Danielle Tenen

A molecular census of arcuate hypothalamus and median eminence cell types

The hypothalamic arcuate–median eminence complex (Arc-ME) controls energy balance, fertility and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a new leptin-sensing neuron population, multiple agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) subtypes, and an orexigenic somatostatin neuron population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type–specific responses to energy status, including distinct responses in AgRP and POMC neuron subtypes. Finally, integrating our data with human genome-wide association study data implicates two previously unknown neuron populations in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred.

The histone chaperone CAF-1 safeguards somatic cell identity

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions

BackgroundTransposable elements (TEs) have significantly influenced the evolution of transcriptional regulatory networks in the human genome. Post-transcriptional regulation of human genes by TE-derived sequences has been observed in specific contexts, but has yet to be systematically and comprehensively investigated. Here, we study a collection of 75 CLIP-Seq experiments mapping the RNA binding sites for a diverse set of 51 human proteins to explore the role of TEs in post-transcriptional regulation of human mRNAs and lncRNAs via RNA-protein interactions.ResultsWe detect widespread interactions between RNA binding proteins (RBPs) and many families of TE-derived sequence in the CLIP-Seq data. Further, alignment coverage peaks on specific positions of the TE consensus sequences, illuminating a diversity of TE-specific RBP binding motifs. Evidence of binding and conservation of these motifs in the nonrepetitive transcriptome suggests that TEs have generally appropriated existing sequence preferences of the RBPs. Depletion assays for numerous RBPs show that TE-derived binding sites affect transcript abundance and splicing similarly to nonrepetitive sites. However, in a few cases the effect of RBP binding depends on the specific TE family bound; for example, the ubiquitously expressed RBP HuR confers transcript stability unless bound to an Alu element.ConclusionsOur meta-analysis suggests a widespread role for TEs in shaping RNA-protein regulatory networks in the human genome.

IRF3 promotes adipose inflammation and insulin resistance and represses browning

The chronic inflammatory state that accompanies obesity is a major contributor to insulin resistance and other dysfunctional adaptations in adipose tissue. Cellular and secreted factors promote the inflammatory milieu of obesity, but the transcriptional pathways that drive these processes are not well described. Although the canonical inflammatory transcription factor NF-κB is considered to be the major driver of adipocyte inflammation, members of the interferon regulatory factor (IRF) family may also play a role in this process. Here, we determined that IRF3 expression is upregulated in the adipocytes of obese mice and humans. Signaling through TLR3 and TLR4, which lie upstream of IRF3, induced insulin resistance in murine adipocytes, while IRF3 knockdown prevented insulin resistance. Furthermore, improved insulin sensitivity in IRF3-deficient mice was associated with reductions in intra-adipose and systemic inflammation in the high fat-fed state, enhanced browning of subcutaneous fat, and increased adipose expression of GLUT4. Taken together, the data indicate that IRF3 is a major transcriptional regulator of adipose inflammation and is involved in maintaining systemic glucose and energy homeostasis.

Dnmt3a is an epigenetic mediator of adipose insulin resistance

Insulin resistance results from an intricate interaction between genetic make-up and environment, and thus may be orchestrated by epigenetic mechanisms like DNA methylation. Here, we demonstrate that DNA methyltransferase 3a (Dnmt3a) is both necessary and sufficient to mediate insulin resistance in cultured mouse and human adipocytes. Furthermore, adipose-specific Dnmt3a knock-out mice are protected from diet-induced insulin resistance and glucose intolerance without accompanying changes in adiposity. Unbiased gene profiling studies revealed Fgf21 as a key negatively regulated Dnmt3a target gene in adipocytes with concordant changes in DNA methylation at the Fgf21 promoter region. Consistent with this, Fgf21 can rescue Dnmt3a-mediated insulin resistance, and DNA methylation at the FGF21 locus was elevated in human subjects with diabetes and correlated negatively with expression of FGF21 in human adipose tissue. Taken together, our data demonstrate that adipose Dnmt3a is a novel epigenetic mediator of insulin resistance in vitro and in vivo.

Brown Adipose Tissue Controls Skeletal Muscle Function via the Secretion of Myostatin

Skeletal muscle and brown adipose tissue (BAT) are functionally linked, as exercise increases browning via secretion of myokines. It is unknown whether BAT affects muscle function. Here, we find that loss of the transcription factor IRF4 in BAT (BATI4KO) reduces exercise capacity, mitochondrial function, ribosomal protein synthesis, and mTOR signaling in muscle and causes tubular aggregate formation. Loss of IRF4 induces myogenic gene expression in BAT, including the secreted factor myostatin, a known inhibitor of muscle function. Reducing myostatin via neutralizing antibodies or soluble receptor rescues the exercise capacity of BATI4KO mice. In addition, overexpression of IRF4 in brown adipocytes reduces serum myostatin and increases exercise capacity in muscle. Finally, mice housed at thermoneutrality have reduced IRF4 in BAT, lower exercise capacity, and elevated serum myostatin; these abnormalities are corrected by excising BAT. Collectively, our data point to an unsuspected level of BAT-muscle crosstalk driven by IRF4 and myostatin.

NCoR1-independent mechanism plays a role in the action of the unliganded thyroid hormone receptor

Significance Thyroid hormone receptors (TRs) mediate the genomic actions of thyroid hormones. In the absence of T3 (3,3′,5-triiodothyronine), the TR recruits a multiprotein repressor complex that decreases histone acetylation in the vicinity of target genes. Nuclear receptor corepressor 1 (NCoR1) is hypothesized to be the main corepressor that interacts with TR. Here we report that the deletion of NCoR1 does not prevent all gene repression and histone deacetylation across a variety of mouse models, whereas only the lack of TR was able to overturn the effects of hypothyroidism. Thus, we conclude that NCoR1 is not sufficient to mediate the actions of the unliganded TR; furthermore, our data suggest that alternative mechanisms of repression may be involved in the action of TRs. Nuclear receptor corepressor 1 (NCoR1) is considered to be the major corepressor that mediates ligand-independent actions of the thyroid hormone receptor (TR) during development and in hypothyroidism. We tested this by expressing a hypomorphic NCoR1 allele (NCoR1ΔID), which cannot interact with the TR, in Pax8-KO mice, which make no thyroid hormone. Surprisingly, abrogation of NCoR1 function did not reverse the ligand-independent action of the TR on many gene targets and did not fully rescue the high mortality rate due to congenital hypothyroidism in these mice. To further examine NCoR1’s role in repression by the unliganded TR, we deleted NCoR1 in the livers of euthyroid and hypothyroid mice and examined the effects on gene expression and enhancer activity measured by histone 3 lysine 27 (H3K27) acetylation. Even in the absence of NCoR1 function, we observed strong repression of more than 43% of positive T3 (3,3′,5-triiodothyronine) targets in hypothyroid mice. Regulation of approximately half of those genes correlated with decreased H3K27 acetylation, and nearly 80% of these regions with affected H3K27 acetylation contained a bona fide TRβ1-binding site. Moreover, using liver-specific TRβ1-KO mice, we demonstrate that hypothyroidism-associated changes in gene expression and histone acetylation require TRβ1. Thus, many of the genomic changes mediated by the TR in hypothyroidism are independent of NCoR1, suggesting a role for additional signaling modulators in hypothyroidism.

Thermoneutrality induces skeletal muscle myopathy via brown adipose tissue in an IRF4- and myostatin-dependent manner

Skeletal muscle and brown adipose tissue (BAT) share a common lineage and have been functionally linked, as exercise increases browning through the actions of various myokines. It is unknown, however, whether BAT can affect skeletal muscle function. Our prior work has shown that loss of the transcription factor IRF4 in BAT (BATI4KO) reduces adaptive thermogenesis. Here, we note that these mice also have reduced exercise capacity relative to wild-type littermates, associated with diminished mitochondrial function, ribosomal protein synthesis, and mTOR signaling in muscle, in addition to the signature ultrastructural abnormalities of tubular aggregate myopathy. Within brown adipose tissue, loss of IRF4 caused the induction of a myogenic gene expression signature, which includes an increase in the secreted factor myostatin, a known inhibitor of muscle function. Reduction of myostatin activity by the injection of neutralizing antibodies or soluble ActRIIB receptor rescued the exercise capacity of BATI4KO mice. Additionally, overexpression of IRF4 in brown adipocytes reduced serum myostatin and increased mitochondrial function and exercise capacity in muscle. A physiological role for this system is suggested by the observation that mice housed at thermoneutrality show lower exercise capacity with increased serum myostatin; both of these abnormalities are corrected by surgical removal of BAT. Collectively, our data point to an unsuspected level of BAT-muscle cross-talk driven by IRF4 and myostatin. Highlights Mice lacking IRF4 in BAT have a decrease in exercise capacity, accompanied by histological, ultrastructural, signaling, gene expression, and bioenergetic evidence of myopathy in white vastus. Loss of IRF4 promotes the expression of a myogenic signature in BAT, including the myokine myostatin. Neutralization of serum myostatin rescues the ability of BATI4KO mice to exercise normally, while overexpression of IRF4 in BAT allows mice to run better than wild-type counterparts. Thermoneutrality reduces the level of IRF4 in BAT of WT mice, resulting in a myopathic phenotype that can be reversed by surgical excision of BAT.