Nunciada Salma

Temporal Recruitment of Transcription Factors and SWI/SNF Chromatin-Remodeling Enzymes during Adipogenic Induction of the Peroxisome Proliferator-Activated Receptor γ Nuclear Hormone Receptor

ABSTRACT The peroxisome proliferator-activated receptor gamma (PPARγ) regulates adipogenesis, lipid metabolism, and glucose homeostasis, and roles have emerged for this receptor in the pathogenesis and treatment of diabetes, atherosclerosis, and cancer. We report here that induction of the PPARγ activator and adipogenesis forced by overexpression of adipogenic regulatory proteins is blocked upon expression of dominant-negative BRG1 or hBRM, the ATPase subunits of distinct SWI/SNF chromatin-remodeling enzymes. We demonstrate that histone hyperacetylation and the binding of C/EBP activators, polymerase II (Pol II), and general transcription factors (GTFs) initially occurred at the inducible PPARγ2 promoter in the absence of SWI/SNF function. However, the polymerase and GTFs were subsequently lost from the promoter in cells expressing dominant-negative SWI/SNF, explaining the inhibition of PPARγ2 expression. To corroborate these data, we analyzed interactions at the PPARγ2 promoter in differentiating preadipocytes. Changes in promoter structure, histone hyperacetylation, and binding of C/EBP activators, Pol II, and most GTFs preceded the interaction of SWI/SNF enzymes with the PPARγ2 promoter. However, transcription of the PPARγ2 gene occurred only upon subsequent association of SWI/SNF and TFIIH with the promoter. Thus, induction of the PPARγ nuclear hormone receptor during adipogenesis requires SWI/SNF enzymes to facilitate preinitiation complex function.

Phenotypic transcription factors epigenetically mediate cell growth control

Ribosomal RNA (rRNA) genes are down-regulated during osteogenesis, myogenesis, and adipogenesis, necessitating a mechanistic understanding of interrelationships between growth control and phenotype commitment. Here, we show that cell fate-determining factors [MyoD, myogenin (Mgn), Runx2, C/EBPβ] occupy rDNA loci and suppress rRNA expression during lineage progression, concomitant with decreased rRNA expression and reciprocal loss of occupancy by c-Myc, a proliferation-specific activator of rRNA transcription. We find interaction of phenotypic factors with the polymerase I activator upstream binding factor UBF-1 at interphase nucleoli, and this interaction is epigenetically retained on mitotic chromosomes at nucleolar organizing regions. Ectopic expression and RNA interference establish that MyoD, Mgn, Runx2, and C/EBPβ each functionally suppress rRNA genes and global protein synthesis. We conclude that epigenetic control of ribosomal biogenesis by lineage-specific differentiation factors is a general developmental mechanism for coordinate control of cell growth and phenotype.

Chromatin Accessibility and Transcription Factor Binding at the PPARγ2 Promoter during Adipogenesis is Protein Kinase A-Dependent

The nuclear hormone receptor peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand‐activated transcription factor that specifies formation of the adipocyte lineage. PPARγ also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARγ expression and function. Here, we characterize molecular changes that occur at the PPARγ2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARγ2 promoter and occupancy of the promoter by the c‐Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/enhancer binding protein beta (C/EBPβ) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation. J. Cell. Physiol. 226: 86–93, 2010.

Transcription Factor Tfe3 Directly Regulates Pgc-1alpha in Muscle.

The microphthalmia (MiT) family of transcription factors is an important mediator of metabolism. Family members Mitf and Tfeb directly regulate the expression of the master regulator of metabolism, peroxisome‐proliferator activated receptor gamma coactivator‐1 alpha (Pgc‐1alpha), in melanomas and in the liver, respectively. Pgc‐1alpha is enriched in tissues with high oxidative capacity and plays an important role in the regulation of mitochondrial biogenesis and cellular metabolism. In skeletal muscle, Pgc‐1alpha affects many aspects of muscle functionally such as endurance, fiber‐type switching, and insulin sensitivity. Tfe3 also regulates muscle metabolic genes that enhance insulin sensitivity in skeletal muscle. Tfe3 has not yet been shown to regulate Pgc‐1alpha expression. Our results reported here show that Tfe3 directly regulates Pgc‐1alpha expression in myotubes. Tfe3 ectopic expression induces Pgc‐1alpha, and Tfe3 silencing suppresses Pgc‐1alpha expression. This regulation is direct, as shown by Tfe3s binding to E‐boxes on the Pgc‐1alpha proximal promoter. We conclude that Tfe3 is a critical transcription factor that regulates Pgc‐1alpha gene expression in myotubes. Since Pgc‐1alpha coactivates numerous biological programs in diverse tissues, the regulation of its expression by upstream transcription factors such Tfe3 implies potential opportunities for the treatment of diseases where modulation of Pgc‐1alpha expression may have important clinical outcomes. J. Cell. Physiol. 230: 2330–2336, 2015.

MITF pathway mutations in melanoma

Microphthalmia-associated transcription factor (MITF), a key regulator of melanocyte development, belongs to the class of basic-helix ⁄ loop ⁄ helixleucine-zipper transcription factors. Microphthalmia-associated transcription factor regulates multiple genes related to melanin synthesis (e.g. TYR, DCT), control of apoptosis (e.g. BCL2) and cell cycle progression (e.g. p21, p16, CDK2, TBX2) as well as factors controlling additional important biological activities (Steingrimsson et al., 2004). In humans, genomic loss-of-function mutations of MITF are associated with 10–15% of Waardenburg Syndrome or Tietz Syndrome characterized by skin hypopigmentation, ocular pigmentation defects and deafness. Transcription factors PAX3, CREB (cAMP-reponsive element binding protein), and SOX10 play important roles in regulating MITF expression in melanocytes. Loss-offunction mutations of SOX10 and PAX3 also occur in Waardenburg Syndrome and phenocopy melanocytic phenotypes of MITF mutation ⁄ deficiency. Signal responsive and tissue restricted regulation of MITF appear to play central roles in the growth, development, differentiation, and survival of melanocytes. Recent studies have identified gain-of-function aberrations of MITF, which have led to its classification as a human oncogene. These include evidence of MITF amplification in 10–20% of melanomas (Garraway et al., 2005). Microphthalmia-associated transcription factor expression was also seen to be dysregulated by the product of the translocation EWS-ATF1, in human Clear Cell Sarcomas where enforced MITF expression could functionally rescue knockdown of EWSATF1. In addition, TFE3 is a very close relative of MITF’s, which has been shown to be genetically redundant with MITF in certain non-melanocytic settings and to participate in translocation breakpoints of several human malignancies (Renal Carcinomas and Alveolar Soft Parts Sarcoma). Translocation ⁄ fusion breakpoints represent less ambiguous oncogenic lesions than genomic amplifications, and it is also notable that TFE3, TFEB, or MITF have been seen capable of replacing one another in a variety of experimental ‘gene-replacement’ studies, suggesting all may confer oncogenic activity. Although MITF amplifications have been previously recognized, somatic mutations within MITF or genes regulating MITF expression have not been identified, until now. Cronin et al. (2009) from the laboratory of Yardena Samuels at NIH now present an important study demonstrating the presence of somatic mutations of MITF or SOX10 in human melanomas. These mutations cause amino acid substitutions in conserved residues in defined functional domains, exon skipping, or truncations. Interestingly, one of the identified MITF mutations, which was caused by a splice-site alteration, exhibits higher activation of the TYR and DCT promoters when cotransfected with SOX10. This MITF 4TD2B mutation is the first evidence of a gain-offunction mutation of MITF in human melanoma. The mutation may function to enhance protein levels by deleting exon 2B which was previously implicated in control of ubiquitin-dependent proteolysis of MITF. Cronin et al. also identify SOX10 mutations, which produce a truncated form of the protein or cause amino acid substitutions. The authors demonstrate that five somatic MITF mutations, including 4TD2B, did not activate the p21 promoter, in contrast to wildtype MITF. This finding raises the possibility that promoter-selective MITF loss-of-function somatic mutation may confer tumorigenic activity (such as diminished expression of a cell cycle negative regulator). Such activity might even be dominant-negative, if the less-active protein were to occupy the target promoters. Additionally, MITF mutant protein may increase the expression of other prooncogenic genes, like BCL2, TBX2, c-MET, or CDK2, or it may diminish prodifferentiation genes, like those involved in pigmentation, in a promoter-specific manner. A question that therefore arises is: how might MITF mutant proteins differentiate a particular promoter, for instance, DCT ⁄ TYR from p21 promoters? One mechanism could rely upon the participation of cofactor(s). Microphthalmia-associated transcription factor can interact with the histone acetyl transferases CBP ⁄ p300 to activate target genes (Price et al., 1998; Sato et al., 1997). Furthermore, it has been shown that BRG1, the ATPase subunit of the SWI ⁄ SNF complex, can physically interact with MITF allowing MITF to transactivate target genes (de la Serna et al., 2006). Because appropriate regulation of MITF is important for differentiation, survival and cell cycle control, specific cofactors might confer regulated expression of different target genes which modulate the biological state of the cell. The identification of MITF transcriptional partners required for MITF activity may thus provide valuable information pertinent to understanding MITF’s activities. Microphthalmia-associated transcription factor genomic mutations occur frequently at the helix-loop-helix leucinezipper domain in the Waardenburg syndrome which participates in DNA binding and dimerization. Although only relatively few somatic mutations of MITF have thus far been identified within primary and metastatic melanomas, it may be notable that their locations more commonly affect the transactivation domain, suggesting that changes in the transcriptional activity of MITF may be sufficient to trigger secondary events required for tumorigenesis. Cronin et al. (2009) also identify somatic mutations within Sox10, an upstream transcriptional regulator of MITF. The precise modes of action of these mutations will remain to be dissected. However, the epistatic relationship between these factors in the lossof-function setting (Waardenburg Syndrome) implies that similar regulatory relationships might plausibly occur in the gain-of-function setting. These studies expand not only our amazement at the activities in which MITF engages, but also the repertoire of molecular aberrations that may contribute to malignant melanoma. Moreover, the mechanistic links to non-melanoma cancers which share genomic abnormalities in MITF-related oncogenes (TFEB and TFE3) suggest that a search for somatic Coverage on: Cronin, J.C., Wunderlich, J., Loftus, S.K. et al. (2009). Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res. 22, 435–444.

Segmentation of subcutaneous fat within mouse skin in 3D OCT image data using random forests.

Cryolipolysis is a well-established cosmetic procedure for non-invasive local fat reduction. This technique selectively destroys subcutaneous fat cells using controlled cooling. Thickness measurements of subcutaneous fat were conducted using a mouse model. For detailed examination of mouse skin optical coherence tomography (OCT) was performed, which is a non-invasive imaging modality. Due to a high number of image slices manual delineation is not feasible. Therefore, automatic segmentation algorithms are required. In this work an algorithm for the automatic 3D segmentation of the subcutaneous fat layer is presented, which is based on a random forest classification followed by a graph-based refinement step. Our approach is able to accurately segment the subcutaneous fat layer with an overall average symmetric surface distance of 11.80±6.05 μm and Dice coefficient of 0.921 ± 0.045. Furthermore, it was shown that the graph-based refining step leads to increased accuracy and robustness of the segmentation results of the random forest classifier.

Tfe3 and Tfeb transcriptionally regulate peroxisome proliferator-activated receptor γ2 expression in adipocytes and mediate adiponectin and glucose levels in mice

ABSTRACT Members of the MiT transcription factor family are pivotal regulators of several lineage-selective differentiation programs. We show that two of these, Tfeb and Tfe3, control the regulator of adipogenesis, peroxisome proliferator-activated receptor γ2 (Pparγ2). Knockdown of Tfeb or Tfe3 expression during in vitro adipogenesis causes dramatic downregulation of Pparγ2 expression as well as adipogenesis. Additionally, we found that these factors regulate Pparγ2 in mature adipocytes. Next, we demonstrated that Tfeb and Tfe3 act directly by binding to consensus E-boxes within the Pparγ transcriptional regulatory region. This transcriptional control also exists in vivo, as we discovered that wild-type mice in the fed state increased their expression of Tfe3, Tf3b, and Pparγ in white adipose tissue. Furthermore, Tfe3 knockout (Tfe3KO) mice in the fed state failed to upregulate Pparγ and the adiponectin gene, a Pparγ-dependent gene, confirming the in vivo role for Tfe3. Lastly, we found that blood glucose is elevated and serum adiponectin levels are suppressed in the Tfe3KO mice, indicating that the Tfe3/Tfeb/Pparγ2 axis may contribute to whole-body energy balance. Thus, we offer new insights into the upstream regulation of Pparγ by Tfe3/Tf3b and propose that targeting these transcription factors may offer opportunities to complement existing approaches for the treatment of diseases that have dysregulated energy metabolism.

Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM

Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.

Phasor fluorescence lifetime microscopy of NADH to analyze metabolic activity of adipocytes (Conference Presentation)

The evaluation of complex metabolic changes of individual live cells and heterogeneous cell cultures is not feasible using traditional methods due to their destructive behavior and lack of spatial information. Two-photon excited fluorescence of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) facilitate a label-free and non-destructive evaluation of metabolic activity. This study explores the phasor approach in combination with two-photon fluorescence lifetime imaging microscopy (FLIM) as a potential method to evaluate pharmacologically induced metabolic changes that occur during the browning of adipocytes. The possibility of browning of white adipose cells is a desirable prospect for the treatment of obesity and related disorders. Here, we compared the results obtained by Fourier-based phasor analysis with the traditional exponential FLIM analysis as well as results of an extracellular flux analyzer. The alteration of glycolytic function and oxidative phosphorylation after treatment with pharmacological reagents significantly shift the contribution of each of the fluorescence lifetime components to the total fluorescence intensity. Further, we showed that the ratios of the lifetime components obtained by the phasor approach reflect the shift in mitochondrial and cytosolic NADH concentration. The phasor analysis agrees with traditional assessments, such as the optical free-to-bound NADH ratio as well as the oxygen consumption rate and extracellular acidification rate as determined by the extracellular flux analyzer. Our results support the concept that non-invasive sensing of fat metabolism and browning of fat may be possible by analyzing the fluorescence lifetime of NADH using the phasor approach.

Abstract: Random-Forest-basierte Segmentierung der subkutanen Fettschicht der Mäusehaut in 3D-OCT-Bilddaten

Die Kryolipolyse ist ein nichtinvasives kosmetisches Verfahren zur lokalen Fettreduktion [1], bei der durch kontrollierte Kuhlung selektiv subkutane Fettzellen zerstӧrt werden. Fur eine quantitative Evaluation des Verfahrens soll die subkutane Fettschicht in Mausen segmentiert werden. Fur eine Darstellung der Mausehaut wurde die Optische Koharenztomographie (OCT) als Bildmodalitat genutzt, die eine detaillierte Aufnahme der subkutanen Fettschicht in Mikrometer-Auflӧsung ermӧglicht.