DNase I behaves as a transcription factor which modulates Fas expression in human cells

Abstract Normal differentiation of red cells, platelets and eosinophils from a myeloid progenitor requires expression of the transcription factor GATA1. Moreover, GATA1 expression level influences lineage output; higher levels promote erythromegakaryocytic differentiation and lower levels eosinophil maturation. Conversely, repression of GATA1 expression is required for monocyte/neutrophil development. GATA1 expression is principally controlled transcriptionally. Thus, dissecting the molecular basis of transcriptional control of GATA1 expression will be one important facet in understanding how myeloid lineages are specified. To address this question we sought to identify all DNA sequences important for GATA1 expression. Previous analysis identified 3 murine (m)Gata1 cis-elements (an upstream enhancer, mHS-3.5, a haematopoietic IE promoter and elements in a GATA1 intron, mHS+3.5) conserved in sequence between human(h) and mouse. These studies also suggested additional unidentified elements were required for erythroid and eosinophil GATA1 expression. We compared sequence, mapped DNase I hypersensitive sites (HS) and determined histone H3/H4 acetylation over ~120 kb flanking the hGATA1 locus and corresponding region in mouse to pinpoint cis-elements. Remarkably, despite lying in a ~10 MB conserved syntenic segment, the chromatin structures of both GATA1 loci are strikingly different. Two previously unidentified haematopoietic cis-elements, one in each species (mHS-25 and hHS+14), are not conserved in position and sequence and have enhancer activity in erythroid cells. Chromatin immunoprecipitation studies show both mHS-25 and hHS+14 are bound in vivo in red cells by the transcription factors GATA1, SCL, LMO2, Ldb1. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. Further analysis of in vivo transcription factor occupancy at GATA1 cis-elements in primary mouse eosinophils and red cells, megakaryocytic cells (L8057) and control fibroblasts show lineage- and cis-element-specific patterns of regulator binding (see table below). In red cells and megakaryocytes, GATA1, SCL, LMO2 and Ldb1 bind at two regulatory elements (mhHS-25 and mHS-3.5). Interestingly, the megakaryocyte transcriptional regulator Fli1 factor binds to mHS+3.5 specifically in megakaryocytes. In eosinophils, a different pattern of DNase I HS and transcription factor binding is seen. GATA1, PU.1 and C/EBPe (all regulate eosinophil gene expression) bind IE promoter and/or mHS+3.5. Collectively, these results suggest lineage-specific GATA1 expession is dependent on combinations of cis-elements and haematopoietic trans-acting factors that are unique for each lineage. DNase I Hypersensitive sites and transcription factor occupancy at mGATA1 cis-elements. mHS-26/-25* mHS-3.5 mIE mHS+3.5 m: mouse, h: human, *: HS identified in this study, TF: transcription factor Primary erythroid cells HS present, GATA1, SCL, LMO2, Ldb1 HS present, GATA1, SCL, LMO2, Ldb1 HS present, GATA1 HS present, GATA1 Megakaryocytic cells HS present, GATA1, SCL, LMO2, Ldb1 HS present, GATA1, SCL, LMO2, Ldb1 HS present, GATA1 HS present, GATA1 and Fli1 Primary eosinophils HS absent HS present, No TF detected HS present, GATA1 and C/EBPε HS present, GATA1, C/EBP ε and PU.1 Fibroblasts HS absent HS absent HS absent HS absent

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Fine-Mapping and Genome Editing Reveal An Essential Erythroid Enhancer At The HbF-Associated BCL11A Locus

Blood ◽

10.1182/blood.v122.21.437.437 ◽

2013 ◽

Vol 122 (21) ◽

pp. 437-437 ◽

Cited By ~ 1

Author(s):

Daniel E. Bauer ◽

Sophia C. Kamran ◽

Samuel Lessard ◽

Jian Xu ◽

Yuko Fujiwara ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Genome Editing ◽

Fine Mapping ◽

Dnase I ◽

Reporter Activity ◽

Functional Variants ◽

Allele Specific ◽

Hbf Level ◽

Regulatory Dna

Abstract Introduction Genome-wide association studies (GWAS) have ascertained numerous trait-associated common genetic variants localized to regulatory DNA. The hypothesis that regulatory variation accounts for substantial heritability has undergone scarce experimental evaluation. Common variation at BCL11A is estimated to explain ∼15% of the trait variance in fetal hemoglobin (HbF) level but the functional variants remain unknown. Materials and Methods We use chromatin immunoprecipitation (ChIP), DNase I sensitivity and chromosome conformation capture to evaluate the BCL11A locus in mouse and human primary erythroblasts. We extensively genotype 1,263 samples from the Collaborative Study of Sickle Cell Disease within three HbF-associated erythroid DNase I hypersensitive sites (DHSs) at BCL11A. We pyrosequence heterozygous erythroblasts to assess allele-specific transcription factor binding and gene expression. We conduct transgenic analysis by mouse zygotic microinjection and genome editing with transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) RNA-guided nucleases. Results Common genetic variation at BCL11A associated with HbF level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping this putative regulatory DNA uncovers a motif-disrupting common variant associated with reduced GATA1 and TAL1 transcription factor binding, modestly diminished BCL11A expression and elevated HbF. This variant, rs1427407, accounts for the HbF association of the previously reported sentinel SNPs. The composite element functions in vivo as a developmental stage-specific lineage-restricted enhancer. Genome editing reveals that the enhancer is required in erythroid but dispensable in B-lymphoid cells for expression of BCL11A. We demonstrate species-specific functional components of the composite enhancer in mouse as compared to human erythroid precursor cells. The mouse sequences homologous to the human DHS sufficient to drive reporter activity are dispensable from the mouse composite element, whereas the adjacent DHS, whose human homolog does not direct reporter activity, is absolutely required for BCL11A expression. Conclusions We describe a comprehensive and widely applicable approach, including chromatin mapping followed by fine-mapping, allele-specific ChIP and gene expression studies, and functional analyses, to reveal causal variants and critical elements. We assert that functional validation of regulatory DNA ought to include perturbation of the endogenous genomic context by genome editing and not solely rely on in vitro or ectopic surrogate assays. These results validate the hypothesis that common variation modulates cell type-specific regulatory elements, and reveal that although functional variants themselves may be of modest impact, their harboring elements may be critical for appropriate gene expression. We speculate that species-level functional differences in components of the composite enhancer might partially account for differences in timing of globin gene expression among animals. We suggest that the GWAS-marked BCL11A enhancer represents a highly attractive target for therapeutic genome editing for the major b-hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

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Comparative Transcriptomics Highlights the Role of the Activator Protein 1 Transcription Factor in the Host Response to Ebolavirus

Journal of Virology ◽

10.1128/jvi.01174-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 7

Author(s):

James W. Wynne ◽

Shawn Todd ◽

Victoria Boyd ◽

Mary Tachedjian ◽

Reuben Klein ◽

...

Keyword(s):

Transcription Factor ◽

Cell Lines ◽

Human Cells ◽

Activator Protein 1 ◽

Host Gene Expression ◽

Rna Seq ◽

Content Type ◽

Molecular Events ◽

Ebov Infection

ABSTRACT Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors. Furthermore, a significant upregulation of activator protein 1 (AP1) transcription factor complex members FOS and JUN was observed in permissive cell lines. Functional studies focusing on human cells showed that EBOV infection induces protein expression, phosphorylation, and nuclear accumulation of JUN and, to a lesser degree, FOS. Using a luciferase-based reporter, we show that EBOV infection induces AP1 transactivation activity within human cells at 48 and 72 h postinfection. Finally, we show that JUN knockdown decreases the expression of EBOV-induced host gene expression. Taken together, our study highlights the role of AP1 in promoting the host gene expression profile that defines EBOV pathogenesis. IMPORTANCE Many questions remain about the molecular events that underpin filovirus pathophysiology. The rational design of new intervention strategies, such as postexposure therapeutics, will be significantly enhanced through an in-depth understanding of these molecular events. We believe that new insights into the molecular pathogenesis of EBOV may be possible by examining the transcriptomic response of taxonomically diverse cell lines (derived from human, pig, and bat). We first identified the responsive pathways using an RNA-seq-based transcriptomics approach. Further functional and computational analysis focusing on human cells highlighted an important role for the AP1 transcription factor in mediating the transcriptional response to EBOV infection. Our study sheds new light on how host transcription factors respond to and promote the transcriptional landscape that follows viral infection.

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