Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. The use of reporter constructs, cell culture and computational modeling has made enormous contributions to understanding this fundamental question, but analysis of regulatory elements in their natural developmental context is an essential but rarely used complement. Here, we edited Notch-dependent cis-regulatory elements in the endogenous C. elegans sygl-1 gene, which encodes a key stem cell regulator. We then analyzed the in vivo consequences of those mutations – on both gene expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of a single element in a three-element homotypic cluster reduced expression as well as stem cell pool size by about half, while mutation of two elements essentially abolished them. We find that LBS number and LBS neighborhood are both important to activity: elements on separate chromosomes function additively, while elements in the same cluster act synergistically. Our approach of precise CRISPR/Cas9 gene editing coupled with quantitation of both molecular and biological readouts establishes a powerful model for in vivo functional analyses of DNA cis-regulatory elements.

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Dose-Dependent AGO1-Mediated Inhibition of the miRNA165/166 Pathway Modulates Stem Cell Maintenance in Arabidopsis Shoot Apical Meristem

Plant Communications ◽

10.1016/j.xplc.2019.100002 ◽

2020 ◽

Vol 1 (1) ◽

pp. 100002

Author(s):

Fei Du ◽

Wen Gong ◽

Sonia Boscá ◽

Matthew Tucker ◽

Hervé Vaucheret ◽

...

Keyword(s):

Stem Cell ◽

Shoot Apical Meristem ◽

Apical Meristem ◽

Stem Cell Maintenance ◽

Dose Dependent ◽

Cell Maintenance

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Progression from a stem cell–like state to early differentiation in the C. elegans germ line

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0912704107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 2048-2053 ◽

Cited By ~ 67

Author(s):

Olivier Cinquin ◽

Sarah L. Crittenden ◽

Dyan E. Morgan ◽

Judith Kimble

Keyword(s):

Stem Cell ◽

Germ Cells ◽

Cell Biology ◽

Germ Line ◽

Stem Cell Biology ◽

Self Renewal ◽

Early Differentiation ◽

C Elegans ◽

Stem Cell Maintenance ◽

System A

Controls of stem cell maintenance and early differentiation are known in several systems. However, the progression from stem cell self-renewal to overt signs of early differentiation is a poorly understood but important problem in stem cell biology. The Caenorhabditis elegans germ line provides a genetically defined model for studying that progression. In this system, a single-celled mesenchymal niche, the distal tip cell (DTC), employs GLP-1/Notch signaling and an RNA regulatory network to balance self-renewal and early differentiation within the “mitotic region,” which continuously self-renews while generating new gametes. Here, we investigate germ cells in the mitotic region for their capacity to differentiate and their state of maturation. Two distinct pools emerge. The “distal pool” is maintained by the DTC in an essentially uniform and immature or “stem cell–like” state; the “proximal pool,” by contrast, contains cells that are maturing toward early differentiation and are likely transit-amplifying cells. A rough estimate of pool sizes is 30–70 germ cells in the distal immature pool and ≈150 in the proximal transit-amplifying pool. We present a simple model for how the network underlying the switch between self-renewal and early differentiation may be acting in these two pools. According to our model, the self-renewal mode of the network maintains the distal pool in an immature state, whereas the transition between self-renewal and early differentiation modes of the network underlies the graded maturation of germ cells in the proximal pool. We discuss implications of this model for controls of stem cells more broadly.

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Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

Development ◽

10.1242/dev.199629 ◽

2021 ◽

Author(s):

Sophie H. L. Austin ◽

Rut Gabarró-Solanas ◽

Piero Rigo ◽

Oana Paun ◽

Lachlan Harris ◽

...

Keyword(s):

Stem Cell ◽

In Vitro Models ◽

Beta Catenin ◽

Dependent Manner ◽

Direct Role ◽

Adult Nscs ◽

Hippocampal Networks ◽

Dose Dependent

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of NSCs in an active state and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that deletion of β-catenin in NSCs did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

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The C. elegans GSCs: A Powerful Model for In Vivo Study of Adult Stem Cell Regulation

International Journal of Stem cell Research & Therapy ◽

10.23937/2469-570x/1410044 ◽

2016 ◽

Vol 3 (2) ◽

Cited By ~ 2

Author(s):

Patrick Narbonne

Keyword(s):

Stem Cell ◽

Adult Stem Cell ◽

In Vivo Study ◽

Cell Regulation ◽

C Elegans ◽

Stem Cell Regulation

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Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

10.1101/2020.12.29.424694 ◽

2020 ◽

Author(s):

Sophie H. L. Austin ◽

Lachlan Harris ◽

Oana Paun ◽

Piero Rigo ◽

François Guillemot ◽

...

Keyword(s):

Stem Cell ◽

Beta Catenin ◽

Dependent Manner ◽

Direct Role ◽

Adult Nscs ◽

Hippocampal Networks ◽

Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

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The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development

Blood ◽

10.1182/blood-2009-01-200048 ◽

2009 ◽

Vol 113 (22) ◽

pp. 5456-5465 ◽

Cited By ~ 81

Author(s):

Nicola K. Wilson ◽

Diego Miranda-Saavedra ◽

Sarah Kinston ◽

Nicolas Bonadies ◽

Samuel D. Foster ◽

...

Keyword(s):

Stem Cell ◽

Regulatory Networks ◽

Transcriptional Control ◽

Target Genes ◽

Fetal Liver ◽

Bioinformatic Analysis ◽

Regulatory Elements ◽

Hematopoietic Stem ◽

A Genome

The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.

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The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Blood ◽

10.1182/blood.v122.21.1175.1175 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1175-1175

Author(s):

Stefanie Kreutmair ◽

Anna Lena Illert ◽

Rouzanna Istvanffy ◽

Christina Eckl ◽

Christian Peschel ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Spleen Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mitotic Entry ◽

Stem Cell Maintenance ◽

F Box Protein

Abstract Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2+ cells, the balance in NIPA-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells. Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

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CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

eLife ◽

10.7554/elife.32341 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 29

Author(s):

Gabriel N Aughey ◽

Alicia Estacio Gomez ◽

Jamie Thomson ◽

Hang Yin ◽

Tony D Southall

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Ectopic Expression ◽

Stem Cell Differentiation ◽

Cell Types ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Global Changes

During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. Here we show that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). We have profiled chromatin accessibility in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type-specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, we show global changes in the accessibility of chromatin between stem-cells and their differentiated progeny. Our results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development.

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Interaction of the CCAAT displacement protein with shared regulatory elements required for transcription of paired histone genes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3587 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3587-3596 ◽

Cited By ~ 40

Author(s):

H M el-Hodiri ◽

M Perry

Keyword(s):

Bidirectional Promoter ◽

Regulatory Elements ◽

Histone Gene ◽

Single Element ◽

Gene Promoters ◽

Histone Genes ◽

Ccaat Displacement Protein ◽

Intergenic Dna ◽

Specific Complex

The H2A and H2B genes of the Xenopus xlh3 histone gene cluster are transcribed in opposite directions from initiation points located approximately 235 bp apart. The close proximity of these genes to one another suggests that their expression may be controlled by either a single bidirectional promoter or by separate promoters. Our analysis of the transcription of histone gene pairs containing deletions and site-specific mutations of intergenic DNA revealed that both promoters are distinct but that they overlap physically and share multiple regulatory elements, providing a possible basis for the coordinate regulation of their in vivo activities. Using the intergenic DNA fragment as a probe and extracts from mammalian and amphibian cells, we observed the formation of a specific complex containing the CCAAT displacement protein (CDP). The formation of the CDP-containing complex was not strictly dependent on any single element in the intergenic region but instead required the presence of at least two of the three CCAAT motifs. Interestingly, similar CDP-containing complexes were formed on the promoters from the three other histone genes. The binding of CDP to histone gene promoters may contribute to the coordination of their activities during the cell cycle and early development.

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