, developing cell types; photoreceptor precursors, embryonic intestinal epithelium, plasmablasts, and primordial germ cells (PGCs). BLIMP1-binding sites are highly enriched in genomic regions proximal to transcription start sites (TSSs), majority of which are shared among cell types and are highly occupied by BLIMP1, whereas only a small number of associated genes are regulated consistently among cell types. In contrast, BLIMP1 weakly binds to more distal, cell type-specific sites with divergent recognition sequences, which account for gene regulations much more efficiently in proportion to the magnitude of expression level changes, with notably similar impacts per site among cell types. Various TF motifs contained in the cell type-specific binding sites exhibit only moderate impacts on transcription dynamics and BLIMP1-occupancy levels. On the other hand, germ cells uniquely involve the shared binding sites in the specification, and grossly maintain the binding pattern in late PGCs, accounting for vast majority of the repressive targets. Furthermore, we identified new sequence motifs strongly bound to BLIMP1 especially in the late PGCs, GGGAAA repeats, a few of which are located around key regulators of gametogenesis. These findings provide a foundation for understanding the genomic regulation of BLIMP1 across developmental processes. Overall design: 13 EGFP-BLIMP1 ChIP-seq samples in 7 cell types: d2PGCLC (2 replicates), d6PGCLC (2 replicates), E12.5 male and female PGC (E12.5_PGC_M and E12.5_PGC_F, respectively, 1 replicate each), intestinal epithelium (emIE, 2 replicates), Photoreceptor precursor (PRP, 2 replicates), Plasmablast (PB, 2 replicates)., E12.5 male PGC input (E12.5_PGCM, input, 1 replicate)
Project description:Transcription factors (TFs) regulate biological events depending on cellular contexts, precise mechanisms for which are elusive. BLIMP1 has been shown to play key roles in many developmental processes, canonically as a transcriptional repressor that targets to proximities of promoters. Here, we systematically and quantitatively characterized genomic binding patterns of BLIMP1 across four distinct, developing cell types; photoreceptor precursors, embryonic intestinal epithelium, plasmablasts, and primordial germ cells (PGCs). BLIMP1-binding sites are highly enriched in genomic regions proximal to transcription start sites (TSSs), majority of which are shared among cell types and are highly occupied by BLIMP1, whereas only a small number of associated genes are regulated consistently among cell types. In contrast, BLIMP1 weakly binds to more distal, cell type-specific sites with divergent recognition sequences, which account for gene regulations much more efficiently in proportion to the magnitude of expression level changes, with notably similar impacts per site among cell types. Various TF motifs contained in the cell type-specific binding sites exhibit only moderate impacts on transcription dynamics and BLIMP1-occupancy levels. On the other hand, germ cells uniquely involve the shared binding sites in the specification, and grossly maintain the binding pattern in late PGCs, accounting for vast majority of the repressive targets. Furthermore, we identified new sequence motifs strongly bound to BLIMP1 especially in the late PGCs, GGGAAA repeats, a few of which are located around key regulators of gametogenesis. These findings provide a foundation for understanding the genomic regulation of BLIMP1 across developmental processes. Overall design: 26 3' RNAseq samples in 13 cell types: d2PGCLC (2 replicates), E16.5_Embryonic intestinal epithelium (E16.5_emIE, 2 replicates), P3_Embryonic intestinal epithelium (P3_emIE, 2 replicates), P14_Embryonic intestinal epithelium (P14_emIE, 2 replicates), P0_Photoreceptor precursor_GFP+ (P0_PRP_GFP+, 2 replicates), P4_Photoreceptor precursor_GFP+ (P4_PRP_GFP+CD73-, 2 replicates), P4_Photoreceptor precursor_CD73+ (P4_PRP_CD73+, 2 replicates), P6_Photoreceptor precursor_CD73+ (P6_PRP_CD73+, 2 replicates), P10_Photoreceptor precursor_CD73+ (P10_PRP_CD73+, 2 replicates), B cell (B cell, 2 replicates), Blast (Blast, 2 replicates), Pre-plasmablast (prePB, 2 replicates), Plamsablast (PB, 2 replicates)
Project description:The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP1) regulates gene expression and cell fate. The DNA motif bound by BLIMP1 in vitro overlaps with that of interferon regulatory factors (IRFs), which respond to inflammatory/immune signals. At such sites, BLIMP1 and IRFs can antagonistically regulate promoter activity. In vitro motif selection predicts that only a subset of BLIMP1 or IRF sites is subject to antagonistic regulation, but the extent to which antagonism occurs is unknown, since an unbiased assessment of BLIMP1 occupancy in vivo is lacking. To address this, we identified an extended set of promoters occupied by BLIMP1. Motif discovery and enrichment analysis demonstrate that multiple motif variants are required to capture BLIMP1 binding specificity. These are differentially associated with CpG content, leading to the observation that BLIMP1 DNA-binding is methylation sensitive. In occupied promoters, only a subset of BLIMP1 motifs overlap with IRF motifs. Conversely, a distinct subset of IRF motifs is not enriched amongst occupied promoters. Genes linked to occupied promoters containing overlapping BLIMP1/IRF motifs (e.g. AIM2, SP110, BTN3A3) are shown to constitute a dynamic target set which is preferentially activated by BLIMP1 knock-down. These data confirm and extend the competitive model of BLIMP1 and IRF interaction.
Project description:Despite the role of the estrogen receptor alpha (ERalpha) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERalpha into ERalpha-negative cells paradoxically has been growth inhibition. We map the binding profiles of ERalpha and its interacting transcription factors (TFs), FOXA1 and GATA3, in MCF-7 breast carcinoma cells. We observe that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERalpha alone. We demonstrate that these enhanceosome-occupied sites are associated with optimal enhancer characteristics with highest p300 coactivator recruitment, RNA Pol II occupancy, and chromatin opening. The enhancesome binding sites appear to regulate the genes driving core ERalpha function. Most importantly, we show that transfection of all three TFs was necessary to reprogram the ERalpha-negative MDA-MB-231 and BT-459 cells to restore the estrogen responsive growth and to transcriptionally resemble the estrogen-treated ERalpha-positive MCF-7 cells. Cumulatively, these results suggest that all of the enhanceosome components comprising ERalpha, FOXA1 and GATA3 are necessary for the full repertoire of the cancer-associated effects of the ERalpha. The analysis of ERalpha, FOXA1, and GATA3 in MCF-7 cancer cells was done by ChIP-seq data obtained either with estradiol (E2) stimulation or without stimulation using vehicle as a control. Using the ERalpha bindings defined by ChIP-seq (GSE23893), FOXA1 bindings (GSE26831), and GATA3 bindings (this Series), we analyzed the enhanceosome effect of the overlapped binding sites from ERalpha, FOXA1 and GATA3.
Project description:To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.
Project description:Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.
Project description:Transcription factors (TFs) bind to DNA and regulate the transcription of nearby genes. However, only a small fraction of TF binding sites have such regulatory effects. Here we search for the predictors of functional binding sites by carrying out a systematic computational screening of a variety of contextual factors (histone modifications, nuclear lamin-bindings, and cofactor bindings). We used regression analysis to test if contextual factors are associated with upregulation or downregulation of neighboring genes following the induction or knockdown of the 9 TFs in mouse embryonic stem (ES) cells. Functional TF binding sites appeared to be either active (i.e., bound by P300, CHD7, mediator, cohesin, and SWI/SNF) or repressed (i.e., with H3K27me3 histone marks and bound by Polycomb factors). Active binding sites mediated the downregulation of nearby genes upon knocking down the activating TFs or inducing repressors. Repressed TF binding sites mediated the upregulation of nearby genes (e.g., poised developmental regulators) upon inducing TFs. In addition, repressed binding sites mediated repressive effects of TFs, identified by the downregulation of target genes after the induction of TFs or by the upregulation of target genes after the knockdown of TFs. The contextual factors associated with functions of DNA-bound TFs were used to improve the identification of candidate target genes regulated by TFs.
Project description:Genomic imprinting is an epigenetic mechanism that affects a subset of mammalian genes, resulting in monoallelic expression depending on the parental origin of the alleles. Imprinted regions contain regulatory elements that are methylated in the gametes in a sex-specific manner (differentially methylated regions; DMRs). DMRs are present at nonimprinted loci as well, but whereas most regions are equalized after fertilization, methylation at imprinted regions maintains asymmetry. We tested the hypothesis that paternally unmethylated DMRs are occupied by transcription factors (TFs) present during male gametogenesis. Meta-analysis of mouse RNA data to identify DNA-binding proteins expressed in male gametes and motif enrichment analysis of active promoters yielded a list of candidate TFs. We then asked whether imprinted or nonimprinted paternally unmethylated DMRs harbored motifs for these TFs, and found many shared motifs between the two groups. However, DMRs that are methylated in the male germ cells also share motifs with DMRs that remain unmethylated. There are recognition sequences exclusive to the unmethylated DMRs, whether imprinted or not, that correspond with cell-cycle regulators, such as p53. Thus, at least with the current available data, our results indicate a complex scenario in which TF occupancy alone is not likely to play a role in protecting unmethylated DMRs, at least during male gametogenesis. Rather, the epigenetic features of DMRs, regulatory sequences other than DMRs, and the role of DNA-binding proteins capable of endowing sequence specificity to DNA-methylating enzymes are feasible mechanisms and further investigation is needed to answer this question.
Project description:The zinc-finger transcriptional repressor Blimp1 (Prdm1) controls gene expression patterns during differentiation of B lymphocytes and regulates epigenetic changes required for specification of primordial germ cells. Blimp1 is dynamically expressed at diverse tissue sites in the developing mouse embryo, but its functional role remains unknown because Blimp1 mutant embryos arrest at E10.5 due to placental insufficiency. To explore Blimp1 activities at later stages in the embryo proper, here we used a conditional inactivation strategy. A Blimp1-Cre transgenic strain was also exploited to generate a fate map of Blimp1-expressing cells. Blimp1 plays essential roles in multipotent progenitor cell populations in the posterior forelimb, caudal pharyngeal arches, secondary heart field and sensory vibrissae and maintains key signalling centres at these diverse tissues sites. Interestingly, embryos carrying a hypomorphic Blimp1gfp reporter allele survive to late gestation and exhibit similar, but less severe developmental abnormalities, whereas transheterozygous Blimp1(gfp/-) embryos with further reduced expression levels, display exacerbated defects. Collectively, the present experiments demonstrate that Blimp1 requirements in diverse cell types are exquisitely dose dependent.
Project description:Identifying transcription factors (TFs) whose DNA bindings are altered by genetic variants that regulate susceptibility genes is imperative to understand transcriptional dysregulation in disease etiology. Here, we develop a statistical framework to analyze extensive ChIP-seq and GWAS data and identify 22 breast cancer risk-associated TFs. We find that, by analyzing genetic variations of TF-DNA bindings, the interaction of FOXA1 with co-factors such as ESR1 and E2F1, and the interaction of TFs with chromatin features (i.e., enhancers) play a key role in breast cancer susceptibility. Using genetic variants occupied by the 22 TFs, transcriptome-wide association analyses identify 52 previously unreported breast cancer susceptibility genes, including seven with evidence of essentiality from functional screens in breast relevant cell lines. We show that FOXA1 and co-factors form a core TF-transcriptional network regulating the susceptibility genes. Our findings provide additional insights into genetic variations of TF-DNA bindings (particularly for FOXA1) underlying breast cancer susceptibility.