ABSTRACT
Macro long non-coding RNAs (lncRNAs) play major roles in gene silencing in inprinted gene clusters. Within the imprinted Gnas cluster, the paternally expressed Nespas lncRNA downregulates its sense counterpart Nesp. To explore the mechanism of action of Nespas, we generated two new knock-in alleles to truncate Nespas upstream and downstream of the Nesp promoter. We show that Nespas is essential for methylation of the Nesp differentially methylated region (DMR), but higher levels of Nespas are required for methylation than are needed for downregulation of Nesp. Although Nespas is transcribed for over 27 kb, only Nespas transcript/transcription across a 2.6 kb region that includes the Nesp promoter is necessary for methylation of the Nesp DMR. In both mutants, the levels of Nespas were extraordinarily high, due at least in part to increased stability, an effect not seen with other imprinted lncRNAs. However, even when levels were greatly raised, Nespas remained exclusively cis-acting. We propose Nespas regulates Nesp methylation and expression to ensure appropriate levels of expression of the protein coding transcripts Gnasxl and Gnas on the paternal chromosome. Thus, Nespas mediates paternal gene expression over the entire Gnas cluster via a single gene, Nesp.
ABSTRACT
Transcription factors such as Scl/Tal1, Lmo2, and Runx1 are essential for the development of hematopoietic stem cells (HSCs). However, the precise mechanisms by which these factors interact to form transcriptional networks, as well as the identity of the genes downstream of these regulatory cascades, remain largely unknown. To this end, we generated an Scl(-/-) yolk sac cell line to identify candidate Scl target genes by global expression profiling after reintroduction of a TAT-Scl fusion protein. Bioinformatics analysis resulted in the identification of 9 candidate Scl target transcription factor genes, including Runx1 and Runx3. Chromatin immunoprecipitation confirmed that both Runx genes are direct targets of Scl in the fetal liver and that Runx1 is also occupied by Scl in the yolk sac. Furthermore, binding of an Scl-Lmo2-Gata2 complex was demonstrated to occur on the regions flanking the conserved E-boxes of the Runx1 loci and was shown to transactivate the Runx1 element. Together, our data provide a key component of the transcriptional network of early hematopoiesis by identifying downstream targets of Scl that can explain key aspects of the early Scl(-/-) phenotype.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Core Binding Factor Alpha 3 Subunit/metabolism , Liver/embryology , Liver/metabolism , Proto-Oncogene Proteins/metabolism , Yolk Sac/embryology , Yolk Sac/metabolism , Adaptor Proteins, Signal Transducing , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line , Cell Separation , Conserved Sequence , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 3 Subunit/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Humans , LIM Domain Proteins , Metalloproteins/genetics , Metalloproteins/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Protein Binding , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Sequence Alignment , T-Cell Acute Lymphocytic Leukemia Protein 1ABSTRACT
The transcription factor Runx1/AML1 is an important regulator of hematopoiesis and is critically required for the generation of the first definitive hematopoietic stem cells (HSCs) in the major vasculature of the mouse embryo. As a pivotal factor in HSC ontogeny, its transcriptional regulation is of high interest but is largely undefined. In this study, we used a combination of comparative genomics and chromatin analysis to identify a highly conserved 531-bp enhancer located at position + 23.5 in the first intron of the 224-kb mouse Runx1 gene. We show that this enhancer contributes to the early hematopoietic expression of Runx1. Transcription factor binding in vivo and analysis of the mutated enhancer in transient transgenic mouse embryos implicate Gata2 and Ets proteins as critical factors for its function. We also show that the SCL/Lmo2/Ldb-1 complex is recruited to the enhancer in vivo. Importantly, transplantation experiments demonstrate that the intronic Runx1 enhancer targets all definitive HSCs in the mouse embryo, suggesting that it functions as a crucial cis-regulatory element that integrates the Gata, Ets, and SCL transcriptional networks to initiate HSC generation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/physiology , GATA2 Transcription Factor/physiology , Hematopoietic Stem Cells/cytology , Proto-Oncogene Protein c-ets-1/physiology , Proto-Oncogene Proteins/physiology , Transcription, Genetic , Adaptor Proteins, Signal Transducing , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA-Binding Proteins/metabolism , Embryo, Mammalian , Enhancer Elements, Genetic/physiology , GATA2 Transcription Factor/metabolism , LIM Domain Proteins , Metalloproteins/metabolism , Mice , Multiprotein Complexes/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Proto-Oncogene Proteins/metabolism , T-Cell Acute Lymphocytic Leukemia Protein 1ABSTRACT
Genomic imprinting results in allele-specific silencing according to parental origin. Silencing is brought about by imprinting control regions (ICRs) that are differentially marked in gametogenesis. The group of imprinted transcripts in the mouse Gnas cluster (Nesp, Nespas, Gnasxl, Exon 1A and Gnas) provides a model for analyzing the mechanisms of imprint regulation. We previously identified an ICR that specifically regulates the tissue-specific imprinted expression of the Gnas gene. Here we identify a second ICR at the Gnas cluster. We show that a paternally derived targeted deletion of the germline differentially methylated region (DMR) associated with the antisense Nespas transcript unexpectedly affects both the expression of all transcripts in the cluster and methylation of two DMRs. Our results establish that the Nespas DMR is the principal ICR at the Gnas cluster and functions bidirectionally as a switch for modulating expression of the antagonistically acting genes Gnasxl and Gnas. Uniquely, the Nespas DMR acts on the downstream ICR at exon 1A to regulate tissue-specific imprinting of the Gnas gene.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs/genetics , Genomic Imprinting , RNA, Antisense/genetics , RNA, Untranslated/genetics , Transcription, Genetic , Animals , Chromogranins , DNA Methylation , Exons , Female , Male , Mice , Molecular Sequence Data , Multigene Family , Sequence DeletionABSTRACT
Genomic imprinting brings about allele-specific silencing according to parental origin. Silencing is controlled by cis-acting regulatory regions that are differentially marked during gametogenesis and can act over hundreds of kilobases to silence many genes. Two candidate imprinting control regions (ICRs) have been identified at the compact imprinted Gnas cluster on distal mouse chromosome 2, one at exon 1A upstream of Gnas itself and one covering the promoters for Gnasxl and the antisense Nespas (ref. 8). This imprinted cluster is complex, containing biallelic, maternally and paternally expressed transcripts that share exons. Gnas itself is mainly biallelically expressed but is weakly paternally repressed in specific tissues. Here we show that a paternally derived targeted deletion of the germline differentially methylated region at exon 1A abolishes tissue-specific imprinting of Gnas. This rescues the abnormal phenotype of mice with a maternally derived Gnas mutation. Imprinting of alternative transcripts, Nesp, Gnasxl and Nespas (ref. 13), in the cluster is unaffected. The results establish that the differentially methylated region at exon 1A contains an imprinting control element that specifically regulates Gnas and comprises a characterized ICR for a gene that is only weakly imprinted in a minority of tissues. There must be a second ICR regulating the alternative transcripts.
