Discrimination of Dormant and Active Hematopoietic Stem Cells by G₀ Marker Reveals Dormancy Regulation by Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generate a G0 marker (G0M) mouse line that visualizes quiescent cells and identify a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations

Discrimination of Dormant and Active Hematopoietic Stem Cells by G₀ Marker Reveals Dormancy Regulation by Cytoplasmic Calcium

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generate a G0 marker (G0M) mouse line that visualizes quiescent cells and identify a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations

T-Cells Null for the MED23 Subunit of Mediator Express Decreased Levels of KLF2 and Inefficiently Populate the Peripheral Lymphoid Organs

<div><p>MED23, a subunit of the Mediator coactivator complex, is important for the expression of a subset of MAPK/ERK pathway-responsive genes, the constituents of which vary between cell types for reasons that are not completely clear. MAPK/ERK pathway-dependent processes are essential for T-cell development and function, but whether MED23 has a role in this context is unknown. We generated <i>Med23</i> conditional knockout mice and induced <i>Med23</i> deletion in early T-cell development using the lineage specific <i>Lck-Cre</i> transgene. While the total cell number and distribution of cell populations in the thymuses of <i>Med23^flox/flox;Lck-Cre</i> mice were essentially normal, MED23 null T-cells failed to efficiently populate the peripheral lymphoid organs. MED23 null thymocytes displayed decreased expression of the MAPK/ERK-responsive genes <i>Egr1</i>, <i>Egr2</i>, as well as of the membrane glycoprotein <i>Cd52</i> (CAMPATH-1). MED23 null CD4 single-positive thymocytes also showed decreased expression of KLF2 (LKLF), a T-cell master regulatory transcription factor. Indeed, similarities between the phenotypes of mice lacking MED23 or KLF2 in T-cells suggest that KLF2 deficiency in MED23 null T-cells is one of their key defects. Mechanistic experiments using MED23 null MEFs further suggest that MED23 is required for full activity of the MAPK-responsive transcription factor MEF2, which has previously been shown to mediate <i>Klf2</i> expression. In summary, our data indicate that MED23 has critical roles in enabling T-cells to populate the peripheral lymphoid organs, possibly by potentiating MEF2-dependent expression of the T-cell transcription factor KLF2.</p></div

<i>Med23^flox/flox</i>;<i>Lck-Cre</i> mice have normal thymic populations, but MED23 null T-cells poorly populate the peripheral lymphoid tissues.

<p><b>A</b>, FACS analysis of thymus from <i>Med23^flox/flox</i> (control) and <i>Med23^flox/flox;Lck-Cre</i> mice showing CD4 and CD8 subpopulations. <b>B</b>, Total thymocyte counts from 5–8 week old mice. Note that the <i>Lck-Cre</i> transgene alone decreases the total number of thymocytes. <b>C</b>, Flow cytometry for deleted (YFP⁺) T-cells. Since mice have differing overall deletion efficiencies, deletion percentage for each T-cell type and compartment was normalized to the deletion in double positive (DP) thymocytes for each animal. <i>Med23^+/+;Lck-Cre;YFP</i>, N = 2. <i>Med23^flox/flox;Lck-Cre;YFP</i>, N = 7. Mean +/−SEM. Asterisks indicate significance of two-tailed unpaired t test. * P<0.05, ** P<0.01, *** P<0.001. n.s. not statistically significant.</p

Mice with efficient deletion of <i>Med23</i> exhibit decreased T-cell numbers in the peripheral lymphoid organs.

<p>Flow cytometric analysis of spleen, lymph node and blood from <i>Med23^flox/flox</i> and <i>Med23^flox/flox;Lck-Cre</i> mice showing proportions of CD3⁺ T-cells and B220⁺ B-cells. Flow cytometry from mouse shown is representative of phenotype seen in mice with greater than 95% deletion of <i>Med23</i> in the thymus.</p

MEFs lacking MED23 show altered <i>Klf2</i> expression.

<p><b>A</b>, <i>Klf2</i> mRNA expression by qRT-PCR in <i>Med23^+/+</i> (WT) and <i>Med23^{Δflox/Δflox}</i> (MED23 null) MEFs treated with Cre adenovirus, then three days later starved overnight and treated with serum for 1 hour. <i>N</i> = 4, mean +/− SEM, two-tailed t test, <i>P</i> = 0.02 for comparison of either mock or serum-treated samples. <b>B</b>, Activity of a luciferase reporter construct driven by the <i>Klf2</i> promoter (pGL3-<i>Klf2</i>-pro) in WT and MED23 null MEFs (<i>N</i> = 6, mean +/− SEM, two-tailed t test, <i>P</i> = 0.0003). <b>C</b>, Activity of a GAL4-KLF2 fusion construct containing aa 1–88 of KLF2 in WT and MED23 null MEFs (<i>N</i> = 2, triplicates, mean +/− SEM). <b>D</b>, Mutation of a MEF2 site in the <i>Klf2</i> promoter construct (pGL3-<i>Klf2</i>-pro-mut) dramatically decreases activity of the construct (<i>N</i> = 2, duplicates, mean +/− SEM). <b>E</b>, A GAL4-MEF2C fusion construct is dependent upon MED23 for full activity (<i>N</i> = 4–8, mean +/−SEM, two-tailed t test <i>P</i> = 0.0002). pGL3 Basic and pM1 are empty luciferase and GAL4 DNA binding domain (DBD) vectors respectively. n.s., not statistically significant.</p

<i>Med23^flox/flox;Lck-Cre</i> T-cells do not display increased apoptosis.

<p>TUNEL assay was performed on cryosections from thymus and spleen of <i>Med23^flox/flox; Lck-Cre</i> and <i>Lck-Cre</i> control mice. A control section was treated with DNaseI prior to TUNEL staining (+TdT) as a positive control, and a cryosection that was not stained (−TdT) serves as a negative control.</p

MED23 null thymocytes proliferate normally in response to stimulation, but have abnormal gene expression.

<p><b>A</b> Total thymocytes were cultured in the presence of plate bound αCD3 and αCD28 antibodies (as indicated) for 44 hrs, then allowed to incorporate ³H thymidine for 18 hrs. Counts per minute (CPM) were normalized to the <i>Lck-Cre</i> Mock counts for each experiment. N = 2–3, Mean +/− SEM. Statistical analysis shown is Tukey post-test following one way ANOVA, n.s. not statistically significant. <b>B–E</b>, <i>Il2</i> (<b>B</b>), <i>Egr1</i> (<b>C</b>), <i>Egr2</i> (<b>D</b>), and <i>Cd52</i> (<b>E</b>) mRNA expression analyzed by qRT-PCR in <i>Med23^+/+;Lck-Cre</i> (control) and <i>Med23^flox/flox;Lck-Cre</i> (Med23 null) total thymocytes rested 4 hours after harvest, then mock treated or treated 3 hours <i>ex vivo</i> with immobilized αCD3. <i>Med23^flox/flox;Lck-Cre</i> thymocytes showed greater than 90% deletion. N = 2, Mean +/− SEM. <i>P</i> values from two-tailed t-tests between αCD3 (B–D) or Mock (E) treated samples.</p

MED23 null single positive thymocytes express decreased KLF2.

<p><b>A–C</b>, <i>Klf2</i> (<b>A</b>), <i>Cd52</i> (<b>B</b>) and <i>S1pr1</i> (<b>C</b>) mRNA expression in CD4⁺ single positive thymocytes; YFP⁺<i>Med23^+/+;Lck-Cre;YFP</i> or <i>Med23^+/+;Lck-Cre</i> (control) and YFP⁺<i>Med23^flox/flox;Lck-Cre;YFP</i> or <i>Med23^flox/flox;Lck-Cre</i> where deletion was greater than 90% (MED23 null) thymocytes. Two-tailed t test; N = 4–8; Mean +/− SEM. <b>D</b>, Western blot of MED23, KLF2 and ACTB (loading control) in whole cell extracts from YFP⁺ CD4 SP SP thymocytes from <i>Med23^+/+;Lck-Cre;YFP</i> (control) and <i>Med23^flox/flox;Lck-Cre;YFP</i> (mutant) mice.</p

Histone Acetyltransferase CBP Is Vital To Demarcate Conventional and Innate CD8+ T-Cell Development▿ †

Defining the chromatin modifications and transcriptional mechanisms that direct the development of different T-cell lineages is a major challenge in immunology. The transcriptional coactivators CREB binding protein (CBP) and the closely related p300, which comprise the KAT3 family of histone/protein lysine acetyltransferases, interact with over 50 T-lymphocyte-essential transcriptional regulators. We show here that CBP, but not p300, modulates the thymic development of conventional adaptive T cells versus those having unconventional innate functions. Conditional inactivation of CBP in the thymus yielded CD8 single-positive (SP) thymocytes with an effector-, memory-, or innate-like T-cell phenotype. In this regard, CD8 SP thymocytes in CBP mutant mice were phenotypically similar to those reported for Itk and Rlk protein tyrosine kinase mutants, including the increased expression of the T-cell master regulatory transcription factor eomesodermin (Eomes) and the interleukin-2 and -15 receptor beta chain (CD122) and an enhanced ability to rapidly produce gamma interferon. CBP was required for the expression of the Itk-dependent genes Egr2, Egr3, and Il2, suggesting that CBP helps mediate Itk-responsive transcription. CBP therefore defines a nuclear component of the signaling pathways that demarcate the development of innate and adaptive naïve CD8+ T cells in the thymus