POGZ Is Required for Silencing Mouse Embryonic β-like Hemoglobin and Human Fetal Hemoglobin Expression

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked FilesFetal globin genes are transcriptionally silenced during embryogenesis through hemoglobin switching. Strategies to derepress fetal globin expression in the adult could alleviate symptoms in sickle cell disease and β-thalassemia. We identified a zinc-finger protein, pogo transposable element with zinc-finger domain (POGZ), expressed in hematopoietic progenitor cells. Targeted deletion of Pogz in adult hematopoietic cells in vivo results in persistence of embryonic β-like globin expression without affecting erythroid development. POGZ binds to the Bcl11a promoter and erythroid-specific intragenic regulatory regions. Pogz+/- mice show elevated embryonic β-like globin expression, suggesting that partial reduction of Pogz expression results in persistence of embryonic β-like globin expression. Knockdown of POGZ in primary human CD34+ progenitor cell-derived erythroblasts reduces BCL11A expression, a known repressor of embryonic β-like globin expression, and increases fetal hemoglobin expression. These findings are significant, since new therapeutic targets and strategies are needed to treat β-globin disorders.Frederick National Laboratory for Cancer Research, NIH intramural research program of the NHLBI, NIH intramural research program of the NIDDK, NIH USUH

Novel Lymphotoxin Alpha (LTα) Knockout Mice with Unperturbed Tumor Necrosis Factor Expression: Reassessing LTα Biological Functions

Lymphotoxin alpha (LTα) can exist in soluble form and exert tumor necrosis factor (TNF)-like activity through TNF receptors. Based on the phenotypes of knockout (KO) mice, the physiological functions of LTα and TNF are considered partly redundant, in particular, in supporting the microarchitecture of the spleen and in host defense. We exploited Cre-LoxP technology to generate a novel neomycin resistance gene (neo) cassette-free LTα-deficient mouse strain (neo-free LTα KO [LTα(Δ/Δ)]). Unlike the “conventional” LTα(−/−) mice, new LTα(Δ/Δ) animals were capable of producing normal levels of systemic TNF upon lipopolysaccharide (LPS) challenge and were susceptible to LPS/d-galactosamine (D-GalN) toxicity. Activated neutrophils, monocytes, and macrophages from LTα(Δ/Δ) mice expressed TNF normally at both the mRNA and protein levels as opposed to conventional LTα KO mice, which showed substantial decreases in TNF. Additionally, the spleens of the neo-free LTα KO mice displayed several features resembling those of LTβ KO mice rather than conventional LTα KO animals. The phenotype of the new LTα(Δ/Δ) mice indicates that LTα plays a smaller role in lymphoid organ maintenance than previously thought and has no direct role in the regulation of TNF expression

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

<div><p>Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia <i>in vivo</i>. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (<i>TgK18GT</i>_<i>121</i><i>; K18</i> mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.</p></div

Transgene expression by immunostaining in <i>TgK18GT</i>_<i>121</i> (<i>K18</i>) or induced-<i>K18</i> mice.

<p>(A) Transgene cassette consisting of floxed eGFP stop cassette upstream of truncated SV40 large T antigen (first 121 amino acid; T₁₂₁) was inserted into the 1^st exon of K18 gene on a bacterial artificial chromosome (BAC). Transgene eGFP was driven by K18 regulation. Once K18 mice were crossed to a transgenic mice expressing Cre recombinase, T₁₂₁ was expressed directly under K18 regulation. (B) Representative images of K18 IHC staining in cortex (C) and medulla (M) of <i>WT</i> thymus. Inserts are higher magnification of the images. (C) Representative immunofluorescence images of T₁₂₁ (green), K18 (yellow), K5 (red), and DAPI (blue) in cortex (C) and medulla (M) delineated with a white dotted line, in induced-<i>K18;Cre</i> thymus. Middle and right images are higher magnification of areas in white and red boxes of left image, respectively. Right image: * Cell is positive for T₁₂₁, K18, and K5, and # positive for K5 only. (D) Representative images of K18 (red) and eGFP (green) immunostaining in thymic cortex and medulla (data not shown) of uninduced-<i>K18</i> mice (Cre negative). (E) Representative images of K18 (red) and T₁₂₁ (green) immunostaining in thymic cortex and medulla (data not shown) of induced-<i>K18</i> mice (<i>K18;β-actin Cre</i>).</p

Rb-TS inactivation in K18+ TEC led to decreased survival and thymic hyperplasia.

<p>(A) Kaplan-Meier survival curve of <i>K18;β-actin Cre</i> (n = 74), <i>K18;R26CreER</i> (n = 27), and <i>K18;PbCre4</i> (n = 45) mice with median survival of 94, 41, and 231 days, respectively. Uninduced -<i>K18</i> mice (n = 8) did not develop any gross abnormalities. (B) Gross phenotype of thymuses in <i>WT</i> and <i>K18;β-actin Cre</i> mice. (C) Representative images of H.E. stained thymus sections in <i>WT</i>, <i>K18;R26CreER</i> mice. C: cortex; M: medulla. (D) Representative low magnification images of H.E. stained thymuses in <i>WT</i> and <i>K18;β-actin Cre</i> mice. C: cortex; M: medulla.</p