Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of mouse hematopoietic cells

SummaryMembers of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for twenty cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions - along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

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Kit regulatory elements required for expression in developing hematopoietic and germ cell lineages

Blood ◽

10.1182/blood-2003-04-1296 ◽

2003 ◽

Vol 102 (12) ◽

pp. 3954-3962 ◽

Cited By ~ 57

Author(s):

Linda A. Cairns ◽

Emanuela Moroni ◽

Elena Levantini ◽

Alessandra Giorgetti ◽

Francesca G. Klinger ◽

...

Keyword(s):

Stem Cell ◽

Fluorescent Protein ◽

Primordial Germ Cells ◽

Hematopoietic Cells ◽

Cell Types ◽

Regulatory Elements ◽

Mouse Development ◽

Hematopoietic Stem ◽

Hypersensitive Sites ◽

Green Fluorescent

Abstract The Kit (White) gene encodes the transmembrane receptor of stem cell factor/Kit ligand (KL) and is essential for the normal development/maintenance of pluripotent primordial germ cells (PGCs), hematopoietic stem cells (HSCs), melanoblasts, and some of their descendants. The molecular basis for the transcriptional regulation of Kit during development of these important cell types is unknown. We investigated Kit regulation in hematopoietic cells and PGCs. We identified 6 DNase I hypersensitive sites (HS1-HS6) within the promoter and first intron of the mouse Kit gene and developed mouse lines expressing transgenic green fluorescent protein (GFP) under the control of these regulatory elements. A construct driven by the Kit promoter and including all 6 HS sites is highly expressed during mouse development in Kit+ cells including PGCs and hematopoietic progenitors (erythroid blast-forming units and mixed colony-forming units). In contrast, the Kit promoter alone (comprising HS1) is sufficient to drive low-level GFP expression in PGCs, but unable to function in hematopoietic cells. Hematopoietic expression further requires the addition of the intronproximal HS2 fragment; HS2 also greatly potentiates the activity in PGCs. Thus, HS2 acts as an enhancer integrating transcriptional signals common to 2 developmentally unrelated stem cell/progenitor lineages. Optimal hematopoietic expression further requires HS3-HS6.

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Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors

Blood ◽

10.1182/blood-2008-11-187757 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5783-5792 ◽

Cited By ~ 57

Author(s):

Josette-Renée Landry ◽

Nicolas Bonadies ◽

Sarah Kinston ◽

Kathy Knezevic ◽

Nicola K. Wilson ◽

...

Keyword(s):

Regulatory Networks ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Cells ◽

Cell Types ◽

Regulatory Elements ◽

Proximal Promoter ◽

Hematopoietic Stem ◽

Conserved Noncoding Elements

Abstract The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

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Decoding gene regulation in the fly brain

10.1101/2021.08.11.454937 ◽

2021 ◽

Author(s):

Jasper Janssens ◽

Sara Aibar ◽

Ibrahim Ihsan Taskiran ◽

Joy N. Ismail ◽

Katina I. Spanier ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Motif Discovery ◽

Target Genes ◽

Single Cells ◽

Neuronal Cell ◽

Cell Types ◽

Regulatory Elements ◽

Specific Cell ◽

Drosophila Brain

The Drosophila brain is a work horse in neuroscience. Single-cell transcriptome analysis, 3D morphological classification, and detailed EM mapping of the connectome have revealed an immense diversity of neuronal and glial cell types that underlie the wide array of functional and behavioral traits in the fruit fly. The identities of these cell types are controlled by still unknown gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. To characterize the GRN for each cell type in the Drosophila brain, we profiled chromatin accessibility of 240,919 single cells spanning nine developmental timepoints, and integrated this data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which around 70,000 are linked to specific developmental trajectories, involving neurogenesis, reprogramming and maturation. For 40 cell types, their uniquely accessible regions could be associated with their expressed transcription factors and downstream target genes, through a combination of motif discovery, network inference techniques, and deep learning. We illustrate how these enhancer-GRNs can be used to reveal enhancer architectures leading to a better understanding of neuronal regulatory diversity. Finally, our atlas of regulatory elements can be used to design genetic driver lines for specific cell types at specific timepoints, facilitating the characterization of brain cell types and the manipulation of brain function.

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Histone Acetyltransferases and Stem Cell Identity

Cancers ◽

10.3390/cancers13102407 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2407

Author(s):

Ruicen He ◽

Arthur Dantas ◽

Karl Riabowol

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Epigenetic Modification ◽

Cell Types ◽

Histone Acetyltransferases ◽

Specific Cell ◽

Cell Fates ◽

Cell Identity ◽

Hematopoietic Stem

Acetylation of histones is a key epigenetic modification involved in transcriptional regulation. The addition of acetyl groups to histone tails generally reduces histone-DNA interactions in the nucleosome leading to increased accessibility for transcription factors and core transcriptional machinery to bind their target sequences. There are approximately 30 histone acetyltransferases and their corresponding complexes, each of which affect the expression of a subset of genes. Because cell identity is determined by gene expression profile, it is unsurprising that the HATs responsible for inducing expression of these genes play a crucial role in determining cell fate. Here, we explore the role of HATs in the maintenance and differentiation of various stem cell types. Several HAT complexes have been characterized to play an important role in activating genes that allow stem cells to self-renew. Knockdown or loss of their activity leads to reduced expression and or differentiation while particular HATs drive differentiation towards specific cell fates. In this study we review functions of the HAT complexes active in pluripotent stem cells, hematopoietic stem cells, muscle satellite cells, mesenchymal stem cells, neural stem cells, and cancer stem cells.

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Thrombopoietin Induces HOXA9 Nuclear Transport in Immature Hematopoietic Cells: Potential Mechanism by Which the Hormone Favorably Affects Hematopoietic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.24.15.6751-6762.2004 ◽

2004 ◽

Vol 24 (15) ◽

pp. 6751-6762 ◽

Cited By ~ 53

Author(s):

Keita Kirito ◽

Norma Fox ◽

Kenneth Kaushansky

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Hematopoietic Stem Cells ◽

Nuclear Translocation ◽

Hematopoietic Cells ◽

Mitogen Activated Protein Kinase ◽

Mrna Levels ◽

Hematopoietic Stem ◽

Nuclear Localization Signals ◽

Mitogen Activated Protein

ABSTRACT Members of the homeobox family of transcription factors are major regulators of hematopoiesis. Overexpression of either HOXB4 or HOXA9 in primitive marrow cells enhances the expansion of hematopoietic stem cells (HSCs). However, little is known of how expression or function of these proteins is regulated during hematopoiesis under physiological conditions. In our previous studies we demonstrated that thrombopoietin (TPO) enhances levels of HOXB4 mRNA in primitive hematopoietic cells (K. Kirito, N. Fox, and K. Kaushansky, Blood 102:3172-3178, 2003). To extend our studies, we investigated the effects of TPO on HOXA9 in this same cell population. Although overall levels of the transcription factor were not affected, we found that TPO induced the nuclear import of HOXA9 both in UT-7/TPO cells and in primitive Sca-1+/c-kit+/Gr-1− hematopoietic cells in a mitogen-activated protein kinase-dependent fashion. TPO also controlled MEIS1 expression at mRNA levels, at least in part due to phosphatidylinositol 3-kinase activation. Collectively, TPO modulates the function of HOXA9 by leading to its nuclear translocation, likely mediated by effects on its partner protein MEIS1, and potentially due to two newly identified nuclear localization signals. Our data suggest that TPO controls HSC development through the regulation of multiple members of the Hox family of transcription factors through multiple mechanisms.

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The underground life of homeodomain-leucine zipper transcription factors

Journal of Experimental Botany ◽

10.1093/jxb/erab112 ◽

2021 ◽

Author(s):

Perotti M F ◽

Arce A L ◽

R L Chan

Keyword(s):

Transcription Factors ◽

Root Development ◽

Leucine Zipper ◽

Current Knowledge ◽

Expression Patterns ◽

Large Family ◽

Cell Types ◽

Structural Features ◽

Specific Cell ◽

High Plasticity

Abstract Roots are the anchorage organs of plants, responsible for water and nutrient uptake, exhibiting high plasticity. Root architecture is driven by the interactions of biomolecules, including transcription factors (TFs) and hormones that are crucial players regulating root plasticity. Multiple TF families are involved in root development; some, such as ARFs and LBDs, have been well characterized, whereas others remain less investigated. In this review, we synthesize the current knowledge about the involvement of the large family of homeodomain-leucine zipper (HD-Zip) TFs in root development. This family is divided into four subfamilies (I to IV), mainly according to structural features, such as additional motifs aside from HD-Zip, as well as their size, gene structure, and expression patterns. We explored and analyzed public databases and the scientific literature regarding HD-Zip TFs in Arabidopsis and other species. Most members of the four HD-Zip subfamilies are expressed in specific cell types and several ones from each group have assigned functions in root development. Notably, a high proportion of the studied proteins are part of intricate regulation pathways involved in primary and lateral root growth and development.

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The Ly-6E.1 (Sca-1) gene requires a 3' chromatin-dependent region for high-level gamma-interferon-induced hematopoietic cell expression

Blood ◽

10.1182/blood.v87.7.2750.bloodjournal8772750 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2750-2761 ◽

Cited By ~ 17

Author(s):

A Sinclair ◽

B Daly ◽

E Dzierzak

Keyword(s):

Consensus Sequence ◽

Hematopoietic Cells ◽

Regulatory Elements ◽

Dnase I ◽

Gamma Interferon ◽

Hematopoietic Cell ◽

Hematopoietic Stem ◽

Dnase I Hypersensitive Sites ◽

Hypersensitive Sites ◽

High Level

The Ly-6E.1/A.2 gene product recognized by the Sca-1 antibody has been found on murine hematopoietic stem cells and some hematopoietic precursors, T lymphocytes, and nonhematopoietic cell lineages, suggesting a complex array of gene regulatory elements. The ability to use the Ly6E.1/A.2 transcriptional regulatory elements to direct expression of heterologous genes will allow for the manipulation of these cells during development and in hematopoietic cell transplantations. To identify the elements necessary for high-level expression, we have made deletion constructs of Ly-6E.1 gene flanking regions containing DNase I hypersensitive sites, tested them for expression in hematopoietic cells, and have performed kinetic analyses to correlate the appearance of hypersensitive sites with gene transcription and protein expression. We show that a 3′ region containing two DNase I hypersensitive sites at +8.7 and +8.9 kb is required for high-level, gamma-interferon (gamma-IFN)-induced expression of the Ly-6E.1 gene and that a consensus sequence for a gamma-IFN-responsive element localizes to the +8.7 site. We also provide a description of allele- and cell-specific DNase I hypersensitive site patterns of the Ly-6E.1 and Ly-6A.2 genes. Taken together, these data indicate that while both 5′ and 3′ hypersensitive sites are rapidly induced with gamma-IFN, the 3′ most distal hypersensitive sites are involved in directing high levels of expression of Sca-1 in hematopoietic cells.

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Transforming Activity of AML1-ETO Oncoprotein Is Independent of CBFβ and ETO but Requires Homo-Oligomeric Interaction.

Blood ◽

10.1182/blood.v112.11.3348.3348 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3348-3348

Author(s):

Bernd B Zeisig ◽

Colin Kwok ◽

Jihui Qiu ◽

Shuo Dong ◽

Chi Wai Eric So

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Structure Function ◽

Hematopoietic Cells ◽

Primary Cells ◽

Hematopoietic Stem ◽

Endogenous Expression ◽

Function Data ◽

Essential Components ◽

Transforming Activity

Abstract AML1/RUNX1 and CBFb encode two critical transcription factors essential for the generation of hematopoietic stem cells (HSC). In acute myeloid leukemia, where leukemic stem cells (LSCs) have been functionally identified, AML1 and CBFb also represent the most commonly mutated targets. While animal models indicate that AML1 fusions per se are not sufficient to induce full-blown leukemia, they enhance self-renewal and expand targeted HSC and early progenitors, a property also reported for other oncogenic transcription factors involved in acute leukemia. Although attempts had been made to identify the critical domains required for AML-ETO (AE) mediated transformation, conflicting results were presented from most of these studies using exploited wellestablished cell lines, which suffer from the pitfall of carrying irrelevant genetic aberrants that may not reflect the normal biology of the disease. The only available structure/ function data on primary cells was limited to NHR2 of the ETO portion of the fusion, but it does not distinguish the functional contribution between homo-oligomerization and hetero-oligomerization. The lack of comprehensive structure/function data has not only significantly impeded the progress of understanding the biology of the disease, but also hinders the development of specific therapeutics. To this end, we performed extensive functional analysis to identify the key components essential for AE-mediated transformation of primary hematopoietic cells. In spite of the critical role of CBFb for wild type AML1 functions and its direct involvement in chromosomal translocation, we demonstrate that multiple AE single point mutants defective in CBFb interaction were still capable of transforming primary hematopoietic cells. Consistently, shRNA mediated knockdown of the endogenous expression of CBFb in primary cells did not compromise the transforming activity of AE, strongly suggesting a dispensable function of CBFb in AE mediated transformation. On the other hand, we demonstrate that NHR2 as the only domain in the ETO portion of the fusion is essential for transformation, but its heterooligomeric function including interaction with transcriptional repressor ETO family proteins is dispensable for the transforming activity. In contrast, synthetic FKBP homooligomerization modules could functionally replace NHR2, indicating that AE mediated transformation is critically dependent on homo-oligomeric property of the resultant fusion. Moreover, the transformation can also be abolished by a small molecule inhibitor that specifically dissociates homo-oligomerization. Together, these results not only identify the essential components and refine potential avenues for therapeutic targeting of AE oncogenic complexes, but also strongly endorse a common homo-oligomerization dependent mechanism shared by the most prevalent leukemia associated transcription factors.

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Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

10.1101/759936 ◽

2019 ◽

Author(s):

Leila Haery ◽

Benjamin E. Deverman ◽

Katherine Matho ◽

Ali Cetin ◽

Kenton Woodard ◽

...

Keyword(s):

Viral Vectors ◽

Cell Types ◽

Regulatory Elements ◽

Specific Cell ◽

Specific Expression ◽

Adeno Associated Virus ◽

Cell Type Specific Expression ◽

Cell Type Specific ◽

And Function ◽

Novel Applications

AbstractCell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.

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CellWalker integrates single-cell and bulk data to resolve regulatory elements across cell types in complex tissues

10.1101/847657 ◽

2019 ◽

Cited By ~ 1

Author(s):

Pawel F. Przytycki ◽

Katherine S. Pollard

Keyword(s):

Single Cell ◽

Cell Types ◽

Regulatory Elements ◽

Cell Labeling ◽

Open Chromatin ◽

Specific Cell ◽

Rna Seq ◽

Data Types ◽

Bulk Data ◽

Cell Type Specific

Single-cell and bulk genomics assays have complementary strengths and weaknesses, and alone neither strategy can fully capture regulatory elements across the diversity of cells in complex tissues. We present CellWalker, a method that integrates single-cell open chromatin (scATAC-seq) data with gene expression (RNA-seq) and other data types using a network model that simultaneously improves cell labeling in noisy scATAC-seq and annotates cell-type specific regulatory elements in bulk data. We demonstrate CellWalker’s robustness to sparse annotations and noise using simulations and combined RNA-seq and ATAC-seq in individual cells. We then apply CellWalker to the developing brain. We identify cells transitioning between transcriptional states, resolve enhancers to specific cell types, and observe that autism and other neurological traits can be mapped to specific cell types through their enhancers.

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