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Histone Deacetylase (HDAC) Activity Is Critical for Embryonic Kidney Gene Expression, Growth, and Differentiation*

Open AccessPublished:July 21, 2011DOI:https://doi.org/10.1074/jbc.M111.248278
      Histone deacetylases (HDACs) regulate fundamental biological processes such as cellular proliferation, differentiation, and survival via genomic and nongenomic effects. This study examined the importance of HDAC activity in the regulation of gene expression and differentiation of the developing mouse kidney. Class I HDAC1–3 and class II HDAC4, -7, and -9 genes are developmentally regulated. Moreover, HDAC1–3 are highly expressed in nephron precursors. Short term treatment of cultured mouse embryonic kidneys with HDAC inhibitors (HDACi) induced global histone H3 and H4 hyperacetylation and H3K4 hypermethylation. However, genome-wide profiling revealed that the HDAC-regulated transcriptome is restricted and encompasses regulators of the cell cycle, Wnt/β-catenin, TGF-β/Smad, and PI3K-AKT pathways. Further analysis demonstrated that base-line expression of key developmental renal regulators, including Osr1, Eya1, Pax2/8, WT1, Gdnf, Wnt9b, Sfrp1/2, and Emx2, is dependent on intact HDAC activity. Treatment of cultured embryonic kidney cells with HDACi recapitulated these gene expression changes, and chromatin immunoprecipitation assays revealed that HDACi is associated with histone hyperacetylation of Pax2/Pax8, Gdnf, Sfrp1, and p21. Gene knockdown studies demonstrated that HDAC1 and HDAC2 play a redundant role in regulation of Pax2/8 and Sfrp1 but not Gdnf. Long term treatment of embryonic kidneys with HDACi impairs the ureteric bud branching morphogenesis program and provokes growth arrest and apoptosis. We conclude that HDAC activity is critical for normal embryonic kidney homeostasis, and we implicate class I HDACs in the regulation of early nephron gene expression, differentiation, and survival.

      Introduction

      Kidney development depends on reciprocal inductive interactions between the metanephric mesenchyme (MM),
      The abbreviations used are: MM
      metanephric mesenchyme
      HDAC
      histone deacetylase
      HDACi
      HDAC inhibitor
      UB
      ureteric bud
      mK4 cells
      metanephric kidney cell line
      MO
      morpholino
      TSA
      trichostatin A
      ISH
      in situ hybridization.
      a specified region in the caudal intermediate mesoderm, and the ureteric bud (UB), an epithelial outgrowth from the Wolffian (nephric) duct (
      • Yu J.
      • McMahon A.P.
      • Valerius M.T.
      ,
      • Rosenblum N.D.
      ,
      • Dressler G.R.
      ). Recent years have witnessed significant progress in our understanding of the gene regulatory networks of early kidney development (
      • Dressler G.R.
      ,
      • Bouchard M.
      ,
      • Vainio S.
      • Lin Y.
      ,
      • Boyle S.
      • de Caestecker M.
      ). For example, the Osr1/Eya1/Pax2/Six/Sall/WT1/Hoxd11 gene regulatory network specifies the MM and is absolutely required for expression of glia-derived neurotrophic factor (Gdnf) (
      • Gong K.Q.
      • Yallowitz A.R.
      • Sun H.
      • Dressler G.R.
      • Wellik D.M.
      ,
      • Sajithlal G.
      • Zou D.
      • Silvius D.
      • Xu P.X.
      ). Gdnf, in turn, is essential for UB outgrowth and subsequent branching (
      • Pichel J.G.
      • Shen L.
      • Sheng H.Z.
      • Granholm A.C.
      • Drago J.
      • Grinberg A.
      • Lee E.J.
      • Huang S.P.
      • Saarma M.
      • Hoffer B.J.
      • Sariola H.
      • Westphal H.
      ,
      • Sánchez M.P.
      • Silos-Santiago I.
      • Frisén J.
      • He B.
      • Lira S.A.
      • Barbacid M.
      ,
      • Basson M.A.
      • Watson-Johnson J.
      • Shakya R.
      • Akbulut S.
      • Hyink D.
      • Costantini F.D.
      • Wilson P.D.
      • Mason I.J.
      • Licht J.D.
      ). Gdnf acts via activation of a c-Ret/PI3K/ERK-dependent gene network (Wnt11, Spry1, Etv4, Etv5, Cxcr4, Myb, Met, and Mmp14) in UB tip cells to control the branching morphogenesis program (
      • Costantini F.
      ,
      • Sakurai H.
      ,
      • Lu B.C.
      • Cebrian C.
      • Chi X.
      • Kuure S.
      • Kuo R.
      • Bates C.M.
      • Arber S.
      • Hassell J.
      • MacNeil L.
      • Hoshi M.
      • Jain S.
      • Asai N.
      • Takahashi M.
      • Schmidt-Ott K.M.
      • Barasch J.
      • D'Agati V.
      • Costantini F.
      ). Various growth factor/receptor signaling pathways, including FGFs, bone morphogenic proteins, VEGF, semaphorins, hepatocyte growth factor, EGF, among others, share signaling components with the c-Ret pathway and are required for optimal metanephric growth and patterning (
      • Challen G.
      • Gardiner B.
      • Caruana G.
      • Kostoulias X.
      • Martinez G.
      • Crowe M.
      • Taylor D.F.
      • Bertram J.
      • Little M.
      • Grimmond S.M.
      ,
      • Dudley A.T.
      • Godin R.E.
      • Robertson E.J.
      ,
      • Gao X.
      • Chen X.
      • Taglienti M.
      • Rumballe B.
      • Little M.H.
      • Kreidberg J.A.
      ,
      • Gerber H.P.
      • Hillan K.J.
      • Ryan A.M.
      • Kowalski J.
      • Keller G.A.
      • Rangell L.
      • Wright B.D.
      • Radtke F.
      • Aguet M.
      • Ferrara N.
      ,
      • Godin R.E.
      • Robertson E.J.
      • Dudley A.T.
      ,
      • Hains D.
      • Sims-Lucas S.
      • Kish K.
      • Saha M.
      • McHugh K.
      • Bates C.M.
      ,
      • Hartwig S.
      • Bridgewater D.
      • Di Giovanni V.
      • Cain J.
      • Mishina Y.
      • Rosenblum N.D.
      ,
      • Reidy K.J.
      • Villegas G.
      • Teichman J.
      • Veron D.
      • Shen W.
      • Jimenez J.
      • Thomas D.
      • Tufro A.
      ).
      Following induction of the MM, activation of the Wnt/β-catenin signaling pathway plays a key role in nephrogenesis. Release of Wnt9b from the UB branches triggers a β-catenin-dependent morphogenetic program leading to expression of Wnt4, Fgf8, and Pax8 and transition of ventrally located MM cells to epithelial nephron progenitors (pretubular aggregates and renal vesicles) (
      • Carroll T.J.
      • Park J.S.
      • Hayashi S.
      • Majumdar A.
      • McMahon A.P.
      ,
      • Schmidt-Ott K.M.
      • Barasch J.
      ). Wnt9b signaling cooperates with Six2, a transcription factor expressed exclusively in the dorsal metanephric cap region to maintain the proliferation or self-renewal of pluripotent MM cells (
      • Self M.
      • Lagutin O.V.
      • Bowling B.
      • Hendrix J.
      • Cai Y.
      • Dressler G.R.
      • Oliver G.
      ,
      • Kobayashi A.
      • Valerius M.T.
      • Mugford J.W.
      • Carroll T.J.
      • Self M.
      • Oliver G.
      • McMahon A.P.
      ,
      • Karner C.M.
      • Das A.
      • Ma Z.
      • Self M.
      • Chen C.
      • Lum L.
      • Oliver G.
      • Carroll T.J.
      ). Subsequently, the Lim homeodomain transcription factor, Lhx1 (also known as Lim-1), a downstream target of Wnt4/Fgf8 signaling, mediates further maturation to comma- and S-shaped bodies (
      • Chen Y.T.
      • Kobayashi A.
      • Kwan K.M.
      • Johnson R.L.
      • Behringer R.R.
      ,
      • Kobayashi A.
      • Kwan K.M.
      • Carroll T.J.
      • McMahon A.P.
      • Mendelsohn C.L.
      • Behringer R.R.
      ,
      • Potter S.S.
      • Hartman H.A.
      • Kwan K.M.
      • Behringer R.R.
      • Patterson L.T.
      ). Segmentation of the epithelial progenitor to proximal and distal fates is partly accomplished by signaling pathways downstream of Notch and Irx/Brn-1, respectively (
      • Cheng H.T.
      • Kim M.
      • Valerius M.T.
      • Surendran K.
      • Schuster-Gossler K.
      • Gossler A.
      • McMahon A.P.
      • Kopan R.
      ,
      • Alarcón P.
      • Rodríguez-Seguel E.
      • Fernández-González A.
      • Rubio R.
      • Gómez-Skarmeta J.L.
      ,
      • Reggiani L.
      • Raciti D.
      • Airik R.
      • Kispert A.
      • Brändli A.W.
      ,
      • Nakai S.
      • Sugitani Y.
      • Sato H.
      • Ito S.
      • Miura Y.
      • Ogawa M.
      • Nishi M.
      • Jishage K.
      • Minowa O.
      • Noda T.
      ). The canonical Wnt/β-catenin signaling pathway is also required for UB outgrowth and branching (
      • Marose T.D.
      • Merkel C.E.
      • McMahon A.P.
      • Carroll T.J.
      ,
      • Bridgewater D.
      • Cox B.
      • Cain J.
      • Lau A.
      • Athaide V.
      • Gill P.S.
      • Kuure S.
      • Sainio K.
      • Rosenblum N.D.
      ).
      Histone deacetylases (HDACs) are an evolutionarily conserved group of enzymes that regulate chromatin architecture and gene expression by removing acetyl groups from histone tails. To date, 18 mammalian HDACs have been identified. Based on sequence homology to yeast hda genes, they are divided into four classes as follows: class I HDACs (1–3 and 8), class II HDACs (4–7, 9, and 10), class III (Sirtuins 1–7), and class IV (HDAC11) (
      • Smith C.L.
      ,
      • de Ruijter A.J.
      • van Gennip A.H.
      • Caron H.N.
      • Kemp S.
      • van Kuilenburg A.B.
      ,
      • Zhang K.
      • Dent S.Y.
      ). The action of HDACs on histone acetylation is counteracted by histone acetyltransferases, e.g. CBP/p300 and GCN5, which acetylate histone tails.
      Although the fundamental functions of HDACs in cancer and cardiac and immune disorders are well documented, their role in embryogenesis and organogenesis has just begun to be elucidated (
      • Haberland M.
      • Montgomery R.L.
      • Olson E.N.
      ). HDAC1 and HDAC2 are crucial for regulating cell proliferation and are indispensable for embryo survival (
      • Lagger G.
      • O'Carroll D.
      • Rembold M.
      • Khier H.
      • Tischler J.
      • Weitzer G.
      • Schuettengruber B.
      • Hauser C.
      • Brunmeir R.
      • Jenuwein T.
      • Seiser C.
      ,
      • Bhaskara S.
      • Chyla B.J.
      • Amann J.M.
      • Knutson S.K.
      • Cortez D.
      • Sun Z.W.
      • Hiebert S.W.
      ). HDAC1 and HDAC2 perform redundant yet essential functions in cardiac growth and development, in the pathological responses to increased cardiac load, and differentiation of oligodendrocytes (
      • Montgomery R.L.
      • Davis C.A.
      • Potthoff M.J.
      • Haberland M.
      • Fielitz J.
      • Qi X.
      • Hill J.A.
      • Richardson J.A.
      • Olson E.N.
      ,
      • Trivedi C.M.
      • Luo Y.
      • Yin Z.
      • Zhang M.
      • Zhu W.
      • Wang T.
      • Floss T.
      • Goettlicher M.
      • Noppinger P.R.
      • Wurst W.
      • Ferrari V.A.
      • Abrams C.S.
      • Gruber P.J.
      • Epstein J.A.
      ). Class II HDACs are important for restricting cell size; HDAC4, −5, and −9 exert antihypertrophic properties in chondrocytes and heart, respectively (
      • Vega R.B.
      • Matsuda K.
      • Oh J.
      • Barbosa A.C.
      • Yang X.
      • Meadows E.
      • McAnally J.
      • Pomajzl C.
      • Shelton J.M.
      • Richardson J.A.
      • Karsenty G.
      • Olson E.N.
      ,
      • Chang S.
      • McKinsey T.A.
      • Zhang C.L.
      • Richardson J.A.
      • Hill J.A.
      • Olson E.N.
      ). In addition, HDAC7 null mice are embryonic lethal by E11.0 secondary to loss of vascular integrity (
      • Chang S.
      • Young B.D.
      • Li S.
      • Qi X.
      • Richardson J.A.
      • Olson E.N.
      ). The role of HDACs in mammalian kidney development is unknown, but there is evidence that HDACs regulate the progenitor cell population in the pronephric kidney of zebrafish (
      • de Groh E.D.
      • Swanhart L.M.
      • Cosentino C.C.
      • Jackson R.L.
      • Dai W.
      • Kitchens C.A.
      • Day B.W.
      • Smithgall T.E.
      • Hukriede N.A.
      ).
      In this study, we show that several members of class I and II HDACs are developmentally regulated in the kidney. Furthermore, using pharmacological inhibitors of HDACs in ex vivo cultured embryonic kidneys, we show that HDAC activity is required for expression of key developmental pathways. Moreover, gene knockdown studies and ChIP assays revealed a redundant role for HDAC1 and HDAC2 in MM cell gene expression. Finally, we show that HDAC activity is necessary for growth and survival of the developing nephron.

      DISCUSSION

      This study investigated the developmental expression of HDACs and the effects of HDAC inhibition on gene expression and development of the embryonic kidney. Our major findings are as follows. 1) The developing kidney expresses nine members of the class I and II HDAC family of enzymes. 2) Based on their spatiotemporal expression patterns and the effects of HDACi and gene knockdown, class I HDACs are candidate regulators of metanephric development. 3) HDACs regulate essential genes involved in renal growth and differentiation. 4) Genome-wide analysis revealed HDAC inhibition can either stimulate or repress metanephric gene transcription.

      Developmental Expression of HDACs

      Our results demonstrate that several HDAC genes are developmentally regulated. These include HDAC1–4, -7, and -9. In comparison, HDAC5, -6, and -8 are constitutively expressed. The developmental down-regulation of class I HDAC genes correlates with the known reduction in proliferative activity during nephron differentiation, reminiscent of intestinal villus differentiation. In the mouse intestinal epithelium, HDAC2 and HDAC3 are highest in abundance in the proliferating crypt cells and decline during villus maturation (
      • Mariadason J.M.
      • Nicholas C.
      • L'Italien K.E.
      • Zhuang M.
      • Smartt H.J.
      • Heerdt B.G.
      • Yang W.
      • Corner G.A.
      • Wilson A.J.
      • Klampfer L.
      • Arango D.
      • Augenlicht L.H.
      ). Overexpression of HDAC1 and HDAC2 in cultured intestinal explants delays expression of differentiation markers, whereas HDACi provokes premature differentiation (
      • Tou L.
      • Liu Q.
      • Shivdasani R.A.
      ). In this regard, our data indicate that high HDAC activity in the embryonic kidney is required for expression of cell proliferation and survival pathway genes, such as c-myc, cyclin D1, and thymidylate kinase, and for suppression of tumor suppressors and cell cycle inhibitors, such as p21, p15, and p16.

      Effect of HDACi on the Embryonic Renal Transcriptome

      Genome-wide profiling revealed that 12% of the embryonic kidney transcriptome is HDAC-regulated (using a cutoff of 1.5-fold, p < 0.05). Bioinformatic analysis revealed several interesting observations. First, most differentially expressed genes fall under the cell cycle, Wnt/β-catenin, TGF-β/Smad, and PI3K-AKT, pathways. Considering the importance of these biological pathways in organ growth and morphogenesis, it is not surprising that exposure of kidney explants to HDACi led to hypoplasia accompanied by suppressed cellular proliferation and enhanced apoptosis. Second, ∼60% of the differentially expressed genes in response to HDACi were repressed. It is commonly assumed that HDACs function as repressors of gene transcription. However, there is increasing evidence that HDACs exhibit both repressive and activating effects on gene transcription (
      • Smith C.L.
      ,
      • Gui C.Y.
      • Ngo L.
      • Xu W.S.
      • Richon V.M.
      • Marks P.A.
      ). In this regard, global HDAC inhibition or deletion of individual HDAC genes in mice or embryonic stem cells affects expression of up to 10% of the expressed cellular genes, and nearly as many genes are down-regulated as are up-regulated (
      • Haberland M.
      • Montgomery R.L.
      • Olson E.N.
      ,
      • Van Lint C.
      • Emiliani S.
      • Verdin E.
      ,
      • Chambers A.E.
      • Banerjee S.
      • Chaplin T.
      • Dunne J.
      • Debernardi S.
      • Joel S.P.
      • Young B.D.
      ,
      • Haberland M.
      • Mokalled M.H.
      • Montgomery R.L.
      • Olson E.N.
      ). Our findings are consistent with the notion that HDACs can either stimulate or repress gene transcription, and the widely held view that HDACs function predominantly as transcriptional repressors should be reconsidered. Mechanistically, specific patterns of histone lysine acetylation may cooperate with other forms of histone modifications (e.g. methylation) to create a “histone signature” for recruitment of transcriptional regulators (
      • Agalioti T.
      • Chen G.
      • Thanos D.
      ,
      • Ferguson M.
      • Henry P.A.
      • Currie R.A.
      ,
      • Sakamoto S.
      • Potla R.
      • Larner A.C.
      ). Our ChIP analysis and gene knockdown studies support the idea that HDACs regulate directly at least a subset of genes involved in nephron differentiation. However, HDACs also act via nongenomic mechanisms as well. For example, HDACs deacetylate transcription factors, such as p53, STAT3, YY1, GATA1, E2F1, and other proteins like tubulin and Hsp90 (
      • Mehnert J.M.
      • Kelly W.K.
      ). Thus, the effects of HDACi on gene expression may be direct (i.e. epigenetic) or indirect (via nongenomic effect) or both.
      The third important finding of our study is that HDAC activity is required for basal expression of key renal developmental regulators, such as Eya1, WT1, Pax2, Hox11 paralogs, HNF1β, FoxD1, Wnt9b, Gdnf, and downstream epithelial differentiation effectors, e.g. Pax8, Fgf8, Wnt4, Lhx1. Time course analysis in organ culture revealed that some developmental genes, e.g. Pax2, WT1, Eya1, Wnt9b, and Wnt4, are more sensitive to HDACi than others, e.g. c-ret or Bmp4, despite the widespread expression of HDACs in the developing kidney. Although the exact reason is presently unknown, this finding suggests that HDACs may differentially bind to and/or regulate different target genes in the developing nephron.
      Our organ culture studies suggest that HDAC activity is required to maintain active canonical Wnt signaling in metanephric kidneys. We speculate that this function is mediated via Wnt9b. Our data show that Wnt9b is among the earliest genes down-regulated in HDACi-treated metanephroi and may account for repression of β-catenin-target genes, Axin2, c-myc, and cyclin D1, as well as renal vesicle genes Fgf8, Pax8, Wnt4, and Lhx1. In a recent study, De Groh et al. (
      • de Groh E.D.
      • Swanhart L.M.
      • Cosentino C.C.
      • Jackson R.L.
      • Dai W.
      • Kitchens C.A.
      • Day B.W.
      • Smithgall T.E.
      • Hukriede N.A.
      ) reported in zebrafish that HDACi expands the domain of pronephric developmental regulators and renal progenitor cells in a retinoic acid-dependent manner.

      HDACs and Metanephric Cell Proliferation/Apoptosis

      This study demonstrates that sustained inhibition of HDAC activity (>12 h) blocks cell proliferation and induces cell apoptosis in cultured kidney explants. The mechanisms of HDACi-induced growth arrest and apoptosis have been extensively investigated. In cancer cell lines, HDACi-induced cell cycle arrest at G1/S and G2/M is commonly associated with activation of CDK inhibitors p21, p19, and p1, and repression of cyclin D1, c-myc, and thymidylate synthase. Also, HDACi can transcriptionally activate the extrinsic and intrinsic pathways of apoptosis (
      • de Ruijter A.J.
      • van Gennip A.H.
      • Caron H.N.
      • Kemp S.
      • van Kuilenburg A.B.
      ,
      • Mehnert J.M.
      • Kelly W.K.
      ).
      In this study, we observed that CDK inhibitors/tumor suppressor genes, including p21, p15, p19, Bop1, and Htra1, were significantly up-regulated by HDACi. Moreover, many oncogenes, such as c-myc, N-myc, cyclin D1, cyclin J, cyclin B2, and thymidylate synthase, were dramatically down-regulated. Thus, the imbalance between growth promoters and suppressors can collectively contribute to metanephric growth arrest induced by HDACi. The apoptosis-regulating genes, Bcl-2 and caspase 9, exhibited modest down- and up-regulation, respectively, in response to HDACi. Although we cannot completely rule out nonspecific effects related to the use of pharmacological inhibitors, morpholino-mediated knockdown of HDAC1 and HDAC2 strongly suggests that class I HDACs regulate gene expression in MM cells. Ongoing studies in our laboratory utilizing nephric lineage-specific inactivation of individual HDACs will be paramount in delineating the role of individual HDACs in renal development.
      In summary, multiple lines of evidence derived from our study and others support the hypothesis that HDACs play important roles in nephrogenesis. HDAC1–3, which play key roles in regulating cell proliferation and cell survival in other organs such as intestines, liver, and brain, are also enriched in nephron progenitors and UB branches. Utilizing two class I/II HDAC inhibitors and a more selective class I HDAC inhibitor, we show that inhibition of HDAC activity impairs renal growth. Inhibition of HDAC activity alters expression of developmental regulators involved in MM competence and survival, mesenchyme-to-epithelium differentiation, and proliferation. Finally, our findings support the notion that HDAC1 and HDAC2 perform important yet redundant functions in regulation of embryonic kidney cell gene expression, as described previously in other cell types.

      Acknowledgments

      We are grateful to O. Wessely (Louisiana State University Health Sciences Center) and members of the El-Dahr laboratory for advice, reagents, and discussions.

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