FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

Background Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. Methods We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR–Cas9 genome editing in samples from patients. Results Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR–Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. Conclusions Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.)National Institutes of Health (U.S.) (R01HG004037)National Institutes of Health (U.S.) (R01GM113708)National Institutes of Health (U.S.) (R01HG008155)National Institutes of Health (U.S.) (RC1HG005334

Antagonistic and cooperative actions of Kif7 and Sufu define graded intracellular Gli activities in Hedgehog signaling.

Graded Hedgehog (Hh) signaling governs the balance of Gli transcriptional activators and repressors to specify diverse ventral cell fates in the spinal cord. It remains unclear how distinct intracellular Gli activity is generated. Here, we demonstrate that Sufu acts universally as a negative regulator of Hh signaling, whereas Kif7 inhibits Gli activity in cooperation with, and independent of, Sufu. Together, they deter naïve precursors from acquiring increasingly ventral identity. We show that Kif7 is also required to establish high intracellular Gli activity by antagonizing the Sufu-inhibition of Gli2. Strikingly, by abolishing the negative regulatory action of Sufu, diverse ventral cell fates can be specified in the absence of extracellular Hh signaling. These data suggest that Sufu is the primary regulator of graded Hh signaling and establish that the antagonistic and cooperative actions of Kif7 and Sufu are responsible for setting up distinct Gli activity in ventral cell fate specification

Kif7 promotes ectopic FP induction by restricting Sufu in <i>Ptch1</i> mutants.

<p>(<b>A–D</b>) Nkx2.2 and Olig2 expression shows an increased number of Olig2⁺ pMN cells by <i>Kif7</i> inactivation in the <i>Ptch1</i>^−/− background. (<b>E–H</b>') Shh and Foxa2 expression shows that <i>Kif7</i> inactivation abolishes ectopic FP induction in <i>Ptch1</i>^−/− embryos, which is restored by reducing one gene dosage of <i>Sufu</i>. Arrowheads indicate the dorsal limit of the neural tube. Scale bar, 25 µm. (<b>I–L</b>) In situ hybridization analysis of <i>Shh</i> RNA expression from the FP in anterior spinal cord sections. (<b>M</b>) Graphs indicate the number of FP cells, represented as the mean ± SEM, n≥5.</p

Diverse ventral fates form despite the absence of graded Shh signaling when Sufu function is abolished.

<p>(<b>A–D</b>) Nkx2.2 and Pax6 expression shows that <i>Kif7</i> or <i>Sufu</i> inactivation rescues dorsalization of the <i>Smo</i>^−/− neural tube. (<b>E–H</b>) Nkx2.2 and Olig2 expression shows that in <i>Smo</i>^−/− embryos, <i>Kif7</i> inactivation rescues Olig2⁺ pMN cell specification, but rarely that of Nkx2.2⁺ p3 cells, while <i>Sufu</i> inactivation leads to robust induction of both cell fates. (<b>I–L</b>') Shh and Foxa2 expression shows that only <i>Sufu</i> inactivation, but not that of <i>Kif7</i>, rescues FP development in <i>Smo</i>^−/− embryos. Scale bar, 25 µm. (<b>M</b>) Graphs indicate the number of pMN, p3 and FP cells, represented as the mean ± SEM, n≥4. (<b>N</b>) Western blot analysis shows reduced Gli2 expression in <i>Sufu</i>^−/− embryos are restored when <i>Smo</i> is simultaneously inactivated; little to no Gli3^83kD repressor is expressed in <i>Sufu</i>^−/− and <i>Smo</i>^−/−;<i>Sufu</i>^−/− embryos.</p

A model of Kif7 and Sufu regulation of graded Gli activity in ventral cell fate specification.

<p>The concerted actions of Kif7 and Sufu alter the Gli activator to repressor ratio to regulate intracellular Gli activity for the specification of ventral cell fates (pMN, p3 and FP) from naïve neuroepithelial precursor cells. As Shh signaling increases Smo activity, Gli repressor formation and the inhibition of activators become less efficient as denoted (<b>i–iii</b>), while robust Smo activation results in Kif7 antagonizing the inhibition of Gli activators by Sufu for p3 and FP induction (<b>iv</b>).</p

Opposing roles of Kif7 and Sufu in FP development.

<p>(<b>A–D</b>) Nkx2.2 and Olig2 immunofluorescence labeling shows increasing neural tube ventralization in E9.5 <i>Kif7</i>^−/−, <i>Sufu</i>^−/− and <i>Kif7</i>^−/−;<i>Sufu</i>^−/− embryos. (<b>E–H</b>') Shh and Foxa2 expression contrasts the FP deficiencies <i>Kif7</i>^−/− embryos to ectopic FP induction in <i>Sufu</i>^−/− and <i>Kif7</i>^−/−;<i>Sufu</i>^−/− embryos. <i>Kif7</i>^−/−;<i>Sufu</i>^−/− and <i>Sufu</i>^−/− embryos exhibit similar FP defects, indicating that <i>Kif7</i> is epistatic to <i>Sufu</i> in FP induction. Scale bar, 25 µm. (<b>I–L</b>) In situ hybridization analysis of <i>Shh</i> RNA expression verifies FP phenotypes. (<b>M</b>) Graphs indicate the number of Olig2⁺ pMN, Nkx2.2⁺ p3 and Shh⁺/Foxa2⁺ FP cells, represented as the mean ± SEM, n≥4, n.s., not significant.</p

The COMPASS complex maintains the metastatic capacity imparted by a subpopulation of cells in UPS

Summary: Intratumoral heterogeneity is common in cancer, particularly in sarcomas like undifferentiated pleomorphic sarcoma (UPS), where individual cells demonstrate a high degree of cytogenic diversity. Previous studies showed that a small subset of cells within UPS, known as the metastatic clone (MC), as responsible for metastasis. Using a CRISPR-based genomic screen in-vivo, we identified the COMPASS complex member Setd1a as a key regulator maintaining the metastatic phenotype of the MC in murine UPS. Depletion of Setd1a inhibited metastasis development in the MC. Transcriptome and chromatin sequencing revealed COMPASS complex target genes in UPS, such as Cxcl10, downregulated in the MC. Deleting Cxcl10 in non-MC cells increased their metastatic potential. Treating mice with human UPS xenografts with a COMPASS complex inhibitor suppressed metastasis without affecting tumor growth in the primary tumor. Our data identified an epigenetic program in a subpopulation of sarcoma cells that maintains metastatic potential

<i>Sufu</i>^{<i>T396I/T396I</i>} shows reduction of Gli3 activity but not Gli2 activity.

<p>(<b>A–H</b>) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), <i>Sufu</i>^{<i>T396I/T396I</i>} (B and F), and <i>Smo</i>^{<i>G457X/G457X</i>}; <i>Sufu</i>^{<i>T396I/T396I</i>} (C and G) embryos at E10.5 and <i>Smo</i>^{<i>G457X/G457X</i>} embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s007" target="_blank">S7 Fig</a>.</p