Loss of the Hematopoietic Stem Cell Factor GATA2 in the Osteogenic Lineage Impairs Trabecularization and Mechanical Strength of Bone

The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.Peer reviewe

The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae

The acetylation state of histones plays a central role in determining gene expression in chromatin. The reestablishment of the acetylation state of nucleosomes after DNA replication and chromatin assembly requires both deacetylation and acetylation of specific lysine residues on newly incorporated histones. In this study, the MYST family acetyltransferase Sas2 was found to interact with Cac1, the largest subunit of Saccharomyces cerevisiae chromatin assembly factor-I (CAF-I), and with the nucleosome assembly factor Asf1. The deletions of CAC1 (cac1Δ), ASF1 (asf1Δ), and SAS2 (sas2Δ) had similar effects on gene silencing and were partially overlapping. Furthermore, Sas2 was found in a nuclear protein complex that included Sas4 and Sas5, a homolog of TAF(II)30. This complex, termed SAS-I, was also found to contribute to rDNA silencing. Furthermore, the observation that a mutation of H4 lysine 16 to arginine displayed the identical silencing phenotypes as sas2Δ suggested that it was the in vivo target of Sas2 acetylation. In summary, our data present a novel model for the reestablishment of acetylation patterns after DNA replication, by which SAS-I is recruited to freshly replicated DNA by its association with chromatin assembly complexes to acetylate lysine 16 of H4

Y2H screen for GR isoform-specific interactions.

<p>(a) Domain structure of GR highlighting the ligand binding domain (LBD), Activation Function 1 (AF1) and the DNA binding domain (DBD) which includes the lever arm that diverges between GRα and GRγ. (b) Selective Y2H plate containing 1 μM desoxycorticosterone. Colonies indicate that both GRα and GRγ interact with known GR interaction partners UBE2I and SMARCB1 whereas GRγ shows some autoactivation at this hormone concentration (prey -: empty prey plasmid) (c) Schematic representation of the screen. Isoform-specific interactions are identified by comparing pairwise interactions between each prey with either GRα, GRγ, or FEZ (control for specificity of the interaction) as bait. Red marked boxes represent interactions, white boxes a lack of interaction between bait and prey. (d) Selective Y2H plates either without (left) or with (right) hormone (300 nM desoxycorticosterone (cort)). Colonies indicate an interacting bait-prey pair between the bait as indicated and macroH2A2. (e) Same as for (d) except that prey is BATF3 (JDP1).</p

Y2H screen for GR isoform-specific interactions.

<p>(a) Domain structure of GR highlighting the ligand binding domain (LBD), Activation Function 1 (AF1) and the DNA binding domain (DBD) which includes the lever arm that diverges between GRα and GRγ. (b) Selective Y2H plate containing 1 μM desoxycorticosterone. Colonies indicate that both GRα and GRγ interact with known GR interaction partners UBE2I and SMARCB1 whereas GRγ shows some autoactivation at this hormone concentration (prey -: empty prey plasmid) (c) Schematic representation of the screen. Isoform-specific interactions are identified by comparing pairwise interactions between each prey with either GRα, GRγ, or FEZ (control for specificity of the interaction) as bait. Red marked boxes represent interactions, white boxes a lack of interaction between bait and prey. (d) Selective Y2H plates either without (left) or with (right) hormone (300 nM desoxycorticosterone (cort)). Colonies indicate an interacting bait-prey pair between the bait as indicated and macroH2A2. (e) Same as for (d) except that prey is BATF3 (JDP1).</p

The Ligand Binding Domain Controls Glucocorticoid Receptor Dynamics Independent of Ligand Release▿

Ligand binding to the glucocorticoid receptor (GR) results in receptor binding to glucocorticoid response elements (GREs) and the formation of transcriptional regulatory complexes. Equally important, these complexes are continuously disassembled, with active processes driving GR off GREs. We found that cochaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD). Next, we examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Overall, these studies showed that activities impinging on the LBD regulate GR exchange with GREs but that the dissociation of GR from GREs is independent from ligand dissociation

Co-IP assays comparing protein:protein interactions between GRα and GRγ.

<p>(a) Fold change over control co-IPs (protein A only) for the binding of the GR isoform as indicated with BATF3 (fused to firefly luciferase). Averages ± standard deviation from at least triplicate transfections are shown for experiments done in either the presence (1 μM dexamethasone (dex)) or absence (no hormone) of GR ligand. The p-values were calculated using a two-tailed Student’s t-test (n = 6). (b) Western blot analysis of the lysates used for co-IP experiments showing similar levels for GRα- and GRγ-protein A fusion proteins. Lysates were from cells transfected with the fusion protein as indicated; cells were either treated with dexamethasone (1 μM, “+”) or with ethanol vehicle (“-“). (c) Same as for (a) except that the interaction between JDP2 and GRα or GRγ was quantified. The p-values were calculated using a two-tailed Student’s t-test (n = 9).</p

BATF3 is a context-specific co-activator of GR.

<p>(a) U2OS cells were cotransfected with an expression construct for GRα, a luciferase reporter encompassing a minimal promoter and a ~1kb GR region derived from the GR-responsive <i>GILZ</i> gene and increasing amounts of an expression construct for FLAG-tagged BATF3. Average normalized luciferase activity ± S.E.M. (n = 3) is shown for cells treated overnight with either 1 μM dexamethasone (dex) or ethanol (-) as vehicle control. (b, top) Same as for (a) except that a different luciferase reporter containing a minimal promoter and the CGT GBS was used. (b, bottom) Western blot analysis of dexamethasone-treated cells transfected with increasing amounts of FLAG-tagged BATF3 expression construct. Expression of FLAG-tagged BATF3 and actin (loading control) are shown. (c) U2OS cells stably expressing comparable amounts of either GRα or GRγ were transfected with the CGT luciferase reporter and increasing amounts of FLAG-tagged BATF3 expression construct. Average normalized luciferase activity ± S.E.M. (n = 3) is shown for cells treated overnight with either 1 μM dexamethasone (dex) or ethanol (-) as vehicle control. The p-values were calculated using a two-tailed Student’s t-test. (d) Western blot analysis of cells U2OS cells stably expressing comparable amounts of either GRα or GRγ that were transfected as described for with 15ng FLAG-tagged BATF3 expression construct and treated overnight with either 1 μM dexamethasone (+) or ethanol (-) as vehicle control. Expression of GR, FLAG-tagged BATF3 and actin (loading control) are shown.</p

Primers used for cloning.

<p>Primers used for cloning.</p