Biomedical subjects

Mitchell A Lazar

Publications and source records attributed to Mitchell A Lazar.

15 recordsLinked to original sources

PubMed2003

A SANT motif in the SMRT corepressor interprets the histone code and promotes histone deacetylation.

Nuclear receptor corepressors SMRT (silencing mediator of retinoid and thyroid receptors) and N-CoR (nuclear receptor corepressor) recruit histone deacetylase (HDAC) activity to targeted regions of chromatin. These corepressors contain a closely spaced pair of SANT motifs whose sequence and organization is highly conserved. The N-terminal SANT is a critical component of a deacetylase activation domain (DAD) that binds and activates HDAC3. Here, we show that the second SANT motif functions as part of a histone interaction domain (HID). The HID enhances repression by increasing the affinity of the DAD-HDAC3 enzyme for histone substrate. The two SANT motifs synergistically promote histone deacetylation and repression through unique functions. The HID contribution to repression is magnified by its ability to inhibit histone acetyltransferase enzyme activity. Remarkably, the SANT-containing HID preferentially binds to unacetylated histone tails. This implies that the SMRT HID participates in interpreting the histone code in a feed-forward mechanism that promotes and maintains histone deacetylation at genomic sites of SMRT recruitment.

Acetylation↗

PubMed2003

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response.

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

Carrier Proteins↗

PubMed2003

Resistin: molecular history and prognosis.

Obesity and diabetes have reached epidemic proportions worldwide. The antidiabetic thiazolidinedione (TZD) drugs are insulin-sensitizing agents now widely used in the treatment of type 2 diabetes. TZDs are ligands for the nuclear hormone receptor peroxisome proliferator activated receptor gamma, which is a master regulator of adipogenesis and adipocyte metabolism. The molecular mechanisms by which TZDs improve insulin sensitivity have not been fully identified. Here we consider a novel secreted factor first identified as a TZD-suppressible gene in mouse adipocytes, called resistin, and discuss what is currently known about resistin regulation and function in mouse and human.

Amino Acid Sequence↗

PubMed2003

The N-CoR/histone deacetylase 3 complex is required for repression by thyroid hormone receptor.

Nuclear receptor corepressors (N-CoR) and silencing mediator for retinoid and thyroid receptors (SMRT) have both been implicated in thyroid hormone receptor (TR)-mediated repression. Here we show that endogenous N-CoR, TBL1, and histone deacetylase 3 (HDAC3), but not HDAC1, -2, or -4, are recruited to a stably integrated reporter gene repressed by unliganded TR as well as the orphan receptor RevErb. Unliganded TR also recruits this complex to a transiently transfected reporter, and transcriptional repression is associated with local histone deacetylation that is reversed by the presence of thyroid hormone. Knockdown of N-CoR using small interfering RNAs markedly reduces repression by the TR ligand binding domain in human 293T cells, whereas knockdown of SMRT has little effect. RevErb repression appears to involve both corepressors in this system. Knockdown of HDAC3 markedly reduces repression by both TR and RevErb, while knockdown of HDAC1 or 2 has more modest, partly nonspecific effects. Thus, HDAC3 is critical for repression by multiple nuclear receptors and the N-CoR HDAC3 complex plays a unique and necessary role in TR-mediated gene repression in human 293T cells.

Cell Line↗

PubMed2003

Thyroid hormone action: a binding contract.

Thyroid hormones are critical for differentiation, growth, and metabolism. A new study investigating the biological role of the TH receptor TR-beta has demonstrated that DNA binding is critical for most of its functions, but also suggests that novel mechanisms independent of DNA binding may contribute to regulation of auditory function by TR-beta.

Animals↗

PubMed2002

Assembly of the SMRT-histone deacetylase 3 repression complex requires the TCP-1 ring complex.

The acetylation of histone tails is a primary determinant of gene activity. Histone deacetylase 3 (HDAC3) requires the nuclear receptor corepressor SMRT for HDAC enzyme activity. Here we report that HDAC3 interacts with SMRT only after priming by cellular chaperones including the TCP-1 ring complex (TRiC), which is required for proper folding of HDAC3 in an ATP-dependent process. SMRT displaces TRiC from HDAC3, yielding an active HDAC enzyme. The SMRT-HDAC3 repression complex thus joins the VHL-elongin BC tumor suppression complex and the cyclin E-Cdk2 cell cycle regulation complex as critical cellular machines requiring TRiC for proper assembly and function. The strict control of HDAC3 activity underscores the cellular imperative that histone deacetylation occur only in targeted regions of the genome.

Adenosine Triphosphate↗

PubMed2002

A futile metabolic cycle activated in adipocytes by antidiabetic agents.

Thiazolidinediones (TZDs) are effective therapies for type 2 diabetes, which has reached epidemic proportions in industrialized societies. TZD treatment reduces circulating free fatty acids (FFAs), which oppose insulin actions in skeletal muscle and other insulin target tissues. Here we report that TZDs, acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma, markedly induce adipocyte glycerol kinase (GyK) gene expression. This is surprising, as standard textbooks indicate that adipocytes lack GyK and thereby avoid futile cycles of triglyceride breakdown and resynthesis from glycerol and FFAs. By inducing GyK, TZDs markedly stimulate glycerol incorporation into triglyceride and reduce FFA secretion from adipocytes. The 'futile' fuel cycle resulting from expression of GyK in adipocytes is thus a novel mechanism contributing to reduced FFA levels and perhaps insulin sensitization by antidiabetic therapies.

Adipocytes↗

PubMed2002

Thiazolidinedione activation of peroxisome proliferator-activated receptor gamma can enhance mitochondrial potential and promote cell survival.

Thiazolidinediones (TZDs) are widely used for treatment of type 2 diabetes mellitus. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is the molecular target of TZDs and is believed to mediate the apoptotic effects of this class of drugs in a variety of cell types, including B and T lymphocytes. The finding that TZDs induce lymphocyte death has raised concerns regarding whether TZDs might further impair immune functions in diabetics. To address this issue, we investigated the roles of PPAR gamma and TZDs in lymphocyte survival. PPAR gamma was up-regulated upon T cell activation. As previously reported, PPAR gamma agonists induced T cell death in a dose-dependent manner. However, the concentrations of TZD needed to cause T cell death were above those needed to induce PPAR gamma-dependent transcription. Surprisingly, at concentrations that induce optimal transcriptional activation, TZD activation of PPAR gamma protected cells from apoptosis following growth factor withdrawal. The survival-enhancing effects depended on both the presence and activation of PPAR gamma. Measurements of mitochondrial potential revealed that PPAR gamma activation enhanced the ability of cells to maintain their mitochondrial potential. These data indicate that activation of PPAR gamma with TZDs can promote cell survival and suggest that PPAR gamma activation may potentially augment the immune responses of diabetic patients.

Animals↗

PubMed2002

Cooperative function of Aml1-ETO corepressor recruitment domains in the expansion of primary bone marrow cells.

AML1-ETO is an oncoprotein that can promote self-renewal of primary hematopoietic cells by opposing the activity of AML1. Two domains, Nervy-homology(NH) 2 and NH4, have been implicated in the recruitment of corepressors by AML1-ETO, but the relative roles of NH2 and NH4 vary in different cell lines and have not been examined in nonimmortalized cells. Here, we have used a series of differentiation, proliferation, and self-renewal assays in an effort to determine the roles of the NH domains using progenitor-enriched primary bone marrow cells. In these assays, deletion of NH2 or NH4, individually, has a minimal effect on AML1-ETO function, and the mutants retain the ability to promote long-term expansion of cells. In contrast, the double deletion of NH2 and NH4 eliminates the activity of the fusion protein. Thus, the double deletion of NH2 and NH4 produces a functional deficit greater than the sum of the individual deletions. These findings suggest that the NH2 and NH4 domains function cooperatively in the corepressor environment of primary bone marrow cells.

Animals↗

PubMed2002

Mechanisms regulating adipocyte expression of resistin.

Resistin, also known as Adipocyte Secreted Factor (ADSF) and Found in Inflammatory Zone 3 (FIZZ3), is a mouse protein with potential roles in insulin resistance and adipocyte differentiation. The resistin gene is expressed almost exclusively in adipocytes. Here we show that a proximal 264-base pair fragment of the mouse resistin promoter is sufficient for expression in adipocytes. Ectopic expression of the adipogenic transcription factor CCAAT/enhancer-binding protein (C/EBPalpha) was sufficient for expression in non-adipogenic cells. C/EBPalpha binds specifically to a site that is essential for expression of the resistin promoter. Chromatin immunoprecipitation studies of the endogenous gene demonstrated adipocyte-specific association of C/EBPalpha with the proximal resistin promoter in adipocytes but not preadipocytes. C/EBPalpha binding was associated with the recruitment of coactivators p300 and CREB-binding protein and a dramatic increase in histone acetylation in the vicinity of the resistin promoter. The antidiabetic thiazolidinedione (TZD) drug rosiglitazone reduced resistin expression with an ED(50) similar to its K(d) for binding to peroxisome proliferator activated receptor gamma (PPARgamma). Other TZD- and non-TZD PPARgamma ligands also down-regulated resistin expression. However, no functional PPARgamma binding site was found within 6.2 kb of the transcriptional start site, suggesting that if PPARgamma is involved, it is either acting at a long distance from the start site, in an intron, or indirectly. Nevertheless, rosiglitazone treatment selectively decreased histone acetylation at the resistin promoter without a change in occupation by C/EBPalpha, CREB-binding protein, or p300. Thus, adipocyte specificity of resistin gene expression is because of C/EBPalpha binding, leading to the recruitment of transcriptional coactivators and histone acetylation that is characteristic of an active chromatin environment. TZD reduces resistin gene expression at least in part by reducing histone acetylation associated with the binding of C/EBPalpha in mature adipocytes.

Adipocytes↗

PubMed2002

The dawn of the SPPARMs?

Thiazolidinediones (TZDs) are used as antidiabetic agents in the treatment of type II diabetes. These compounds are ligands for the nuclear hormone receptor PPARgamma, which is highly expressed in adipose tissue. PPARgamma acts as a molecular switch in the process of fat cell development. The quest for the ideal antidiabetic agent is challenged by the need to develop PPARgamma ligands that improve insulin sensitivity, but do not promote fat cell formation. A newly described PPARgamma ligand may represent an initial step in this direction and could lead to improved agents for treating insulin resistance in type II diabetes.

Adipocytes↗

PubMed2002

Becoming fat.

Explore the source record for details and available documents.

Adipocytes↗

PubMed2002

Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR.

Histone deacetylases (HDACs) play a key role in regulating eukaryotic gene expression. The HDAC domain, homologous to the yeast repressors RPD3 and HDA1, is considered necessary and sufficient for enzymatic activity. Here, we show that the catalytic domain of HDAC4 interacts with HDAC3 via the transcriptional corepressor N-CoR/SMRT. All experimental conditions leading to the suppression of HDAC4 binding to SMRT/N-CoR and to HDAC3 result in the loss of enzymatic activity associated with HDAC4. In vitro reconstitution experiments indicate that HDAC4 and other class II HDACs are inactive in the context of the SMRT/N-CoR-HDAC3 complex and do not contribute to its enzymatic activity. These observations indicate that class II HDACs regulate transcription by bridging the enzymatically active SMRT/N-CoR-HDAC3 complex and select transcription factors independently of any intrinsic HDAC activity.

Amino Acid Sequence↗

PubMed2002

Differential gene regulation by PPARgamma agonist and constitutively active PPARgamma2.

The PPARgamma is a key adipogenic determination factor. Ligands for PPARgamma such as antidiabetic thiazolidinedione (TZD) compounds are adipogenic, and many adipocyte genes that are activated by TZDs contain binding sites for PPARgamma. Like ligands for other nuclear receptors, TZDs can regulate genes positively or negatively. Here, we sought to understand the importance of positive regulation of gene expression by PPARgamma in adipogenesis. Fusion of the potent viral transcriptional activator VP16 to PPARgamma2 (VP16-PPARgamma) created a transcription factor that constitutively and dramatically activated transcription of PPARgamma-responsive genes in the absence of ligand. Forced expression of VP16-PPARgamma in 3T3-L1 preadipocytes using retroviral vectors led to adipogenesis in the absence of standard differentiating medium or any exogenous PPARgamma ligand. Gene microarray analysis revealed that VP16-PPARgamma induced many of the genes associated with adipogenesis and adipocyte function. Thus, direct up-regulation of gene expression by PPARgamma is sufficient for adipogenesis. TZD-induced adipogenesis up-regulated many of the same genes, although some were divergently regulated, including resistin, whose gene expression was reduced inVP16-PPARgamma adipocytes treated with TZDs. These results show that, although activation of PPARgamma by a heterologous activation domain is sufficient for adipogenesis, it is not equivalent to TZD treatment. This conclusion has important implications for understanding biological effects of the TZDs on adipogenesis and insulin sensitization.

3T3 Cells↗

PubMedDate not supplied

Resistin and obesity-associated insulin resistance.

Obesity is a major risk factor for insulin resistance and type 2 diabetes mellitus. Adipocytes secrete numerous substances that might contribute to peripheral insulin sensitivity. These include leptin, tumor necrosis factor alpha, Acrp30/adiponectin/adipoQ and interleukin 6, the potential roles of which are briefly reviewed here. Thiazolidinedione (TZD) antidiabetic drugs regulate gene transcription by binding to peroxisome proliferator activated receptor gamma, a nuclear hormone receptor found at its highest levels in adipocytes. A search for genes that are downregulated by TZDs in mouse adipocytes led to the discovery of an adipose-specific secreted protein called resistin. Resistin circulates in the mouse, with increased levels in obesity, and has effects on glucose homeostasis that oppose those of insulin. Thus, resistin is a potential link between TZDs, obesity and insulin resistance in the mouse. Future studies must address the mechanism of action and biological role of resistin and related family members in mice and humans.

Adipose Tissue↗